Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin K1 2,3-epoxide reduction

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.     

BACTIBASE: a new web-accessible database for bacteriocin characterization

Background  

Bacteriocins are very diverse group of antimicrobial peptides produced by a wide range of bacteria and known for their inhibitory activity against various human and animal pathogens. Although many bacteriocins are now well characterized, much information is still missing or is unavailable to potential users. The assembly of such information in one central resource such as a database would therefore be of great benefit to the exploitation of these bioactive molecules in the present context of increasing antibiotic resistance and natural bio-preservation need.     

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

Comparison of the chemical composition and the organoleptic profile of virgin olive oil from two wild and two cultivated Tunisian Olea europaea

With the aim to select new olive cultivars with superior physical and chemical properties than the cultivar Chemlali Sfax, the present study focused on the comparison of the chemical composition and the sensory profile of the virgin olive oils (VOOs) of two wild olive trees (Oleasters K and M) with those of VOOs obtained from Chemlali Sfax and Neb Jmel olive cultivars, all growing in the coastal region of Tunisia. Despite the variability in the chemical composition (fatty acids, pigments, and phenolic and volatile compounds) and the organoleptic profile of the VOOs of the oleasters and the cultivars, the quality indices (free fatty acids, peroxide value, and spectrophotometric indices K232 and K270) as well as the fatty acid composition of all VOOs studied met the commercial standards. Both the α-tocopherol and phenol contents varied between the genotypes. The Neb Jmel and Oleaster K VOOs had more than two times higher total phenol levels than the Chemlali Sfax and Oleaster M VOOs. Also the contents of volatile compounds differed between the olive oils studied. Chemlali Sfax and Oleaster K oils were more abundant in aldehydes, whereas Oleaster M VOO had higher contents of alcohols. These results were confirmed by a sensorial analysis showing that the later oil was deprived for consumption despite its abundance in α-tocopherol. In conclusion, the oleasters studied revealed to be interesting, since they produced oils with good quality characteristics in terms of minor compounds (phenols and volatiles) compared to the Chemlali Sfax cultivar.     

The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats

The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.     

Colistin A and colistin B among inhibitory substances of <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JB05-01-1

Recently, we isolated and reported the antagonism of Paenibacillus polymyxa JB05-01-1 (P. polymyxa JB05-01-1) against Gram-negative bacteria. Here, we provide more insights and attribute the abovementioned antagonism to the production of colistins A and B, which were purified by Amberlite column exchange, C18 column hydrophobicity, superdex 75 16/60 gel filtration chromatography connected to fast protein liquid chromatography and identified by MALDI TOF/TOF, and manual nanospray analysis. The amount of colistin A and colistin B recovered from 500 ml of culture supernatant was about 0.05 mg. The specific activity and the average recovery of the eluted substances were 5,120 AU/mg and 1.1%, respectively. The minimal inhibitory concentrations of the purified colistins against Escherichia coli O157:H7 and Pseudomonas fluorescens LRC R73 were 0.13 and 0.62 μg/ml, respectively.     

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Aim

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Methods and Results

The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.

Conclusion

The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.

Significance and Impact of the Study

This technique allowed rapid C. trachomatis genotype identification.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> bacteriocin Bac 14B with a broad inhibitory spectrum: Purification,amino acid sequence analysis,and physicochemical characterization

Bacillus subtilis strain 14B was used to produce a novel antimicrobial peptide (bacteriocin) called Bac 14B. Pure bacteriocin was obtained after heat and acidic treatments (80°C and pH 4), precipitation by ammonium sulfate, and chromatography on Sephadex G-50 and Mono Q Sepharose columns. Based on MALDI-TOF mass spectrometry analysis, purified Bac 14B is a monomer protein with a molecular mass of 20110.13 Da. N-terminal sequencing allowed for the straightforward identification of its first 12 residues, which were of a pure bacteriocin. It also revealed that this bacteriocin contained a unique sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the identification of a novel compound. Bac 14B was stable for 1 h at temperatures up to 80°C and pH of 4 ∼ 8. It also proved sensitive to various proteases, which demonstrated its protein nature. Bac 14B displayed a bacteriolytical mode of action and a broad range of inhibitory spectra toward Gram-positive and -negative pathogens. Interestingly, based on conventional agronomic seed vigor parameters, the application of Bac 14B (500 activity units/mL) to various crops revealed that this bacteriocin was a potent exogenous enhancer of growth that stimulated the seedling vigor of tomatoes and muskmelons. Compared to those of the control, the germination percentage, shoot weight, shoot height, and root length were all significantly enhanced in Bac 14B-treated plant seeds. Bac 14B also exhibited effective disinfectant properties against a wide range of seedborne diseases and significant effects on the control of damping off diseases, particularly at the pregermination stage. It also proved to be effective against root rot diseases caused by Alternaria solani and other bacterial seedborne pathogens such as wilt diseases. The findings indicate that Bac 14B is the first B. subtilis-produced bacteriocin ever reported to exhibit such promising biological properties.