Relationship between the Magnitude of Intraocular Pressure during an Episode of Acute Elevation and Retinal Damage Four Weeks later in Rats

Purpose

To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats.

Methods

Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; n = 4 to 8 each, 10–100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (−6.04 to 2.72 log cd.s.m⁻²). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m⁻²) after 15 min of light adaptation (150 cd/m²). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology.

Results

All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density.

Conclusions

Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a ‘threshold’ for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more.     

Modulation of iron homeostasis in macrophages by bacterial intracellular pathogens

Background  

Intracellular bacterial pathogens depend on acquisition of iron for their success as pathogens. The host cell requires iron as an essential component for cellular functions that include innate immune defense mechanisms. The transferrin receptor TfR1 plays an important part for delivering iron to the host cell during infection. Its expression can be modulated by infection, but its essentiality for bacterial intracellular survival has not been directly investigated.     

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.     

Cellular Interactions of the Cytolethal Distending Toxins from Escherichia coli and Haemophilus ducreyi

The cytolethal distending toxins (CDTs) compose a subclass of intracellularly acting genotoxins produced by many Gram-negative pathogenic bacteria that disrupt the normal progression of the eukaryotic cell cycle. Here, the intoxication mechanisms of CDTs from Escherichia coli (Ec-CDT) and Haemophilus ducreyi (Hd-CDT), which share limited amino acid sequence homology, were directly compared. Ec-CDT and Hd-CDT shared comparable in vitro DNase activities of the CdtB subunits, saturable cell surface binding with comparable affinities, and the requirement for an intact Golgi complex to induce cell cycle arrest. In contrast, disruption of endosome acidification blocked Hd-CDT-mediated cell cycle arrest and toxin transport to the endoplasmic reticulum and nucleus, while having no effects on Ec-CDT. Phosphorylation of the histone protein H2AX, as well as nuclear localization, was inhibited for Hd-CdtB, but not Ec-CdtB, in cells expressing dominant negative Rab7 (T22N), suggesting that Hd-CDT, but not Ec-CDT, is trafficked through late endosomal vesicles. In support of this idea, significantly more Hd-CdtB than Ec-CdtB co-localized with Rab9, which is enriched in late endosomal compartments. Competitive binding studies suggested that Ec-CDT and Hd-CDT bind to discrete cell surface determinants. These results suggest that Ec-CDT and Hd-CDT are transported within cells by distinct pathways, possibly mediated by their interaction with different receptors at the cell surface.     

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.     

Biosynthesis of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 from jatropha oil as carbon source

Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that can be synthesized through bacterial fermentation. In this study, Cupriavidus necator H16 is used to synthesize PHB by using Jatropha oil as its sole carbon source. Different variables mainly jatropha oil and urea concentrations, and agitation rate were investigated to determine the optimum condition for microbial fermentation in batch culture. Based on the results, the highest cell dry weight and PHB concentrations of 20.1 and 15.5 g/L, respectively, were obtained when 20 g/L of jatropha oil was used. Ethanol was used as external stress factor and the addition of 1.5 % ethanol at 38 h had a positive effect with a high PHB yield of 0.987 g PHB/g jatropha oil. The kinetic studies for cell growth rate and PHB production were conducted and the data were fitted with Logistic and Leudeking–Piret models. The rate constants were evaluated and the theoretical values were in accordance with the experimental data obtained.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.     

Mangiferin Antagonizes Rotenone: Induced Apoptosis Through Attenuating Mitochondrial Dysfunction and Oxidative Stress in SK-N-SH Neuroblastoma Cells

In the present study, using a human neuroblastoma SK-N-SH cells, we explored antioxidant, mitochondrial protective and antiapoptotic properties of mangiferin against rotenone-mediated cytotoxicity. SK-N-SH cells are divided into four experimental groups based on 3-(4,5-dimethyl2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay—untreated cells, cells incubated with rotenone (100 nM), cells treated with mangiferin (20 μg) (pretreatment 4 h before) + rotenone (100 nM) and mangiferin alone treated. 24 h after treatment with rotenone and 28 h after treatment with mangiferin, levels of ATP thiobarbituricacid reactive substances and reduced glutathione and activities of enzymatic antioxidants including superoxide dismutase, catalase and glutathione peroxidise were measured. Finally mitochondrial transmembrane potential and expressions of apoptotic protein were also analysed. Pre-treatment with mangiferin significantly enhanced cell viability, ameliorated decrease in mitochondrial membrane potential and decreased rotenone-induced apoptosis in the cellular model of Parkinson’s disease. Moreover oxidative imbalance induced by rotenone was partially rectified by mangiferin. Our results indicated that anti-apoptotic properties of this natural compound due to its antioxidant and mitochondrial protective function protect rotenone induced cytotoxicity.