Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O₂ bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

High‐performance liquid chromatography with diode array detection coupled to electrospray time‐of‐flight and ion‐trap tandem mass spectrometry to identify phenolic compounds from a Cistus ladanifer aqueous extract

Introduction – Cistus ladanifer is an aromatic shrub that is widespread in the Mediterranean region. The labdanum exudate is used in the fragrance industry and has been characterised. However, there is not enough information about the phenolic content of the raw plant, the aerial part of it being a very rich source of bioactive compounds. Objective – Characterisation of the bioactive compounds of the raw plant and its aerial parts. Methodology – High‐performance liquid chromatography with diode array and electrospray ionisation mass spectrometric detection was used to carry out the comprehensive characterisation of a Cistus ladanifer shrub aqueous extract. Two different MS techniques were coupled to HPLC: time‐of‐flight mass spectrometry and tandem mass spectrometry. Results – Many well‐known compounds present in Cistus ladanifer were characterised, such as flavonoids, phenolic acids, ellagitanins, hexahydroxydiphenoyl and derivatives, and other compounds. Conclusion – The method described simultaneously separated a wide range of phenolic compounds and the proposed characterisation of the major compounds of this extract was carried out. It is important to highlight that, to our knowledge, this is the first time that a Cistus ladanifer aqueous extract from the raw plant has been characterised. Copyright © 2009 John Wiley & Sons, Ltd.     

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.     

Metabolic engineering,new antibiotics and biofilm viscoelasticity

In the following highlight we refer to a number of new advances in the field of Biotechnology that address issues relating to the synthesis of new antibiotics, new biocatalysts and matrices in biofilms.     

Effects of habitat fragmentation and disturbance on howler monkeys: a review

We examined the literature on the effects of habitat fragmentation and disturbance on howler monkeys (genus Alouatta) to (1) identify different threats that may affect howlers in fragmented landscapes; (2) review specific predictions developed in fragmentation theory and (3) identify the empirical evidence supporting these predictions. Although howlers are known for their ability to persist in both conserved and disturbed conditions, we found evidence that they are negatively affected by high levels of habitat loss, fragmentation and degradation. Patch size appears to be the main factor constraining populations in fragmented habitats, probably because patch size is positively related to food availability, and negatively related to anthropogenic pressures, physiological stress and parasite loads. Patch isolation is not a strong predictor of either patch occupancy or population size in howlers, a result that may be related to the ability of howlers to move among forest patches. Thus, we propose that it is probable that habitat loss has larger consistent negative effects on howler populations than habitat fragmentation per se. In general, food availability decreases with patch size, not only due to habitat loss, but also because the density of big trees, plant species richness and howlers' home range size are lower in smaller patches, where howlers' population densities are commonly higher. However, it is unclear which vegetation attributes have the biggest influence on howler populations. Similarly, our knowledge is still limited concerning the effects of postfragmentation threats (e.g. hunting and logging) on howlers living in forest patches, and how several endogenous threats (e.g. genetic diversity, physiological stress, and parasitism) affect the distribution, population structure and persistence of howlers. More long‐term studies with comparable methods are necessary to quantify some of the patterns discussed in this review, and determine through meta‐analyses whether there are significant inter‐specific differences in species' responses to habitat loss and fragmentation. Am. J. Primatol. 72:1–16, 2010. © 2009 Wiley‐Liss, Inc.     

Newly discovered penicillin acylase activity of aculeacin A acylase from Actinoplanes utahensis

Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM(-1) s(-1). Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5 degrees C.     

Crh1p and Crh2p are required for the cross-linking of chitin to beta(1-6)glucan in the Saccharomyces cerevisiae cell wall

In budding yeast, chitin is found in three locations: at the primary septum, largely in free form, at the mother-bud neck, partially linked to beta(1-3)glucan, and in the lateral wall, attached in part to beta(1-6)glucan. By using a recently developed strategy for the study of cell wall cross-links, we have found that chitin linked to beta(1-6)glucan is diminished in mutants of the CRH1 or the CRH2/UTR2 gene and completely absent in a double mutant. This indicates that Crh1p and Crh2p, homologues of glycosyltransferases, ferry chitin chains from chitin synthase III to beta(1-6)glucan. Deletion of CRH1 and/or CRH2 aggravated the defects of fks1Delta and gas1Delta mutants, which are impaired in cell wall synthesis. A temperature shift from 30 degrees C to 38 degrees C increased the proportion of chitin attached to beta(1-6)glucan. The expression of CRH1, but not that of CRH2, was also higher at 38 degrees C in a manner dependent on the cell integrity pathway. Furthermore, the localization of both Crh1p and Crh2p at the cell cortex, the area where the chitin-beta(1-6)glucan complex is found, was greatly enhanced at 38 degrees C. Crh1p and Crh2p are the first proteins directly implicated in the formation of cross-links between cell wall components in fungi.     

Geographical variation in cloacal microflora and bacterial antibiotic resistance in a threatened avian scavenger in relation to diet and livestock farming practices

The impact on wildlife health of the increase in the use of antimicrobial agents with the intensification of livestock production remains unknown. The composition, richness and prevalence of cloacal microflora as well as bacterial resistance to antibiotics in nestlings and full-grown Egyptian vultures Neophron percnopterus were assessed in four areas of Spain in which the degree of farming intensification differs. Differences in diet composition, especially the role of stabled livestock carrion, appear to govern the similarities of bacterial flora composition among continental populations, while the insular vulture population (Fuerteventura, Canary Islands) showed differences attributed to isolation. Evidence of a positive relationship between the consumption of stabled livestock carrion and bacterial resistance to multiple antibiotics was found. Bacterial resistance was high for semisynthetic penicillins and enrofloxacin, especially in the area with the most intensive stabled livestock production. The pattern of antibiotic resistance was similar for the different bacterial species within each area. Bacterial resistance to antibiotics may be determined by resistance of bacteria present in the livestock meat remains that constituted the food of this species, as indicated by the fact that resistance to each antibiotic was correlated in Escherichia coli isolated from swine carrion and Egyptian vulture nestlings. In addition, resistance in normal faecal bacteria (present in the microflora of both livestock and vultures) was higher than in Staphylococcus epidermidis, a species indicator of the transient flora acquired presumably through the consumption of wild rabbits. Potential negative effects of the use of antimicrobials in livestock farming included the direct ingestion of these drug residues and the effects of bacterial antibiotic resistance on the health of scavengers.