A New Scaffold of an Old Protein Fold Ensures Binding to the Bisintercalator Thiocoraline

Thiocoraline is a thiodepsipeptide with potent antitumor activity. TioX, a protein with an unidentified function, is encoded by a gene of the thiocoraline biosynthetic gene cluster. The crystal structure of the full-length TioX protein at 2.15 Å resolution reveals that TioX protomer shares an ancient βαβββ fold motif with glyoxalase I and bleomycin resistance protein families, despite a very low sequence homology. Intriguingly, four TioX monomers form a unique 2-fold symmetric tetrameric assembly that is stabilized by four intermolecular disulfide bonds formed cyclically between Cys60 and Cys66 of adjacent monomers. The arrangement of two of the four monomers in the TioX tetramer is analogous to that in dimeric bleomycin resistance proteins. This analogy indicates that this novel higher-order structural scaffold of TioX may have evolved to bind thiocoraline. Our equilibrium titration studies demonstrate the binding of a thiocoraline chromophore analog, quinaldic acid, to TioX, thereby substantiating this model. Furthermore, a strain of Streptomyces albus containing an exogenous thiocoraline gene cluster devoid of functional tioX maintains thiocoraline production, albeit with a lower yield. Taken together, these observations rule out a direct enzymatic function of TioX and suggest that TioX is involved in thiocoraline resistance or secretion.     

Proteases production by two Vibrio species on residuals marine media

A comparative study was carried out on the growth and production of alkaline proteases by two Vibrio species using different marine peptones from fish viscera residues. The bacteria tested, Vibrio anguillarum and Vibrio splendidus, are producers of high levels of proteolytic enzymes which act as factors of virulence in fish cultures, causing high mortality rates. The kinetic assays and subsequent comparison with the parameters obtained from the adjustment to various mathematical models, highlighted the potential interest of the media formulated, for their possible production on an industrial scale, particularly the production of proteases by V. anguillarum growing in rainbow trout and squid peptones.     

Biocatalytic properties of Baeyer–Villiger monooxygenases in aqueous–organic media

The biocatalytic properties of three Baeyer–Villiger monooxygenases (phenylacetone monooxygenase, 4-hydroxyacetophenone monooxygenase and ethionamide monooxygenase) in a variety of aqueous–organic media were studied using organic sulfides as substrates. The influence of the nature and the concentration of the solvents, as well as of the substrates, on the activity and enantioselectivity of the enzymes was investigated in detail. Solvents were found to decrease, to a different extent, enzyme activity. High increases of enantioselectivity and also reversal of enantiopreference were observed depending on the enzyme and on the nature of the solvent and the substrate employed.     

New convenient defined media for [(35)S]methionine labelling and proteomic analyses of probiotic lactobacilli

AIMS: To develop experimental conditions for efficient protein radiolabelling and two-dimensional (2D) polyacrylamide gel electrophoresis for investigation of stress proteomes of probiotic Lactobacillus spp. METHODS AND RESULTS: Three chemically defined media (CDM) optimized from a commercial medium supported rapid growth of the probiotic Lactobacillus rhamnosus E97800, Lactobacillus brevis ATCC 8287 and Lactobacillus reuteri E97849, and a broad range of other lactic acid bacteria. These CDM allowed efficient protein radiolabelling, requiring as little as 200 mul of logarithmic culture and pulse-chase labelling of 20 min to detect c. 300 distinct protein spots in a mini-scale 2D-gel. Proteins including DnaK, GroEL and ClpATPases were identified from the 2D-gels by immunoblotting. CONCLUSIONS: Radiolabelling coupled with 2D gel electrophoresis provides a sensitive means to monitor changes in protein synthesis rates in probistic lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient tools for proteomic analyses of probiotic Lactobacillus were developed and applied for stress-response studies.     

Complicaciones de la otitis media con parálisis del sexto par craneal contralateral en pediatría

Otitis media is a frequent infection during childhood. Complications may be present in up to 4 of 100 children including serious neurological complications, particularly in developing countries.We report the case of a 9-year-old girl with no disease history who presented with otitis media, otorrhea, intracranial hypertension syndrome, and paralysis of the VI cranial nerve contralateral to the lesion. A computed tomography scan of the skull and a brain magnetic resonance imaging revealed chronic otomastoiditis, petrous apicitis, and thrombosis of the transverse and sigmoid sinus, the jugular bulb, and the right internal jugular vein. She received antibiotics and surgical treatment.This case shows the spectrum of intra and extracranial complications associated with acute otitis media in the antibiotic era. The physical examination allows early identification of intracranial hypertension with signs such as papilledema and sixth contralateral nerve palsy as an unusual finding.     

Construction and elementary mode analysis of a metabolic model for Shewanella oneidensis MR-1

A stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed and used in elementary mode analysis (EMA). Shewanella oneidensis MR-1 can anaerobically respire a diverse pool of electron acceptors, and may be applied in several biotechnology settings, including bioremediation of toxic metals, electricity generation in microbial fuel cells, and whole-cell biocatalysis. The metabolic model presented here was adapted and verified by comparing the growth phenotypes of 13 single- and 1 double-knockout strains, while considering respiration via aerobic, anaerobic fumarate, and anaerobic metal reduction (Mtr) pathways, and utilizing acetate, n-acetylglucosamine (NAG), or lactate as carbon sources. The gene ppc, which encodes phosphoenolpyruvate carboxylase (Ppc), was determined to be necessary for aerobic growth on NAG and lactate, while not essential for growth on acetate. This suggests that Ppc is the only active anaplerotic enzyme when cultivated on lactate and NAG. The application of regulatory and substrate limitations to EMA has enabled creation of metabolic models that better reflect biological conditions, and significantly reduce the solution space for each condition, facilitating rapid strain optimization. This wild-type model can be easily adapted to include utilization of different carbon sources or secretion of different metabolic products, and allows the prediction of single- and multiple-knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells.     

Chromatographic comparison of atenolol separation in reaction media on cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase using ultra fast liquid chromatography

Because chiral liquid chromatography (LC) could become a powerful tool to estimate racemic atenolol quantity, excellent enantiomeric separation should be produced during data acquisition for satisfactory observation of atenolol concentrations throughout the racemic resolution processes. Selection of chiral LC column and analytical protocol that fulfill demands of the ultra fast LC analysis is essential. This article describes the characteristics of atenolol chromatographic separation that resulted from different resolution media and analytical protocols with the use of a Chiralcel® OD column. The chromatograms showed quite different characteristics of the separation process. The single enantiomer and racemic atenolol could be recognized by the Chiralcel® OD column in less than 20 min. Symmetrical peaks were obtained; however, several protocols produced peaks with wide bases and slanted baselines. Observations showed that efficient enantioresolution of racemic atenolol was obtained at slow mobile phase flow rate, decreased concentration of amine‐type modifier but increased alcohol content in mobile phase and highest ultraviolet detection wavelength were required. The optimal ultra fast LC protocol enables to reduce and eliminate the peaks of either the atenolol solvent or the buffers and provided the highest peak intensities of both atenolol enantiomers. Chirality 24:356–367, 2012. © 2012 Wiley Periodicals, Inc.     

Relationships between in vivo and in vitro aflatoxin production: reliable prediction of fungal ability to contaminate maize with aflatoxins

Aflatoxins are highly carcinogenic mycotoxins frequently produced by Aspergillus flavus. Contamination of maize with aflatoxins imposes both economic and health burdens in many regions. Identification of the most important etiologic agents of contamination is complicated by mixed infections and varying aflatoxin-producing potential of fungal species and individuals. In order to know the potential importance of an isolate to cause a contamination event, the ability of the isolate to produce aflatoxins on the living host must be determined. Aflatoxin production in vitro (synthetic and natural media) was contrasted with in vivo (viable maize kernels) in order to determine ability of in vitro techniques to predict the relative importance of causal agents to maize contamination events. Several media types and fermentation techniques (aerated, non-aerated, fermentation volume) were compared. There was no correlation between aflatoxin production in viable maize and production in any of the tested liquid fermentation media using any of the fermentation techniques. Isolates that produced aflatoxins on viable maize frequently failed to produce detectable (limit of detection = 1 ppb) aflatoxin concentrations in synthetic media. Aflatoxin production on autoclaved maize kernels was highly correlated with production on viable maize kernels. The results have important implications for researchers seeking to either identify causal agents of contamination events or characterize atoxigenic isolates for biological control.