Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Bootstrapping phylogenies inferred from rearrangement data

ABSTRACT: Large-scale sequencing of genomes has enabled the inference of phylogenies based on the evolution of genomic architecture, under such events as rearrangements, duplications, and losses. Many evolutionary models and associated algorithms have been designed over the last few years and have found use in comparative genomics and phylogenetic inference. However, the assessment of phylogenies built from such data has not been properly addressed to date. The standard method used in sequence-based phylogenetic inference is the bootstrap, but it relies on a large number of homologous characters that can be resampled; yet in the case of rearrangements, the entire genome is a single character. Alternatives such as the jackknife suffer from the same problem, while likelihood tests cannot be applied in the absence of well established probabilistic models. We present a new approach to the assessment of distance-based phylogenetic inference from whole-genome data; our approach combines features of the jackknife and the bootstrap and remains nonparametric. For each feature of our method, we give an equivalent feature in the sequence-based framework; we also present the results of extensive experimental testing, in both sequence-based and genome-based frameworks. Through the feature-by-feature comparison and the experimental results, we show that our bootstrapping approach is on par with the classic phylogenetic bootstrap used in sequence-based reconstruction, and we establish the clear superiority of the classic bootstrap for sequence data and of our corresponding new approach for rearrangement data over proposed variants. Finally, we test our approach on a small dataset of mammalian genomes, verifying that the support values match current thinking about the respective branches. Our method is the first to provide a standard of assessment to match that of the classic phylogenetic bootstrap for aligned sequences. Its support values follow a similar scale and its receiver-operating characteristics are nearly identical, indicating that it provides similar levels of sensitivity and specificity. Thus our assessment method makes it possible to conduct phylogenetic analyses on whole genomes with the same degree of confidence as for analyses on aligned sequences. Extensions to search-based inference methods such as maximum parsimony and maximum likelihood are possible, but remain to be thoroughly tested.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

In planta production of a candidate vaccine against bovine papillomavirus type 1

Bovine papillomavirus type 1 (BPV-1) is an economically important virus that induces tumourigenic pathologies in horses and cows. Given that the BPV-1 L1 major coat protein can self-assemble into highly immunogenic higher-order structures, we transiently expressed it in Nicotiana benthamiana as a prelude to producing a candidate vaccine. It was found that plant codon optimization of L1 gave higher levels of expression than its non-optimized counterpart. Following protein extraction, we obtained high yields (183 mg/kg fresh weight leaf tissue) of relatively pure L1, which had self-assembled into virus-like particles (VLPs). We found that these VLPs elicited a highly specific and strong immune response, and therefore they may have utility as a potential vaccine. This is the first report demonstrating the viable production of a candidate BPV vaccine protein in plants.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

感染球孢白僵菌后红火蚁体内蛋白质含量的变化

为了解球孢白僵菌Beauveria bassiana成功感染红火蚁Solenopsis invicta的生理生化机制, 本研究在室内条件下测定了经球孢白僵菌感染后红火蚁工蚁、蛹和幼蚁体内蛋白质含量的变化。结果表明: 红火蚁各虫态感染球孢白僵菌后体内蛋白质含量变化存在差异。在接种后12-72 h, 工蚁体内蛋白质含量在24 h时最高, 为47.12 mg/g, 显著低于对照的56.40 mg/g(P＜0.05), 此后呈下降趋势, 72 h时蛋白质含量下降至25.16 mg/g。幼蚁在接种36 h时体内蛋白质含量最高, 为24.13 mg/g, 此后呈下降趋势, 72 h时蛋白质含量为8.95 mg/g, 显著低于对照的24.80 mg/g。蛹感染球孢白僵菌后体内蛋白质含量一直呈下降趋势, 从12 h的36.57 mg/g降到72 h的10.42 mg/g, 与对照的38.61 mg/g相比差异显著(P＜0.05)。结果显示球孢白僵菌感染能显著降低红火蚁各虫态体内蛋白质含量。     

Cicada ear geometry: species and sex effects

Many insect species rely on their sense of audition to find a mate, to localize prey or to escape from a predator. Cicadas are particularly known for their loud call and the conspicuous tympanal hearing system located in their abdomen. The vibration pattern of the tympanal membrane (TM) has been investigated recently revealing mechanical properties specific to species and sex. Although TM size and shape is likely to affect these patterns, the geometry of the cicada ear has never been examined per se. Focusing on three Mediterranean cicada species, namely Cicadatra atra, Cicada orni and Lyristes plebejus, we investigated the structure of TM shape variation at two levels, within and across species. Applying an elliptic Fourier analysis to the outlines of both male and female TMs, we estimated sexual dimorphism and species effects. Cicadatra atra showed a large TM compared with its small size, probably as a result of selective constraints related to the role of the TM in sound production. Sexual dimorphism seemed to be greater than interspecific variation, indicating that constraints operating on sex might be more selective than those acting on species identification. In addition, C. orni appeared to be significantly different from the two other species. This morphological peculiarity could be related to the unique vibrational pattern of its membrane. This would establish for the first time a direct link between the shape and mechanism of a hearing organ. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 922–934.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.