Hypoxia-induced mitochondrial stress granules.

Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining hypoxia-induced mitochondrial protein aggregation in C. elegans, we found that sublethal hypoxia, sodium azide, or heat shock-induced abundant ethidium bromide staining mitochondrial granules that preceded evidence of protein aggregation. Genetic manipulations that reduce cellular and organismal hypoxic death block the formation of these mitochondrial stress granules (mitoSG). Knockdown of mitochondrial nucleoid proteins also blocked the formation of mitoSG by a mechanism distinct from the mitochondrial unfolded protein response. Lack of the major mitochondrial matrix protease LONP-1 resulted in the constitutive formation of mitoSG without external stress. Ethidium bromide-staining RNA-containing mitochondrial granules were also observed in rat cardiomyocytes treated with sodium azide, a hypoxia mimetic. Mitochondrial stress granules are an early mitochondrial pathology controlled by LONP and the nucleoid, preceding hypoxia-induced protein aggregation.

Effect of the mitochondrial unfolded protein response on hypoxic death and mitochondrial protein aggregation.

Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPRmt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPRmt to determine the relationship between hypoxic cell death, UPRmt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPRmt activation invariantly mitigated HIMPA. However, UPRmt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPRmt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.

A screen for protective drugs against delayed hypoxic injury.

Despite longstanding efforts to develop cytoprotective drugs against ischemia/reperfusion (IR) injuries, there remains no effective therapeutics to treat hypoxic injury. The failure of traditional strategies at solving this problem suggests the need for novel and unbiased approaches that can lead to previously unsuspected targets and lead compounds. Towards this end, we report here a unique small molecule screen in the nematode C. elegans for compounds that improve recovery when applied after the hypoxic insult, using a C. elegans strain engineered to have delayed cell non-autonomous death. In a screen of 2000 compounds, six were found to produce significant protection of C. elegans from delayed death. Four of the compounds were tested in an ex vivo mouse heart ischemia/reperfusion model and two, meclocycline and 3-amino-1,2,4-triazole, significantly reduced infarction size. Our work demonstrates the feasibility of this novel C. elegans screen to discover hypoxia protective drugs that are also protective in a mammalian model of hypoxic injury.

Investigation of RNA interference suppression of matrix metalloproteinase-9 in mouse model of atherosclerosis.

OBJECTIVE: To investigate the effect of RNA interference of matrix metalloproteinase (MMP)-9 on atherosclerosis on atherosclerosis in apolipoprotein E (ApoE)-/- mouse. METHODS: ApoE-/- mouse strain and three cell lines (293T, NIH3T3 and Raw264.7) were used in the present study to investigate the effect of MMP-9 silencing by RNA interference. Thirty 10-week-old ApoE-/- mice were randomly assigned to a control group, lentiviruses with naked vector group and Lentiviruses-MMP-9 intervention group (n = 10). Aortic atherosclerotic plaques of the mice were stained with immunohistochemical techniques, the MMP-9 and high-sensitivity C-reactive protein levels of three groups were detected simultaneously. Expression of MMP-9 was significantly down-regulated in interference group. MMP-9 and high-sensitivity C-reactive protein levels in MMP-9 interference group were significantly lower than that of the control group. CONCLUSION: The expression of MMP-9 is closely related to vulnerability of atherosclerotic plaques. Silencing of MMP-9 expression acts as a positive role in maintenance of atherosclerotic plaque stability. The present study provides novel experimental insight for the treatment of vulnerable plaques in atherosclerosis.

Synthesis and Anticonvulsant Activity Evaluation of 3-alkoxy-4-(4-(hexyloxy/heptyloxy)phenyl)-4H-1,2,4 -triazole.

A series of 3-alkoxy-4-(4-(hexyloxy/heptyloxy) phenyl)-4H-1,2,4-triazole was synthesized. The anticonvulsant effect and neurotoxicity of the compounds were calculated with maximal electroshock (MES) test and rotarod tests with intraperitoneally injected mice. Among the synthesized compounds, compound 3-heptyloxy-4-(4-(hexyloxy) phenyl)-4H-1,2,4-triazole (5f) was the most active one and also had the lowest toxicity. In the anti-MES potency test, it showed median effective dose (ED50) of 37.3 mg/Kg, median toxicity dose (TD50) of 422.5 mg/Kg, and the protective index (PI) of 11.3 which is much greater than the reference drug carbamazepine with PI value of 6.4. As well as demonstrating the anti-MES efficacy of compound 5f, its potency against seizures induced by pentylenetetrazole, 3-mercaptopropionic acid, and bicuculline were also established, with the results suggesting that GABA-mediated mechanisms might be involved in its anticonvulsant activity, such as enhancing of GABAergic neurotransmission or activity, activate GAD or inhibit GABA-T, and GABAA-mediated mechanisms.

[Virus spectrum features of adult influenza-like fever in outpatients].

OBJECTIVE: To analyze the results of detection on respiratory virus of influenza-like illness ( ILI ) in Beijing from June 2010 to February 2012 and understand the virus spectrum of adult influenza-like fever. METHODS: A total of 502 swabs were collected and 279 throat swabs tested for 12 respiratory viruses with multiplex reverse transcription-polymerase chain reaction (RT-PCR). And 413 swabs were tested for pH1N1 by virus isolation influenza viruses. And the data were statistically analyzed. RESULTS: One or two viruses were detected in 26.9% (75/279) of the samples. Influenza A virus (FLU-A) accounted for 85.3% of positive samples and 22.9% (64/279) of ILI tested. The positive rate of other viruses was less than 3.0 %. The positive rates among the following subtypes were: 2.7% (11/413) for pH1N1, 2.4% (10/413) for H3 and 6.5% (27/413) for FLU-B. FLU-A was the predominant virus during the 2010-2011 influenza season and the positive rate peaked in January 2011 in Beijing and north China. FLU-B was the primary virus during the 2011-2012 influenza season and the positive rate peaked in January and February 2012. There was a significant reduction in the incidence of ILI in 2010 and 2011 when compared with 2009. During the 2009-2012 influenza seasons, the incidence peaked in December 2009, January 2011 and January and February 2012 in Beijing. CONCLUSIONS: Exposure to pH1N1 had no impact on typical influenza seasonal peaks. Influenza virus was the predominant virus of adult influenza-like fever cases after the pandemic period of influenza A (H1N1) 2009 and the positive rate peaked in January and February during the 2009-2012 influenza seasons.

Role of oxygen consumption in hypoxia protection by translation factor depletion.

The reduction of protein synthesis has been associated with resistance to hypoxic cell death. Which components of the translation machinery control hypoxic sensitivity and the precise mechanism has not been systematically investigated, although a reduction in oxygen consumption has been widely assumed to be the mechanism. Using genetic reagents in Caenorhabditis elegans, we examined the effect on organismal survival after hypoxia of knockdown of 10 factors functioning at the three principal steps in translation. Reduction-of-function of all 10 translation factors significantly increased hypoxic survival to varying degrees, not fully accounted for by the level of translational suppression. Measurement of oxygen consumption showed that strong hypoxia resistance was possible without a significant decrease in oxygen consumption. Hypoxic sensitivity had no correlation with lifespan or reactive oxygen species sensitivity, two phenotypes associated with reduced translation. Resistance to tunicamycin, which produces misfolded protein toxicity, was the only phenotype that significantly correlated with hypoxic sensitivity. Translation factor knockdown was also hypoxia protective for mouse primary neurons. These data show that translation factor knockdown is hypoxia protective in both C. elegans and mouse neurons and that oxygen consumption does not necessarily determine survival; rather, mitigation of misfolded protein toxicity is more strongly associated with hypoxic protection.

Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila.

BACKGROUND: We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process. METHODS: We explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP. RESULTS: We first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger. CONCLUSION: Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry.

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1.

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.

[Expression of novel apoptosis-related protein PDCD5 in granulosa cells of polysystic ovary syndrome].

OBJECTIVE: To detect the novel apoptosis-related protein PDCD5 expression in granulosa cells of polysystic ovary syndrome (PCOS) and normal ovary, and explore the pathogenesis of PCOS. METHODS: The granulosa cells were collected from 30 cases of PCOS and normal ovary in IVF-ET. Expression of PDCD5 was detected by flow cytometry; immunofluorescence and immunohistochemistry. Cell apoptosis was detected by Propidium Iodide (PI) staining. RESULTS: The number of hypodiploidy cells associated with apoptosis in granulosa cells of PCOS was greater than that of the normal control. PDCD5 protein expression in PCOS granulosa cells was significantly higher than that in normal ovary (P<0.05). PDCD5-positive cells were mainly observed in cytoplasm. There was no significant difference between the two groups in terms of age, basal serum FSH concentration, number of ampules of FSH administered. CONCLUSION: Our data suggest that PDCD5 is an important apoptosis regulating factor in granulosa cells of PCOS and normal ovary, which might be involved in the pathogenesis of PCOS.

Potential attenuation of p38 signaling by DDB2 as a factor in acquired TNF resistance.

Our previous study demonstrated that DDB2, a DNA repair protein, attenuates cell surface membrane-associated death signal induced by UV or FasAb; DDB2 is overexpressed in cisplatin-selected cells. However, the molecular mechanism underlying the protective role of DDB2 along the apoptotic pathway remains unknown. Our study identified the cross-resistance of the cisplatin-selected cells to tumor necrosis factor-alpha (TNF-alpha). Since knock-down of the DDB2 level rendered cells (HR18) sensitive to the treatment, the cell sensitivity to TNF-alpha appears inversely proportional to the cellular level of DDB2. Treatment of HeLa cells with TNF-alpha transiently induced activation of p38MAPK signal, but this induction was significantly reduced in the resistant cells. Overexpression of DDB2 attenuated the activation of p38 in cells. TNF-alpha-induced apoptotic signals, represented by caspase-8 and downstream substrate cleavage, were reduced in resistant cells compared to their sensitive counterparts. Inhibition of p38 signal by SB202190 clearly attenuated TNF-alpha-induced apoptotic signals. Moreover, overexpression of DDB2 in HR18 cells also attenuated TNF-alpha induced caspase activation. These results suggest that p38MAPK activation may be a key upstream signal of TNF-alpha-induced apoptosis and that attenuation of p38 signal by DDB2 overexpression may be responsible for acquired TNF-alpha resistance.

Cross-resistance to death ligand-induced apoptosis in cisplatin-selected HeLa cells associated with overexpression of DDB2 and subsequent induction of cFLIP.

This work reports the involvement of damaged DNA-binding protein 2 (DDB2), a component involved in the genomic repair of UV damage, in the cross-resistance of cisplatin-selected cell lines to death ligand-mediated apoptosis. The cisplatin-resistant cell line (HR3) exhibits enhanced expression of DDB2 and cross-resistance to UV-induced activation of apoptosis and caspases. This investigation further demonstrates that HR3 cells also exhibited cross-resistance to death ligands [Fas-inducing antibody and tumor necrosis factor (TNF)-alpha]. Depletion of the elevated DDB2 in HR3 cells sensitizes Fas-inducing antibody-induced and TNF-alpha-induced apoptosis. In contrast, the overexpression of DDB2 induces cellular FLICE-like inhibitory protein (cFLIP) expression and further attenuates death ligand-induced apoptosis. Moreover, reverse transcription-polymerase chain reaction and reporter assay indicated that DDB2 could increase both endogenous and exogenous cFLIP mRNA levels. Accordingly, the elimination of cFLIP by antisense oligonucleotides suppresses DDB2 protection. These findings reveal that DDB2 regulates TNF signaling-mediated apoptosis via cFLIP and contributes to acquired cross-resistance. DDB2, while participating in DNA repair, functions as a negative regulator of apoptosis and may therefore have a pivotal role in regulating immune response and cancer-therapeutic efficacy.

[The differential expression profile of polycystic ovary syndrome associated genes].

OBJECTIVE: To explore the gene differential expression pattern of polycystic ovary syndrome. METHODS: We carried out microarray analysis to define the gene networks by the PCOS granulosa cells in order to identify differentially expressed genes in PCOS patients. These granulosa cells of five PCOS cases and five control cases which were derived during oocyte retrieval from women undergoing IVF. RESULTS: As compared with control human ovarian granulosa cells, 46 genes were screened out, 25 genes were up-regulated, and 21 genes were down-regulated in PCOS. These differentially expressed genes were involved in various biologic functions, such as regulation of fatty acid metabolism, cell-cell signal transduction, immune and inflammatory response, reflecting the complexity of clinical manifestations of PCOS. CONCLUSION: Microarray analysis technology is an effective method to identify novel PCOS associated candidate genes.