Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells,the cell line of murine Friend leukemia virus‐induced leukemic cells

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α‐helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus‐induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose‐dependent and time‐dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G₀/G₁ phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta

Protein tyrosine phosphatase 1B (PTP1B) appears to be an attractive target for the development of new drugs for type 2 diabetes and obesity. In our preliminary test, a MeOH extract of the stem barks of Sorbus commixta Hedl. (Rosaceae) showed strong PTP1B inhibitory activity. Bioassay?guided fractionation of the MeOH extract resulted in the isolation of two lupane?type triterpenes, lupenone (1) and lupeol (2). Compounds 1 and 2 inhibited PTP1B with IC₅₀ values of 13.7 ± 2.1 and 5.6 ± 0.9 μM, respectively. Kinetic studies revealed that both the compounds 1 and 2 are non?competitive inhibitors of PTP1B that decrease V_max values with no effect on K_m values.     

WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis

Caenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed ‘WormFarm’, an integrated microfluidic device for culturing nematodes. Cohorts of 30–50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof‐of‐principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate‐based assay for both RNAi‐mediated and dietary‐induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age‐associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo‐3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Combination of 1,4-naphthoquinone with benzothiazoles had selective algicidal effects against harmful algae

A series of naphthoquinone-benzothiazole conjugates were synthesized as algicides, and their efficacies against harmful algal blooming species, such as Chattonella marina, Heterosigma akashiwo and Cochlodinium polykrikoides, were examined. The introduction of substituted benzothiazole at the C2 position of 1,4-naphthoquinone (compounds 1–9) resulted in higher algicidal activity against C. polykrikoides than the C6 conjugates (compounds 10–20). On the other hand, of the C6 conjugates, compounds 11 and 12 exhibited better algicidal activity against H. akashiwo, C. marina, and C. polykrikoides than the C2 conjugates. Further structure-activity analysis indicated that a replacement of the methoxy groups with hydroxyl groups (compounds 21–26) decreased the algicidal activity significantly. Among the various synthetic naphthoquinonebezothiazole conjugates tested, compound 12 was found to affect the most significant decrease in the level of C. polykrikoides growth, with an IC₅₀ of 0.19 μM. Compound 11 was found to be the most potent inhibitor against H. akashiwo and C. polykrikoides, with IC₅₀ values of 0.32 and 0.12 μM, respectively. Overall, these results highlight a possible method for controlling and inhibiting red tide forming algae using NQ derivatives.     

The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in HeLa and SiHa cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p27^Kip1 and p21^Cip1, leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.     

Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt^thr308 and ^ser473; these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.     

Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Background

To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results

Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions

These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.