Fish Oil and Oxidative Stress by Inflammatory Leukocytes

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO^?) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of l-arginine or the NOS inhibitor l-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO^? production by the oxymyoglobin oxidation assay, or electrochemically using an NO^? electrode. An apparent NO^? production detected by the Griess assay was identified as an artifact. NO^? generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO^?.

Our data show that NO^? production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Sub-chronic exposure to 1,1-dichloropropene induces frameshift mutations in lambda transgenic medaka

1,1-Dichloropropene (1,1-DCP) is a contaminant present in both ground and surface waters used as sources for drinking water. Structural similarity to several compounds with known mutagenicity and carcinogenicity, and recent demonstration of mutagenicity in vitro, suggest this compound may be similarly mutagenic in vivo. A transgenic fish model, the lamda transgenic medaka, was used to evaluate the potential mutagenicity of this contaminant in vivo following sub-chronic exposure for 6 weeks. Mutant frequencies of the cII target gene (MF) increased six-fold in the livers of fish exposed to the lowest 1,1-DCP exposure concentration (0.44 mg/L, MF = 18.4 x 10(-5), and increased with each treatment, culminating in a 32-fold induction in fish from the highest 1,1-DCP treatment (16.60 mg/L, MF = 96.3 x 10(-5). Mutations recovered from treated fish showed a distinctive mutational spectrum comprised predominantly of +1 frameshift mutations, induced 166-fold above that of untreated animals. The majority of frameshifts were +1 insertions at thiamine and adenine. These results represent the first evidence of mutagenicity of 1,1-DCP in vivo, and of the highly characteristic spectrum of induced mutations dominated by +1 frameshift mutations. Based upon results from previous in vitro studies, the similar role of glutathione S-transferase (GSTT1-1) in the activation of 1,1-DCP to a mutagen in vivo is also suggested. This study further illustrates the utility of the lamda transgenic medaka as a model for identifying and characterizing potential genetic health risks associated with chemical exposures in the environment.     

Protective role of beta-chemokines associated with HIV-specific Th responses against perinatal HIV transmission

To examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as beta-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with beta-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with beta-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.     

Loss of 3-nitrotyrosine on exposure to hypochlorous acid: implications for the use of 3-nitrotyrosine as a bio-marker in vivo

Peroxynitrite (ONOO-) is a reactive nitrogen species which in vivo is often assessed by the measurement of free or protein bound 3-nitrotyrosine. Indeed, 3-nitrotyrosine has been detected in many human diseases. However, at sites of inflammation there is also production of the powerful oxidant hypochlorous acid (HOCl) formed by the enzyme myeloperoxidase. Low concentrations of HOCl (<30 microM) caused significant and rapid loss (<10 minutes) of free and protein bound 3-nitrotyrosine. In contrast, no loss of 3-nitrotyrosine was observed with hydrogen peroxide, hydroxyl radical, or superoxide generating systems. Therefore, under conditions where there is concomitant peroxynitrite and hypochlorous acid formation, such as at sites of chronic inflammation, it is possible that HOCl removes 3-nitrotyrosine. This may have implications when assessing the role of reactive nitrogen species in disease conditions and could account for some of the discrepancies reported between 3-nitrotyrosine levels in tissues.     

Evolution of mammalian X-linked and autosomal Pgk and Pdh E1 alpha subunit genes

The phylogeny and substitution rates of the mammalian X chromosome- located and autosomal phosphoglycerate kinase and pyruvate dehydrogenase genes were investigated. Compatibility analysis was used to show reticulate evolution in these genes. Analysis of the marsupial, mouse, and human phosphoglycerate kinase genes suggests that at least two recombination events have taken place, one occurring about the time of the placental-marsupial split involving exons 1-5 and the other before the primate-rodent split involving exons 9-10. Similar analysis of the pyruvate dehydrogenase genes indicates a recombination event involving exons 2-3 at a time before the primate-rodent split and a gene conversion between exons 3-4 in the human somatic and testis- specific pyruvate dehydrogenase genes after the primate-rodent split. This demonstrates that genetic exchange can occur between paralogous genes at widely separated chromosomal locations. Estimation of nucleotide substitution rates in these genes confirmed a higher substitution rate in the pyruvate dehydrogenase genes. In the phosphoglycerate kinase genes, there is no difference between the substitution rates in mice and humans and between the X chromosome- and autosome-located genes. A greater substitution rate was noted in the mouse autosomal pyruvate dehydrogenase gene when compared with the other mouse and human genes. This may be a result of either directional natural selection or a relaxation of functional constraint at this specific gene.      

Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.     

Blueberry-induced changes in spatial working memory correlate with changes in hippocampal CREB phosphorylation and brain-derived neurotrophic factor (BDNF) levels

Phytochemical-rich foods have been shown to be effective at reversing age-related deficits in memory in both animals and humans. We show that a supplementation with a blueberry diet (2% w/w) for 12 weeks improves the performance of aged animals in spatial working memory tasks. This improvement emerged within 3 weeks and persisted for the remainder of the testing period. Memory performance correlated well with the activation of cAMP-response element-binding protein (CREB) and increases in both pro- and mature levels of brain-derived neurotrophic factor (BDNF) in the hippocampus. Changes in CREB and BDNF in aged and blueberry-supplemented animals were accompanied by increases in the phosphorylation state of extracellular signal-related kinase (ERK1/2), rather than that of calcium calmodulin kinase (CaMKII and CaMKIV) or protein kinase A. Furthermore, age and blueberry supplementation were linked to changes in the activation state of Akt, mTOR, and the levels of Arc/Arg3.1 in the hippocampus, suggesting that pathways involved in de novo protein synthesis may be involved. Although causal relationships cannot be made among supplementation, behavior, and biochemical parameters, the measurement of anthocyanins and flavanols in the brain following blueberry supplementation may indicate that changes in spatial working memory in aged animals are linked to the effects of flavonoids on the ERK-CREB-BDNF pathway.     

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H₂S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H₂S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund''s adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO₂⁻), PGE₂, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.     

Consequences of long-distance swimming and travel over deep-water pack ice for a female polar bear during a year of extreme sea ice retreat

Polar bears (Ursus maritimus) prefer to live on Arctic sea ice but may swim between ice floes or between sea ice and land. Although anecdotal observations suggest that polar bears are capable of swimming long distances, no data have been available to describe in detail long distance swimming events or the physiological and reproductive consequences of such behavior. Between an initial capture in late August and a recapture in late October 2008, a radio-collared adult female polar bear in the Beaufort Sea made a continuous swim of 687 km over 9 days and then intermittently swam and walked on the sea ice surface an additional 1,800 km. Measures of movement rate, hourly activity, and subcutaneous and external temperature revealed distinct profiles of swimming and walking. Between captures, this polar bear lost 22% of her body mass and her yearling cub. The extraordinary long distance swimming ability of polar bears, which we confirm here, may help them cope with reduced Arctic sea ice. Our observation, however, indicates that long distance swimming in Arctic waters, and travel over deep water pack ice, may result in high energetic costs and compromise reproductive fitness.