Geraldine Saavedra

Rapid Activation of Antioxidant Defenses by Nerve Growth Factor Suppresses Reactive Oxygen Species during Neuronal Apoptosis: Evidence for a Role in Cytochrome c Redistribution

Depriving mouse sympathetic neurons of nerve growth factor (NGF) causes their apoptotic death. A Bax-dependent increase of mitochondrial-derived reactive oxygen species (ROS) begins in these cells soon after NGF withdrawal. We investigated the effects on these ROS of adding NGF to cultures of NGF-deprived neurons. ROS levels were monitored with the fluorescent, redox-sensitive dyes CM-H2DCFDA and MitoSOX Red. The intensity of the former dye increases when it is oxidized by H2O2 and free radicals downstream of H2O2. MitoSOX Red is relatively insensitive to oxidation by H2O2 but is sensitive to oxidation by superoxide (O2.−). Withdrawing NGF increased CM-H2DCFDA intensity, indicating elevated H2O2-associated ROS. Re-exposure of cells deprived of NGF to NGF resulted in rapid suppression of these ROS. Neurons deprived of NGF also had increased MitoSOX Red intensities. Readdition of NGF had no effect on MitoSOX Red fluorescence. The suppression of CM-H2DCFDA-detected ROS by NGF was caused by a rapid activation of glutathione redox cycling. The most likely explanation for these findings is that mitochondria increased O2.− production after NGF withdrawal. The O2.− was converted to H2O2 by dismutation, and the H2O2 was detoxified by accelerated glutathione redox cycling. Our previous work shows that H2O2 induces cytochrome c to be released from mitochondria in NGF-supported sympathetic neurons, whereas antioxidants that detoxify H2O2 block cytochrome c redistribution in NGF-deprived neurons. Readdition of NGF also immediately inhibits cytochrome c release. We present evidence that this inhibition is mediated by the rapid activation of glutathione redox cycling by NGF.

Respiratory syncytial virus modifies microRNAs regulating host genes that affect virus replication.

Respiratory syncytial virus (RSV) causes substantial morbidity and life-threatening lower respiratory tract disease in infants, young children and the elderly. Understanding the host response to RSV infection is critical for developing disease-intervention approaches. The role of microRNAs (miRNAs) in post-transcriptional regulation of host genes responding to RSV infection is not well understood. In this study, it was shown that RSV infection of a human alveolar epithelial cell line (A549) induced five miRNAs (let-7f, miR-24, miR-337-3p, miR-26b and miR-520a-5p) and repressed two miRNAs (miR-198 and miR-595), and showed that RSV G protein triggered let-7f expression. Luciferase–untranslated region reporters and miRNA mimics and inhibitors validated the predicted targets, which included cell-cycle genes (CCND1, DYRK2 and ELF4), a chemokine gene (CCL7) and the suppressor of cytokine signalling 3 gene (SOCS3). Modulating let-7 family miRNA levels with miRNA mimics and inhibitors affected RSV replication, indicating that RSV modulates host miRNA expression to affect the outcome of the antiviral host response, and this was mediated in part through RSV G protein expression.

Replication and pathogenesis associated with H5N1, H5N2, and H5N3 low-pathogenic avian influenza virus infection in chickens and ducks.

A comparative study examining replication and disease pathogenesis associated with low-pathogenic H5N1, H5N2, or H5N3 avian influenza virus (AIV) infection of chickens and ducks was performed. The replication and pathogenesis of highly pathogenic AIV (HPAIV) has received substantial attention; however, the behavior of low-pathogenic AIVs, which serve as precursors to HPAIVs, has received less attention. Thus, chickens or ducks were inoculated with an isolate from a wild bird [A/Mute Swan/MI/451072/06 (H5N1)] or isolates from chickens [A/Ck/PA/13609/93 (H5N2), A/Ck/TX/167280-4/02 (H5N3)], and virus replication, induction of a serological response, and disease pathogenesis were investigated, and the hemagglutinin and neuraminidase (NA) gene sequences of the isolates were determined. Virus isolated from tracheal and cloacal swabs showed that H5N1 replicated better in ducks, whereas H5N2 and H5N3 replicated better in chickens. Comparison of the NA gene sequences showed that chicken-adapted H5N2 and H5N3 isolates both have a deletion of 20 amino acids in the NA stalk region, which was absent in the H5N1 isolate. Histopathological examination of numerous organs showed that H5N2 and H5N3 isolates caused lesions in chickens in a variety of organs, but to a greater extent in the respiratory and intestinal tracts, whereas H5N1 lesions in ducks were observed mainly in the respiratory tract. This study suggests that the H5N1, H5N2, and H5N3 infections occurred at distinct sites in chicken and ducks, and that comparative studies in different model species are needed to better understand the factors influencing the evolution of these viruses.

Differential Roles for Cytosolic and Microsomal Ca2+-Independent Phospholipase A2 in Cell Growth and Maintenance of Phospholipids

Physiological roles of microsomal (iPLA2γ) and cytosolic (iPLA2β)Ca2+-independent phospholipase A2 were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA2 inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA2γ and iPLA2β, respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 μM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA2β siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA2γ siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA2β and iPLA2γ siRNA on cell number and protein was also determined, and iPLA2β siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA2γ siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA2β, but not iPLA2γ, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA2β inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA2β inhibition results in greater decreases in cell growth and proliferation than iPLA2γ, identifies specific phospholipids whose expressions are differentially regulated by iPLA2β and iPLA2γ, and suggests novel roles for iPLA2β in cell growth.

Bax Regulates Production of Superoxide in Both Apoptotic and Nonapoptotic Neurons: Role of Caspases

A Bax- and, apparently, mitochondria-dependent increase in superoxide (O2·−) and other reactive oxygen species (ROS) occurs in apoptotic superior cervical ganglion (SCG) and cerebellar granule (CG) neurons. Here we show that Bax also lies upstream of ROS produced in nonapoptotic neurons and present evidence that caspases partially mediate the pro-oxidant effect of Bax. We used the O2·−-sensitive dye MitoSOX to monitor O2·− in neurons expressing different levels of Bax and mitochondrial superoxide dismutase (SOD2). Basal and apoptotic O2·− levels in both SCG and CG neurons were reduced in SOD2 wild-type (WT) cells having lower Bax concentrations. Apoptotic and nonapoptotic neurons from Bax-WT/SOD2-null but not Bax-null/SOD2-null mice had increased O2·− levels. A caspase inhibitor inhibited O2·− in both apoptotic and nonapoptotic SCG neurons. O2·− production increased when WT, but not Bax-null, SCG neurons were permeabilized and treated with active caspase 3. There was no apoptosis and little increase in O2·− in SCG neurons from caspase 3-null mice exposed to an apoptotic stimulus. O2·− levels in nonapoptotic caspase 3-null SCG neurons were lower than in WT cells but not as low as in caspase inhibitor-treated cells. These data indicate that Bax lies upstream of most O2·− produced in neurons, that caspase 3 is required for increased O2·− production during neuronal apoptosis, that caspase 3 is partially involved in O2·− production in nonapoptotic neurons, and that other caspases may also be involved in Bax-dependent O2·− production in nonapoptotic cells.

Seroprevalence of Toxoplasma gondii in Swine From Slaughterhouses in Lima, Peru, and Georgia, U.S.A.

Toxoplasma gondii is an important pathogen transmitted by food, with raw or undercooked meat as the main foodborne source of toxoplasmosis. In Peru, 2–4 million people have antibodies to T. gondii. It is believed that more than 60 million people in the United States are infected with T. gondii. In this study, the prevalence of T. gondii in pigs from Peru and the United States was determined by Western blot. The presence of IgG antibodies to T. gondii from serum samples was determined. Blood samples were collected from 137 pigs at a slaughterhouse in Lima, Peru, and 152 pigs at a slaughterhouse in Georgia. Of the serum samples collected from swine, 27.7% (n = 38) from Peru and 16.4% (n = 25) from the United States were positive for T. gondii. Swine represent a significant source of human infection with T. gondii in Peru and the United States.

Passage of low-pathogenic avian influenza (LPAI) viruses mediates rapid genetic adaptation of a wild-bird isolate in poultry.

For a better understanding of evolution of influenza viruses, a chicken-origin and wild-bird-origin low-pathogenic avian influenza virus (LPAI) was serially passaged in chickens. Sequences of the hemagglutinin (HA) and neuraminidase (NA) genes at each passage level were compared to those of the parental virus. Multiple mutations occurring early during passage were detected, but these were maintained during passages. Interestingly, a number of the observed mutations already existed in the parental virus, as indicated by the presence of single-nucleotide polymorphisms. The greatest numbers of mutations occurred during passage of wild-bird-origin LPAI, where a 20-amino-acid deletion in the NA gene that was observed during the first passage was maintained during subsequent passages. Subsequent experiments showed that this NA deletion was already present as a minority population in the parental virus. These results showed that a selection process favoring a viral subpopulation had occurred.