High glucose and interleukin-1β downregulate interleukin-1 type I receptor (IL-1RI) in retinal endothelial cells by enhancing its degradation by a lysosome-dependent mechanism

Diabetic retinopathy has been considered a low-grade chronic inflammatory disease. The production of interleukin-1β (IL-1β) in the retina is increased, and this finding has been correlated with an increase in blood-retinal barrier permeability, suggesting that IL-1β might have an important role in the pathogenesis of diabetic retinopathy. However, in this context, no attention has been given to interleukin-1 type I receptor (IL-1RI), which is the receptor responsible for IL-1β triggered effects. Therefore, we investigated the effect of high glucose and IL-1β on the IL-1RI regulation in retinal endothelial cells. A time-dependent downregulation of IL-1RI protein levels was detected in retinal endothelial cells exposed (1–24 h) to high glucose, mannitol or IL-1β. Long-term exposure (7 days) to high glucose or mannitol also decreased IL-1RI protein content. IL-1RI downregulation was due to its activation by IL-1β, since it was inhibited by the presence of anti-IL-1RI or anti-IL-1β antibodies. Moreover, IL-1RI downregulation was prevented by lysosome inhibitors, chloroquine and ammonium chloride, but not by proteasome inhibitors, MG132 and lactacystin. We also found that IL-1RI translocates to the nucleus after high glucose or IL-1β treatment. In conclusion, our results indicate that high glucose, probably due to osmotic stress, and IL-1β downregulate IL-1RI in retinal endothelial cells. The downregulation of IL-1RI is triggered by its activation and is due, at least partially, to lysosomal degradation. High glucose and IL-1β also enhance the translocation of IL-1RI to the nucleus.     

Binding of Azole Antibiotics to Staphylococcus aureus Flavohemoglobin Increases Intracellular Oxidative Stress

In this work, we report that flavohemoglobin contributes to the azole susceptibility of Staphylococcus aureus. We first observed that deletion of the flavohemoglobin gene leads to an increase in the viability of imidazole-treated S. aureus cells and that reversion to the wild-type phenotype occurs upon expression of flavohemoglobin from a multicopy plasmid. Further spectroscopic analyses showed that miconazole, the most efficient azole antibiotic against S. aureus, ligates to heme of both oxidized and reduced flavohemoglobin. The binding of miconazole to oxidized flavohemoglobin, with an association constant of 1.7 × 10⁶ M⁻¹, typical of a tight, specific binding equilibrium, results in augmentation of the superoxide production by the enzyme. These results are corroborated by in vivo studies showing that imidazole-treated S. aureus cells expressing flavohemoglobin contain a larger amount of reactive oxygen species. Moreover, it was observed that the survival of miconazole-treated S. aureus internalized by murine macrophages is higher for cells lacking flavohemoglobin. Altogether, the present data revealed that in S. aureus, flavohemoglobin enhances the antimicrobial activity of imidazoles via an increase of intracellular oxidative stress.Staphylococcus aureus is an opportunistic pathogen responsible for a large number of human infections that cause systemic diseases from a mild to life-threatening character. The increasing incidence of methicillin-resistant S. aureus (MRSA) strains observed in the past few years makes S. aureus infections a leading threat to public health, causing more deaths in the United States and Europe than human immunodeficiency virus (AIDS) (11). Like other Gram-positive bacteria, staphylococci are sensitive to imidazoles (27). Imidazoles (such as clotrimazole, miconazole, ketoconazole, and sulconazole) (Fig. ​(Fig.1)1) represent one of the major classes of azole antifungal that are useful in the treatment of infections, including cutaneous and vaginal candidiasis (8). The activity of these antifungal drugs derives primarily from inhibition of the biosynthesis of ergosterol, an essential component of the fungal plasma membrane, at the level of lanosterol 14-alpha demethylase. Furthermore, in fungi and yeast, azole treatment leads to an increase in the endogenous production of reactive oxygen species (ROS) (12, 25). For example, in Candida albicans and Saccharomyces cerevisiae, the miconazole inhibition of cytochrome c oxidase, peroxidase, and catalase has been reported to be responsible for a high level of ROS production (3, 4). It has also been reported that clotrimazole inhibition of Plasmodium falciparum hemoperoxidase leads to ROS accumulation in this protozoan pathogen (26). For S. cerevisiae, C. albicans, and Escherichia coli, the action of imidazoles was also correlated with the inhibition of the nitric oxide (NO) scavenger activity of flavohemoglobin (7).Open in a separate windowFIG. 1.Structures of the azole (imidazole; 1,2,4-triazole) antibiotics investigated.Flavohemoglobins (Hmp) are widespread among bacteria and yeast and contain three domains: C-terminal NAD- and flavin adenine dinucleotide (FAD)-binding domains, which together constitute a ferredoxin-NADP⁺ oxidoreductase-like domain, and an N-terminal globin domain, which harbors a single B-type heme. The high-spin heme contains one axial histidine and binds small molecules like NO, carbon monoxide (CO), and dioxygen (O₂). The heme can also bind bulky aromatic bases, since it is inserted in a large hydrophobic pocket (7). We observed that the binding of imidazoles to S. aureus flavohemoglobin results in an increase in the amount of deleterious reactive oxygen species produced by flavohemoglobin that contributes to the bactericidal effect of azole antibiotics toward S. aureus.     

Characterization of missense mutations and large deletions in the ALPL gene by sequencing and quantitative multiplex PCR of short fragments

Hypophosphatasia is a rare inherited bone disorder characterized by defective bone and dental mineralization and deficiency of serum and liver/bone/kidney alkaline phosphatase activity. The disease is due to mutations in the alkaline phosphatase liver-type (ALPL) gene. Gross deletions or insertions have not previously been reported in this gene. We report here the characterization of nine novel ALPL gene mutations in a series of 8 patients affected by various forms of hypophosphatasia. The newly discovered mutations included five missense mutations (c.368C --> A, c.814C--> T, c.1196C--> T, c.1199C--> T, c.1283G--> C), two small deletions (c.797_802del, c.1044_1055del), and two large deletions. The large deletions were detected by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF). We conclude that QMPSF slightly reduces the proportion of undetected mutations in hypophosphatasia and improves genetic counselling in the affected families.     

Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein

A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation.     

A novel membrane-bound Ech [NiFe] hydrogenase in Desulfovibrio gigas

In the present study, we report the identification of an operon with six coding regions for a multisubunit membrane-bound [NiFe] hydrogenase in the genome of Desulfovibrio gigas. Sequence analysis of the deduced polypeptides reveals a high similarity to subunits of proteins belonging to the family of Ech hydrogenases. The operon is organised similarly to the operon coding for the Ech hydrogenase from Methanosarcina barkeri, suggesting that both encode very similar hydrogenases. Expression of the operon was detected by Northern blot and RT-PCR analyses, and the presence of the encoded proteins was examined by Western blotting. The possible role of this hydrogenase is discussed, relating it with a potential function in the H(2) cycling as a mechanism for energy conservation in D. gigas. The present study provides therefore valuable insights into the open question of the energy conserving mechanism in D. gigas.     

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.     

A Unique Pool of Compatible Solutes on Rhodopirellula baltica,Member of the Deep-Branching Phylum Planctomycetes

The intracellular accumulation of small organic solutes was described in the marine bacterium Rhodopirellula baltica, which belongs to the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. Sucrose, α-glutamate, trehalose and mannosylglucosylglycerate (MGG) are the main solutes involved in the osmoadaptation of R. baltica. The ratio and total intracellular organic solutes varied significantly in response to an increase in salinity, temperature and nitrogen content. R. baltica displayed an initial response to both osmotic and thermal stresses that includes α-glutamate accumulation. This trend was followed by a rather unique and complex osmoadaptation mechanism characterized by a dual response to sub-optimal and supra-optimal salinities. A reduction in the salinity to sub-optimal conditions led primarily to the accumulation of trehalose. In contrast, R. baltica responded to salt stress mostly by increasing the intracellular levels of sucrose. The switch between the accumulation of trehalose and sucrose was by far the most significant effect caused by increasing the salt levels of the medium. Additionally, MGG accumulation was found to be salt- as well as nitrogen-dependent. MGG accumulation was regulated by nitrogen levels replacing α-glutamate as a K⁺ counterion in nitrogen-poor environments. This is the first report of the accumulation of compatible solutes in the phylum Planctomycetes and of the MGG accumulation in a mesophilic organism.     

A simple framework to describe the regulation of gene expression in prokaryotes

Based on the bimolecular mass action law and the derived mass conservation laws, we propose a mathematical framework in order to describe the regulation of gene expression in prokaryotes. It is shown that the derived models have all the qualitative properties of the activation and inhibition regulatory mechanisms observed in experiments. The basic construction considers genes as templates for protein production, where regulation processes result from activators or repressors connecting to DNA binding sites. All the parameters in the models have a straightforward biological meaning. After describing the general properties of the basic mechanisms of positive and negative gene regulation, we apply this framework to the self-regulation of the trp operon and to the genetic switch involved in the regulation of the lac operon. One of the consequences of this approach is the existence of conserved quantities depending on the initial conditions that tune bifurcations of fixed points. This leads naturally to a simple explanation of threshold effects as observed in some experiments.     

A nhaD Na+/H+ antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo1 to nqo7 (nqoA) and nqo10 to nqo14 (nqoB)] and two independent genes (nqo8 and nqo9). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4alpha-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo13 and nqo14. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.