Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells.     

Bacteria and protozoa differentially modulate the expression of rab proteins

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.     

Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain

Inflammatory lipid mediators derived from arachidonic acid (AA) and docosahexaenoic acid (DHA) modify the pathophysiology of brain ischemia. The goal of this work was to investigate the formation of eicosanoids and docosanoids generated from AA and DHA, respectively, during no-flow cerebral ischemia. Rats were subjected to head-focused microwave irradiation 5 min following decapitation (complete ischemia) or prior to decapitation (controls). Brain lipids were extracted and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry. After complete ischemia, brain AA, DHA, and docosapentaenoic acid concentrations increased 18-, 5- and 4-fold compared with controls, respectively. Prostaglandin E(2) (PGE(2)) and PGD(2) could not be detected in control microwaved rat brain, suggesting little endogenous PGE(2)/D(2) production in the brain in the absence of experimental manipulation. Concentrations of thromboxane B(2), E(2)/D(2)-isoprostanes, 5-hydroxyeicosatetraenoic acid (5-HETE), 5-oxo-eicosatetraenoic acid, and 12-HETE were significantly elevated in ischemic brains. In addition, DHA products such as mono-, di- and trihydroxy-DHA were detected in control and ischemic brains. Monohydroxy-DHA, identified as 17-hydroxy-DHA and thought to be the immediate precursor of neuroprotectin D(1), was 6.5-fold higher in ischemic than in control brain. The present study demonstrated increased formation of eicosanoids, E(2)/D(2)-IsoPs, and docosanoids following cerebral ischemia. A balance of these lipid mediators may mediate immediate events of ischemic injury and recovery.     

Aminoacyl-tRNA synthetase classes and groups in prokaryotes

Knowledge on the evolution of aminoacyl-tRNA synthetases is crucial to studies on the origins of life. The relationships between the different aminoacyl-tRNA synthetase specificities in prokaryotic organisms are studied in this work. We reconstructed the ancestor sequences and the phylogenetic relationships utilizing the Maximum Likelihood method. The results suggest that in class I the evolution of the N-terminal segment was strongly influenced by the amino acid hydropathy in both domains of prokaryotes. The results for the C-terminal segments of class I were different in the two domains, indicating that its evolution was strongly influenced by the specific types of tRNA modification in each domain. The class II groups in Archaea were more heterogeneous with respect to the hydropathy of amino acids, indicating the interference of other influences. In bacteria, the configuration was also complex but the overall consensual division in two groups was maintained, group IIa forming a single branch with the five hydroapathetic amino acid specificities and group IIb containing the specificities for the moderately hydrophobic together with the hydrophilic amino acids. It is indicated that the aminoacyl-tRNA synthetase in both domains were subjected to different selective forces in diverse parts of the proteins, resulting in complex phylogenetic patterns.     

Isolation and characterization of microsatellite markers for the ornamental discus fish Symphysodon discus and cross-species amplification in other Heroini cichlid species

The discus fishes (Symphysodon spp.) are economically important ornamental species. Thirteen microsatellite markers were developed from a CT(12) - and CA(12) -enriched whole genomic DNA library of Symphysodon discus. Allelic variability was tested on 44 individuals of two species (S. discus and S. aequifasciatus). Allelic richness ranged from two to 11 alleles per locus and observed heterozygosities from 0.083 to 0.998. All loci were at Hardy-Weinberg equilibrium, and no pair of loci showed linkage disequilibrium within a species. Cross-species amplification was also successfully performed in the Neotropical cichlids Uaru amphiacanthoides, Hoplarchus psittacus, Hypselecara coryphaenoides, Pterophyllum sp., Mesonauta sp. and Heros sp.     

Hyperbaric oxygen prevents early death caused by experimental cerebral malaria

Background

Cerebral malaria (CM) is a syndrome characterized by neurological signs, seizures and coma. Despite the fact that CM presents similarities with cerebral stroke, few studies have focused on new supportive therapies for the disease. Hyperbaric oxygen (HBO) therapy has been successfully used in patients with numerous brain disorders such as stroke, migraine and atherosclerosis.

Methodology/Principal Findings

C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were exposed to daily doses of HBO (100% O₂, 3.0 ATA, 1–2 h per day) in conditions well-tolerated by humans and animals, before or after parasite establishment. Cumulative survival analyses demonstrated that HBO therapy protected 50% of PbA-infected mice and delayed CM-specific neurological signs when administrated after patent parasitemia. Pressurized oxygen therapy reduced peripheral parasitemia, expression of TNF-α, IFN-γ and IL-10 mRNA levels and percentage of γδ and αβ CD4⁺ and CD8⁺ T lymphocytes sequestered in mice brains, thus resulting in a reduction of blood-brain barrier (BBB) dysfunction and hypothermia.

Conclusions/Significance

The data presented here is the first indication that HBO treatment could be used as supportive therapy, perhaps in association with neuroprotective drugs, to prevent CM clinical outcomes, including death.     

The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation

The antioxidant activity of β-carotene and oxygenated carotenoids lutein, canthaxanthin, and astaxanthin was investigated during spontaneous and peroxyl-radical-induced cholesterol oxidation. Cholesterol oxidation, measured as generation of 7-keto-cholesterol (7-KC), was evaluated in a heterogeneous solution with cholesterol, AAPH, and carotenoids solubilized in tetrahydrofuran and in water, and in a homogeneous solution of chlorobenzene, with AIBN as a prooxidant. The formation of 7-KC was dependent on temperature and on cholesterol and prooxidant concentrations. All the carotenoids tested, exhibited significant antioxidant activity by inhibiting spontaneous, AAPH- and AIBN-induced formation of 7-KC, although the overall order of efficacy of these compounds was astaxanthin > canthaxanthin > lutein = β-carotene. The finding that carotenoids exert protective effects on spontaneous and free radical-induced cholesterol oxidation may have important beneficial effects on human health, by limiting the formation of atheroma and by inhibiting cholesterol oxidation in food processing or storage.     

Algal contact as a trigger for coral disease

Diseases are causing alarming declines in reef‐building coral species, the foundation blocks of coral reefs. The emergence of these diseases has occurred simultaneously with large increases in the abundance of benthic macroalgae. Here, we show that physical contact with the macroalga Halimeda opuntia can trigger a virulent disease known as white plague type II that has caused widespread mortality in most Caribbean coral species. Colonies of the dominant coral Montastraea faveolata exposed to algal transplants developed the disease whereas unexposed colonies did not. The bacterium Aurantimonas coralicida, causative agent of the disease, was present on H. opuntia sampled close to, and away from diseased corals, indicating that the alga serves as a reservoir for this pathogen. Our results suggest that the spread of macroalgae on coral reefs could account for the elevated incidence of coral diseases over past decades and that reduction of macroalgal abundance could help control coral epizootics.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)