Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.     

Reliability of egg morphology to detect conspecific brood parasitism in goldeneyes Bucephala clangula examined using protein fingerprinting

Eadie (1989) developed a method based on variation between females in egg length, width and weight to detect conspecific brood parasitism in the field: using these three egg measures, Euclidean distance between all pairs of eggs within a clutch is calculated, and if maximum Euclidean distance (MED) between any two eggs exceeds a threshold value the nest is considered parasitized. The MED method has been tested in Finnish and Scottish common goldeneye Bucephala clangula populations but the results have been contradicting. Here we use protein fingerprinting to assess the validity of the MED method. Data comprised 35 clutches of which we knew, based on protein fingerprinting, how many different females laid the clutch (range 1–5 females). The mean MED of non-parasitized clutches (laid by 1 female only) was 1.470 (95% CI: lower 1.169, upper 1.771; n=21) and that of parasitized clutches (laid by 2 or more females) was 3.654 (95% CL: lower 3.083, upper 4.225; n=14). Using a MED>3.0 as a criterion to identify parasitized clutches 89% of all clutches were classified correctly either parasitized or non-parasitized when compared to the identification based on protein fingerprinting. Clutch size and the number of females (beyond 2 females) did not affect the clutch MED, whereas the status of parasitism did. Repeatability of egg length, width and weight were: 0.63, 0.76 and 0.80, respectively, implying that, variation in these egg measures occurs among rather than within females. Our new results confirm that the MED method is reliable enough to detect parasitism in common goldeneye.     

Maternal investment in relation to sex ratio and offspring number in a small mammal – a case for Trivers and Willard theory?

1.  Optimal parental sex allocation depends on the balance between the costs of investing into sons vs. daughters and the benefits calculated as fitness returns. The outcome of this equation varies with the life history of the species, as well as the state of the individual and the quality of the environment.
2.  We studied maternal allocation and subsequent fecundity costs of bank voles, Myodes glareolus , by manipulating both the postnatal sex ratio (all-male/all-female litters) and the quality of rearing environment (through manipulation of litter size by −2/+2 pups) of their offspring in a laboratory setting.
3.  We found that mothers clearly biased their allocation to female rather than male offspring regardless of their own body condition. Male pups had a significantly lower growth rate than female pups, so that at weaning, males from enlarged litters were the smallest. Mothers produced more milk for female litters and also defended them more intensively than male offspring.
4.  The results agree with the predictions based on the bank vole life history: there will be selection for greater investment in daughters rather than sons, as a larger size seems to be more influencial for female reproductive success in this species. Our finding could be a general rule in highly polygynous, but weakly dimorphic small mammals where females are territorial.
5.  The results disagree with the narrow sense Trivers & Willard hypothesis, which states that in polygynous mammals that show higher variation in male than in female reproductive success, high-quality mothers are expected to invest more in sons than in daughters.     

1H, 13C and 15N resonance assignments of the major extracytoplasmic domain of the cell shape-determining protein MreC from Bacillus subtilis

MreB, MreC and MreD are essential cell shape-determining morphogenetic proteins in Gram-positive and in Gram-negative bacteria. While MreB, the bacterial homologue of the eukaryotic cytoskeletal protein actin, has been extensively studied, the roles of MreC and MreD are less well understood. They both are transmembrane proteins. MreC has a predicted single transmembrane domain and the C-terminal part outside the cell membrane. MreC probably functions as a link between the intracellular cytoskeleton and the cell wall synthesizing machinery which is located at the outer surface of the cell membrane. Also proteins involved in cell wall synthesis participate in cell morphogenesis. How these two processes are coordinated is, however, poorly understood. Bacillus subtilis (BS), a non-pathogenic Gram-positive bacterium, is widely used as a model for Gram-positive pathogens, e.g. Staphylococcus aureus (SA). Currently, the structures of MreC from BS and SA are not known. As part of our efforts to elucidate the structure–function relationships of the morphogenetic protein complexes in Gram-positive bacteria, we present the backbone and side chain resonance assignments of the extracytoplasmic domain of MreC from BS.     

Polyketide derivatives active against Botrytis cinerea in Gerbera hybrida

A previously isolated cDNA molecule from Gerbera hybrida (Asteraceae) codes for a new chalcone synthase-like polyketide synthase, 2-pyrone synthase (2PS). 2PS is able to synthesise 4-hydroxy-6-methyl-2-pyrone (triacetolactone), a putative precursor for gerberin and parasorboside, two abundant glucosides in gerbera. In this study, we show that gerbera plants transformed with the gene for 2PS in an antisense orientation and unable to synthesise gerberin and parasorboside are susceptible to Botrytis cinerea infection. In addition to the preformed glucosides, the transgenic plants also lack several compounds that are induced in control plants when infected with the mould. Some of these induced substances are effective in inhibiting fungal growth both in vitro and in vivo. Two of the phytoalexins were identified as the aglycones of gerberin and trans-parasorboside. The third phytoalexin is a rare coumarin, 4-hydroxy-5-methylcoumarin; however, it is typical of many plants of the sunflower family Asteraceae. The coumarin cannot be structurally derived from either gerberin or parasorboside, but may be derived from a related polyketide intermediate.     

Micronuclei, hemoglobin adducts and respiratory tract irritation in mice after inhalation of toluene diisocyanate (TDI) and 4,4'-methylenediphenyl diisocyanate (MDI)

Toluene diisocyanate (TDI) and 4,4'-methylenediphenyl diisocyanate (MDI), used in the production of polyurethane foam, are well known for their irritating and sensitizing properties. Contradictory results have been obtained on their genotoxicity. We investigated the genotoxicity and protein binding of inhaled TDI and MDI in mice by examining micronucleated polychromatic erythrocytes (PCEs) in bone marrow and peripheral blood and TDI- and MDI-derived adducts in hemoglobin. Male C57Bl/6J mice (8 per group) were exposed head-only to TDI vapour (mean concentrations 1.1, 1.5, and 2.4mg/m(3); the mixture of isomers contained, on the average, 63% 2,4-TDI and 37% 2,6-TDI) or MDI aerosol (mean concentrations 10.7, 20.9 and 23.3mg/m(3)), during 1h/day for 5 consecutive days. Bone marrow and peripheral blood were collected 24h after the last exposure. Inhalation of TDI caused sensory irritation (SI) in the upper respiratory tract, and cumulative effects were observed at the highest exposure level. Inhalation of MDI produced SI and airflow limitation, and influx of inflammatory cells into the lungs. Hemoglobin adducts detected in the exposed mice resulted from direct binding to globin of 2,4- and 2,6-TDI and MDI, and dose-dependent increases were observed especially for 2,4-TDI-derived adducts. Adducts originating from the diamines of TDI (toluene diamine) or MDI (methylene dianiline) were not observed. No significant increase in the frequency of micronucleated PCEs was detected in the bone marrow or peripheral blood of the mice exposed to TDI or MDI. The ratio of PCEs and normochromatic erythrocytes (NCEs) was reduced at the highest concentration of MDI, and a slight reduction of the PCE/NCE ratio, dependent on cumulative inhaled dose, was also seen with TDI. Our results indicate that inhalation of TDI or MDI (1h/day for 5 days), at levels that induce toxic effects and formation of TDI- or MDI-specific adducts in hemoglobin, does not have detectable genotoxic effects in mice, as studied with the micronucleus assay.     

Daily Liquorice Consumption for Two Weeks Increases Augmentation Index and Central Systolic and Diastolic Blood Pressure

Background

Liquorice ingestion often elevates blood pressure, but the detailed haemodynamic alterations are unknown. We studied haemodynamic changes induced by liquorice consumption in 20 subjects versus 30 controls with average blood pressures of 120/68 and 116/64 mmHg, respectively.

Methods

Haemodynamic variables were measured in supine position before and after two weeks of liquorice consumption (daily glycyrrhizin dose 290–370 mg) with tonometric recording of radial blood pressure, pulse wave analysis, and whole-body impedance cardiography. Thirty age-matched healthy subjects maintaining their normal diet were studied as controls.

Results

Two weeks of liquorice ingestion elevated peripheral and central systolic and diastolic blood pressure (by 7/4 and 8/4 mmHg, 95% confidence intervals [CI] 2-11/1-8 and 3-13/1-8, respectively, P<0.05), and increased extracellular volume by 0.5 litres (P<0.05 versus controls). Also augmentation index adjusted to heart rate 75/min (from 7% to 11%, 95% CI for change 0.3-7.5, P<0.05) and aortic pulse pressure (by 4 mmHg, 95% CI 1-7, P<0.05) were elevated indicating increased wave reflection from the periphery. In contrast, peripheral (−3/−0.3 mmHg) and central blood pressure (−2/−0.5 mmHg), aortic pulse pressure (−1 mmHg), and augmentation index adjusted to heart rate 75/min (from 9% to 7%) decreased numerically but not statistically significantly without changes in extracellular volume in the control group. Heart rate, systemic vascular resistance, cardiac output, and pulse wave velocity did not differ between the groups.

Conclusions

Two weeks of daily liquorice consumption increased extracellular volume, amplified pressure wave reflection from the periphery, and elevated central systolic and diastolic blood pressure.

Trial Registration

EU Clinical Trials Register EudraCT 2006-002065-39</url>ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01742702","term_id":"NCT01742702"}}NCT01742702     

N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys²⁷ to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys²⁷ reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys²⁷, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys²⁷ precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys²⁷ variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.     

Co-Exposure with Fullerene May Strengthen Health Effects of Organic Industrial Chemicals

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C₆₀ fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C₆₀ and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C₆₀ and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C₆₀ that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C₆₀ that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1β and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C₆₀ co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C₆₀ molecules aggregate together, a portion of organic molecules remains in water.