Hypochlorous acid-mediated mitochondrial dysfunction and apoptosis in human hepatoma HepG2 and human fetal liver cells: role of mitochondrial permeability transition

Liver cirrhosis is often preceded by overt signs of hepatitis, including parenchymal cell inflammation and infiltration of polymorphonuclear (PMN) leukocytes. Activated PMNs release both reactive oxygen species and reactive halogen species, including hypochlorous acid (HOCl), which are known to be significantly cytotoxic due to their oxidizing potential. Because the role of mitochondria in the hepatotoxicity attributed to HOCl has not been elucidated, we investigated the effects of HOCl on mitochondrial function in the human hepatoma HepG2 cell line, human fetal liver cells, and isolated rat liver mitochondria. We show here that HOCl induced mitochondrial dysfunction, and apoptosis was dependent on the induction of the mitochondrial permeability transition (MPT), because HOCl induced mitochondrial swelling and collapse of the mitochondrial membrane potential with the concomitant release of cytochrome c. These biochemical events were inhibited by the classical MPT inhibitor cyclosporin A (CSA). Cell death induced by HOCl exhibited several classical hallmarks of apoptosis, including annexin V labeling, caspase activation, chromatin condensation, and cell body shrinkage. The induction of apoptosis by HOCl was further supported by the finding that CSA and caspase inhibitors prevented cell death. For the first time, these results show that HOCl activates the MPT, which leads to the induction of apoptosis and provides a novel insight into the mechanisms of HOCl-mediated cell death at sites of chronic inflammation.     

Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 10(7) PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.     

Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains

The introduction of West Nile virus (WNV) into North America has been associated with relatively high rates of neurological disease and death in humans, birds, horses, and some other animals. Previous studies identified strains in both genetic lineage 1 and genetic lineage 2, including North American isolates of lineage 1, that were highly virulent in a mouse neuroinvasion model, while other strains were avirulent or significantly attenuated (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. T. Barrett, Virology 296:17-23, 2002). To begin to elucidate the basis for these differences, we compared a highly virulent New York 1999 (NY99) isolate with a related Old World lineage 1 strain, An4766 (ETH76a), which is attenuated for mouse neuroinvasion. Genomic sequencing of ETH76a revealed a relatively small number of nucleotide (5.1%) and amino acid (0.6%) differences compared with NY99. These differences were located throughout the genome and included five amino acid differences in the envelope protein gene. Substitution of premembrane and envelope genes of ETH76a into a NY99 infectious clone backbone yielded a virus with altered in vitro growth characteristics and a mouse virulence phenotype comparable to ETH76a. Further site-specific mutagenesis studies revealed that the altered phenotype was primarily mediated via loss of envelope protein glycosylation and that this was associated with altered stability of the virion at mildly acidic pH. Therefore, the enhanced virulence of North American WNV strains compared with other Old World lineage 1 strains is at least partly mediated by envelope protein glycosylation.     

Evolutionary ecology of facultative paedomorphosis in newts and salamanders

Facultative paedomorphosis is an environmentally induced polymorphism that results in the coexistence of mature, gilled, and fully aquatic paedomorphic adults and transformed, terrestrial, metamorphic adults in the same population. This polymorphism has been of interest to scientists for decades because it occurs in a large number of caudate amphibian taxa as well as in a large diversity of habitats. Numerous experimental and observational studies have been conducted to explain the proximate and ultimate factors affecting these heterochronic variants in natural populations. The production of each alternative phenotype is based on a genotypexenvironment interaction and research suggests that differences in the environment can produce paedomorphs through several ontogenetic pathways. No single advantage accounts for the maintenance of this polymorphism. Rather, the interplay of different costs and benefits explains the success of the polyphenism across variable environments. Facultative paedomorphosis allows individuals to cope with habitat variation, to take advantage of environmental heterogeneity in the presence of open niches, and to increase their fitness. This process is expected to constitute a first step towards speciation events, and is also an example of biodiversity at the intraspecific level. The facultative paedomorphosis system is thus ripe for future studies encompassing ecology, evolution, behaviour, endocrinology, physiology, and conservation biology. Few other systems have been broad enough to provide varied research opportunities on topics as diverse as phenotypic plasticity, speciation, mating behaviour, and hormonal regulation of morphology. Further research on facultative paedomorphosis will provide needed insight into these and other important questions facing biologists.     

A G1127S change in calcium-binding epidermal growth factor-like domain 13 of human fibrillin-1 causes short range conformational effects

Human fibrillin-1, an extracellular matrix glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats. A missense mutation that changes a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissue disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13 but not cbEGF14. We have used limited proteolysis methods and two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construct. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified cleavage sites showed calcium-dependent protection from proteolysis. However, small changes in the conformation of cbEGF13 G1127S, revealed by the presence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to the wild-type, but was native-like. Additional cleavage sites identified in cbEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains. We have concluded the following in this study. (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13. This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.     

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods

The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities.     

Symptoms of Obstructive Sleep Apnea,Gastroesophageal Reflux and the Risk of Barrett’s Esophagus in a Population-Based Case-Control Study

Background

Gastroesophageal reflux is overrepresented in people with obstructive sleep apnea (OSA) and it has been suggested that OSA worsens gastroesophageal reflux symptoms. Aggravated reflux might lead to an increased risk of Barrett’s esophagus.

Aim

To assess the association between sleep apnea symptoms and Barrett’s esophagus.

Methods

Included in a case-control study in Brisbane, Australia were 237 patients with histologically confirmed Barrett’s esophagus and 247 population controls. The controls were randomly selected from the electoral roll and frequency-matched to the cases by age and sex. Information on OSA symptoms (excessive daytime sleepiness and sleep related apnea symptoms), gastroesophageal reflux symptoms and anthropometric measures were collected through interviews and written questionnaires. Multivariable logistic regression provided odds ratios (OR) and 95% confidence intervals (CI), adjusted for potential confounding by BMI and gastroesophageal reflux.

Results

The prevalence of Barrett’s esophagus was higher among people with excessive daytime sleepiness than those without (24% vs. 18%; p-value 0.1142) and in participants with sleep-related apnea symptoms (20% vs. 13%; p-value 0.1730). However, there were non-significantly increased ORs of Barrett’s esophagus among people with excessive daytime sleepiness (OR 1.42, 95% CI 0.90–2.34) and sleep related apnea symptoms (OR 1.32, 95% CI 0.74–2.36) when adjusting for age, sex and BMI. After further adjustment for gastroesophageal reflux symptoms, the point ORs were no longer increased (OR 1.02, 95% CI 0.61–1.70 for daytime sleepiness and OR 0.72, 95% CI 0.38–1.38 for sleep related apnea symptoms).

Conclusions

Symptoms of OSA are possibly associated with an increased risk of Barrett’s esophagus, an association that appears to be mediated entirely by gastroesophageal reflux.     

Pathogen-Triggered Ethylene Signaling Mediates Systemic-Induced Susceptibility to Herbivory in Arabidopsis

Multicellular eukaryotic organisms are attacked by numerous parasites from diverse phyla, often simultaneously or sequentially. An outstanding question in these interactions is how hosts integrate signals induced by the attack of different parasites. We used a model system comprised of the plant host Arabidopsis thaliana, the hemibiotrophic bacterial phytopathogen Pseudomonas syringae, and herbivorous larvae of the moth Trichoplusia ni (cabbage looper) to characterize mechanisms involved in systemic-induced susceptibility (SIS) to T. ni herbivory caused by prior infection by virulent P. syringae. We uncovered a complex multilayered induction mechanism for SIS to herbivory. In this mechanism, antiherbivore defenses that depend on signaling via (1) the jasmonic acid–isoleucine conjugate (JA-Ile) and (2) other octadecanoids are suppressed by microbe-associated molecular pattern–triggered salicylic acid (SA) signaling and infection-triggered ethylene signaling, respectively. SIS to herbivory is, in turn, counteracted by a combination of the bacterial JA-Ile mimic coronatine and type III virulence-associated effectors. Our results show that SIS to herbivory involves more than antagonistic signaling between SA and JA-Ile and provide insight into the unexpectedly complex mechanisms behind a seemingly simple trade-off in plant defense against multiple enemies.     

Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H?0?), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H?0? production. H?0? gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0? was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0?. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H?0? generated by ascorbate and phenolic compounds, but there was still substantial H?0? generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H?0? generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H?0? generation is discussed.