Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2)

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N ¹-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded. This revised version was published online in July 2006 with corrections to the Cover Date.     

Positive selection is the main driving force for evolution of citrus canker-causing Xanthomonas

Understanding the evolutionary history and potential of bacterial pathogens is critical to prevent the emergence of new infectious bacterial diseases. Xanthomonas axonopodis subsp. citri (Xac) (synonym X. citri subsp. citri), which causes citrus canker, is one of the hardest-fought plant bacterial pathogens in US history. Here, we sequenced 21 Xac strains (14 XacA, 3 XacA* and 4 XacA^w) with different host ranges from North America and Asia and conducted comparative genomic and evolutionary analyses. Our analyses suggest that acquisition of beneficial genes and loss of detrimental genes most likely allowed XacA to infect a broader range of hosts as compared with XacA^w and XacA*. Recombination was found to have occurred frequently on the relative ancient branches, but rarely on the young branches of the clonal genealogy. The ratio of recombination/mutation ρ/θ was 0.0790±0.0005, implying that the Xac population was clonal in structure. Positive selection has affected 14% (395 out of 2822) of core genes of the citrus canker-causing Xanthomonas. The genes affected are enriched in ‘carbohydrate transport and metabolism'' and ‘DNA replication, recombination and repair'' genes (P<0.05). Many genes related to virulence, especially genes involved in the type III secretion system and effectors, are affected by positive selection, further highlighting the contribution of positive selection to the evolution of citrus canker-causing Xanthomonas. Our results suggest that both metabolism and virulence genes provide advantages to endow XacA with higher virulence and a wider host range. Our analysis advances our understanding of the genomic basis of specialization by positive selection in bacterial evolution.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

Regulation and function of xanthophyll cycle-dependent photoprotection in algae

The xanthophyll cycle represents one of the important photoprotection mechanisms in plant cells. In the present review, we summarize current knowledge about the violaxanthin cycle of vascular plants, green and brown algae, and the diadinoxanthin cycle of the algal classes Bacillariophyceae, Xanthophyceae, Haptophyceae, and Dinophyceae. We address the biochemistry of the xanthophyll cycle enzymes with a special focus on protein structure, co-substrate requirements and regulation of enzyme activity. We present recent ideas regarding the structural basis of xanthophyll cycle-dependent photoprotection, including different models for the mechanism of non-photochemical quenching of chlorophyll a fluorescence. In a dedicated chapter, we also describe the unique violaxanthin antheraxanthin cycle of the Prasinophyceae, together with its implication for the mechanism of xanthophyll cycle-dependent heat dissipation. The interaction between the diadinoxanthin cycle and alternative electron flow pathways in the chloroplasts of diatoms is an additional topic of this review, and in the last chapter we cover aspects of the importance of xanthophyll cycle-dependent photoprotection for different algal species in their natural environments.     

Winter Distribution,Movements, and Annual Survival of Radiomarked Vancouver Canada Geese in Southeast Alaska

ABSTRACT Management of Pacific Flyway Canada geese (Branta canadensis) requires information on winter distribution of different populations. Recoveries of tarsus bands from Vancouver Canada geese (B. canadensis fulva) marked in southeast Alaska, USA, ≥4 decades ago suggested that ≥83% of the population was non-migratory and that annual adult survival was high (Ŝ = 0.836). However, recovery distribution of tarsus bands was potentially biased due to geographic differences in harvest intensity in the Pacific Flyway. Also, winter distribution of Vancouver Canada geese could have shifted since the 1960s, as has occurred for some other populations of Canada geese. Because winter distribution and annual survival of this population had not recently been evaluated, we surgically implanted very high frequency radiotransmitters in 166 adult female Canada geese in southeast Alaska. We captured Vancouver Canada geese during molt at 2 sites where adults with goslings were present (breeding areas) and 2 sites where we observed nonbreeding birds only. During winter radiotracking flights in southeast Alaska, we detected 98% of 85 females marked at breeding areas and 83% of 70 females marked at nonbreeding sites, excluding 11 females that died prior to the onset of winter radiotracking. We detected no radiomarked females in coastal British Columbia, or western Washington and Oregon, USA. Most (70%) females moved ≤30 km between November and March. Our model-averaged estimate of annual survival (Ŝ = 0.844, SE = 0.050) was similar to the estimate of annual survival of geese marked from 1956 to 1960. Likely <2% of Vancouver Canada geese that nest in southeast Alaska migrate to winter areas in Oregon or Washington where they could intermix with Canada geese from other populations in the Pacific Flyway. Because annual survival of adult Vancouver Canada geese was high and showed evidence of long-term consistency, managers should examine how reproductive success and recruitment may affect the population.     

Inter‐regional comparison of land‐use effects on stream metabolism

1. Rates of whole‐system metabolism (production and respiration) are fundamental indicators of ecosystem structure and function. Although first‐order, proximal controls are well understood, assessments of the interactions between proximal controls and distal controls, such as land use and geographic region, are lacking. Thus, the influence of land use on stream metabolism across geographic regions is unknown. Further, there is limited understanding of how land use may alter variability in ecosystem metabolism across regions. 2. Stream metabolism was measured in nine streams in each of eight regions (n = 72) across the United States and Puerto Rico. In each region, three streams were selected from a range of three land uses: agriculturally influenced, urban‐influenced, and reference streams. Stream metabolism was estimated from diel changes in dissolved oxygen concentrations in each stream reach with correction for reaeration and groundwater input. 3. Gross primary production (GPP) was highest in regions with little riparian vegetation (sagebrush steppe in Wyoming, desert shrub in Arizona/New Mexico) and lowest in forested regions (North Carolina, Oregon). In contrast, ecosystem respiration (ER) varied both within and among regions. Reference streams had significantly lower rates of GPP than urban or agriculturally influenced streams. 4. GPP was positively correlated with photosynthetically active radiation and autotrophic biomass. Multiple regression models compared using Akaike’s information criterion (AIC) indicated GPP increased with water column ammonium and the fraction of the catchment in urban and reference land‐use categories. Multiple regression models also identified velocity, temperature, nitrate, ammonium, dissolved organic carbon, GPP, coarse benthic organic matter, fine benthic organic matter and the fraction of all land‐use categories in the catchment as regulators of ER. 5. Structural equation modelling indicated significant distal as well as proximal control pathways including a direct effect of land‐use on GPP as well as SRP, DIN, and PAR effects on GPP; GPP effects on autotrophic biomass, organic matter, and ER; and organic matter effects on ER. 6. Overall, consideration of the data separated by land‐use categories showed reduced inter‐regional variability in rates of metabolism, indicating that the influence of agricultural and urban land use can obscure regional differences in stream metabolism.