Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 : primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c 3

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8?Å electron density maps. The refinement at 1.8?Å resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c ₃, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229–514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c ₃ from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c ₃ and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c ₃ upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.     

In vitro effects of citral on trypanosoma cruzi metacyclogenesis

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

Evidence of cylindrospermopsin uptake and clearance in fish (Oreochromis niloticus) under laboratory conditions

Cylindrospermopsin (CYN) is a cyanotoxin that has raised serious concerns about public health in many parts of the world. It can bioaccumulate and affect the health of aquatic organisms, but despite this, few studies have been conducted on CYN uptake and clearance in fish. In this paper, the authors evaluate the uptake and clearance of CYN in the muscle tissue and viscera of juvenile tilapia (Oreochromis niloticus) after exposure to aqueous extracts and whole cells of Cylindrospermopsis raciborskii (CYN-producer). CYN blended with commercial fish food, and three experiments were conducted. In the first trial, fish food, and aqueous extracts containing 0.31 μg CYN g⁻¹ of food per day, was administered to tilapia for 15 days. In the second trial, fish were provided food and intact cells (5.4 μg CYN g⁻¹ of food per day) for 15 days. In the last trial, they were provided fish food and aqueous extracts (0.8 μg CYN g⁻¹ of food per day) for 12 days, and for the next 10 days, the animals were fed food without toxic cell extracts (to simulate a clearance period). The concentration of CYN in muscle tissue and viscera was analysed using ELISA. In the case of juvenile tilapia, the presence of CYN was higher in viscera than in muscle tissue, and the toxin remained in the tissues even after 10 days without the addition of contaminated food. The results suggest that tilapia represents a potential source of CYN transfer through the food web, and this shows the need for a continuous monitoring of this compound in organisms that are used for human and animal consumption.     

Evaluation of the Role of Glutathione in the Lead-Induced Toxicity in Saccharomyces cerevisiae

A Gram-negative, aerobic, short-rod-shaped, non-motile bacterium designated Rs7^T, was isolated from peat soil collected from Russia and was characterized to determine its taxonomic position. 16S rRNA gene sequence analysis revealed that the strain Rs7^T belongs to the class Betaproteobacteria. The highest degree of sequence similarities were determined to be with Burkholderia tropica Ppe8^T (98.4 %), Burkholderia unamae MTI-641^T (97.8 %), Burkholderia bannensis E25^T (97.7 %), Burkholderia heleia SA41^T (97.0 %), and Burkholderia sacchari IPT101^T (97.0 %). Chemotaxonomic data revealed that the strain Rs7^T possesses ubiquinone Q-8. The polar lipid profile of strain Rs7^T contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unknown amino phospholipid. The predominant fatty acids were C_16:0, C_19:0 cyclo ω8c, and C_17:0 cyclo, all of which corroborated the assignment of the strain to the genus Burkholderia. The DNA G+C content was 63.2 mol%. DNA–DNA hybridization experiments showed less than 37.8 % DNA relatedness with closely related type strains, thus confirming separate species status. The results of physiological and biochemical tests allowed phenotypic differentiation of strain Rs7^T from the members of the genus Burkholderia. Based on these data, Rs7^T (=KEMC 7302-068^T = JCM 18069^T) should be classified as the type strain for a novel Burkholderia species, for which the name Burkholderia humi sp. nov. is proposed.     

Influence of Genetic Variation on Plasma Protein Levels in Older Adults Using a Multi-Analyte Panel

Proteins, widely studied as potential biomarkers, play important roles in numerous physiological functions and diseases. Genetic variation may modulate corresponding protein levels and point to the role of these variants in disease pathophysiology. Effects of individual single nucleotide polymorphisms (SNPs) within a gene were analyzed for corresponding plasma protein levels using genome-wide association study (GWAS) genotype data and proteomic panel data with 132 quality-controlled analytes from 521 Caucasian participants in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort. Linear regression analysis detected 112 significant (Bonferroni threshold p = 2.44×10⁻⁵) associations between 27 analytes and 112 SNPs. 107 out of these 112 associations were tested in the Indiana Memory and Aging Study (IMAS) cohort for replication and 50 associations were replicated at uncorrected p<0.05 in the same direction of effect as those in the ADNI. We identified multiple novel associations including the association of rs7517126 with plasma complement factor H-related protein 1 (CFHR1) level at p<1.46×10⁻⁶⁰, accounting for 40 percent of total variation of the protein level. We serendipitously found the association of rs6677604 with the same protein at p<9.29×10⁻¹¹². Although these two SNPs were not in the strong linkage disequilibrium, 61 percent of total variation of CFHR1 was accounted for by rs6677604 without additional variation by rs7517126 when both SNPs were tested together. 78 other SNP-protein associations in the ADNI sample exceeded genome-wide significance (5×10⁻⁸). Our results confirmed previously identified gene-protein associations for interleukin-6 receptor, chemokine CC-4, angiotensin-converting enzyme, and angiotensinogen, although the direction of effect was reversed in some cases. This study is among the first analyses of gene-protein product relationships integrating multiplex-panel proteomics and targeted genes extracted from a GWAS array. With intensive searches taking place for proteomic biomarkers for many diseases, the role of genetic variation takes on new importance and should be considered in interpretation of proteomic results.     

Limosilactobacillus fermentum,Current Evidence on the Antioxidant Properties and Opportunities to be Exploited as a Probiotic Microorganism

Probiotics and Antimicrobial Proteins - The unbalance in the production and removal of oxygen-reactive species in the human organism leads to oxidative stress, a physiological condition commonly...