Conformational analysis and clustering of short and medium size loops connecting regular secondary structures: a database for modeling and prediction.

Loops are regions of nonrepetitive conformation connecting regular secondary structures. We identified 2,024 loops of one to eight residues in length, with acceptable main-chain bond lengths and peptide bond angles, from a database of 223 protein and protein-domain structures. Each loop is characterized by its sequence, main-chain conformation, and relative disposition of its bounding secondary structures as described by the separation between the tips of their axes and the angle between them. Loops, grouped according to their length and type of their bounding secondary structures, were superposed and clustered into 161 conformational classes, corresponding to 63% of all loops. Of these, 109 (51% of the loops) were populated by at least four nonhomologous loops or four loops sharing a low sequence identity. Another 52 classes, including 12% of the loops, were populated by at least three loops of low sequence similarity from three or fewer nonhomologous groups. Loop class suprafamilies resulting from variations in the termini of secondary structures are discussed in this article. Most previously described loop conformations were found among the classes. New classes included a 2:4 type IV hairpin, a helix-capping loop, and a loop that mediates dinucleotide-binding. The relative disposition of bounding secondary structures varies among loop classes, with some classes such as beta-hairpins being very restrictive. For each class, sequence preferences as key residues were identified; those most frequently at these conserved positions than in proteins were Gly, Asp, Pro, Phe, and Cys. Most of these residues are involved in stabilizing loop conformation, often through a positive phi conformation or secondary structure capping. Identification of helix-capping residues and beta-breakers among the highly conserved positions supported our decision to group loops according to their bounding secondary structures. Several of the identified loop classes were associated with specific functions, and all of the member loops had the same function; key residues were conserved for this purpose, as is the case for the parvalbumin-like calcium-binding loops. A significant number, but not all, of the member loops of other loop classes had the same function, as is the case for the helix-turn-helix DNA-binding loops. This article provides a systematic and coherent conformational classification of loops, covering a broad range of lengths and all four combinations of bounding secondary structure types, and supplies a useful basis for modelling of loop conformations where the bounding secondary structures are known or reliably predicted.     

Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.     

Artery Wall Assessment Helps Predict Kidney Transplant Outcome

Background

Kidney transplant recipients have high cardiovascular risk, and vascular inflammation may play an important role. We explored whether the inflammatory state in the vessel wall was related to carotid intima-media thickness (c-IMT) and patient survival following kidney transplantation.

Methods

In this prospective observational cohort study we measured c-IMT and expression of proinflammatory cytokines and adhesion molecules in the inferior epigastric artery in 115 kidney transplant candidates. Another c-IMT measurement was done 1-year post-transplantation in 107. By stepwise multiple regression analysis we explored factors associated with baseline c-IMT and their changes over time. Multivariate Cox regression analysis was constructed to identify risk factors for mortality.

Results

A worse cardiovascular profile (older age, smoker, diabetic, carotid plaque, systolic blood pressure and vascular calcification) and higher VCAM-1 levels were found in patients in the highest baseline c-IMT tertile, who also had a worse survival. Factors independently related to baseline c-IMT were age (β=0.369, P<0.0001), fasting glucose (β=0.168, P=0.045), smoking (β=0.228, P=0.003) and VCAM-1 levels (β=0.244, P=0.002). Independent factors associated with c-IMT measurement 1-year post-transplantation were baseline c-IMT (β=-0.677, P<0.0001), post-transplant diabetes (β=0.225, P=0.003) and triglycerides (β=0.302, P=0.023). Vascular VCAM-1 levels were associated with increased risk of mortality in bivariate and multivariate Cox regression. Notably, nearly 50% of patients showed an increase or maintenance of high c-IMT 1 year post-transplantation and these patients experienced a higher mortality (13 versus 3.5%; P=0.021).

Conclusion

A worse cardiovascular profile and a higher vascular VCAM-1 protein levels at time of KT are related to subclinical atheromatosis. This could lead to a higher post-transplant mortality. Pre-transplant c IMT, post-transplant diabetes and triglycerides at 1-year post-transplantation may condition a high c-IMT measurement post-transplantation, which may decrease patient survival.     

Cystic echinococcosis in Spain: current situation and relevance for other endemic areas in Europe

Cystic echinococcosis (CE) remains an important health problem in many regions of the world, both where no control measures have been implemented, and where control programs have been incompletely successful with ensuing re-emergence of the disease. In Spain, official data on CE show an increase in the proportion of intermediate hosts with CE during the last few years, and autochthonous pediatric patients have been reported, a sign of active local transmission of disease. A similar picture emerges from data reported to the European Food Safety Authority by other European countries. Nevertheless, several crucial aspects related to CE that would help better understand and control the disease have not been tackled appropriately, in particular the emergence of infection in specific geographical areas. In this respect, while some data are missing, other data are conflicting because they come from different databases. We review the current situation of CE in Spain compared with areas in which similar problems in the CE field exist, and offer recommendations on how to overcome those limitations. Specifically, we believe that the introduction of national registries for CE with online data entry, following the example set by the European Registry for Alveolar Echinococcosis, would help streamline data collection on CE by eliminating the need for evaluating and integrating data from multiple regions, by avoiding duplication of data from patients who access several different health facilities over time, and by providing much needed clinical and epidemiological data that are currently accessible only to clinicians.     

Culture conditions determine the balance between two different exopolysaccharides produced by Lactobacillus pentosus LPS26

Lactobacillus pentosus LPS26, isolated from a natural fermentation of green olives, produces a capsular polymer constituted of two exopolysaccharides (EPS): EPS A, a high-molecular-weight (high-Mw) polysaccharide (1.9x10(6) Da) composed of glucose and rhamnose (3:1), and EPS B, a low-Mw polysaccharide (3.3x10(4) Da) composed of glucose and mannose (3:1). Fermentation experiments in a chemically semidefined medium with different carbon sources (glucose, fructose, mannitol, and lactose) showed that all of them except fructose supported EPS A production rather than EPS B production. The influence of temperature and pH was further analyzed. As the temperature dropped, increased synthesis of both EPS was detected. The control of pH especially enhanced EPS B production. With regard to this, the maximum total EPS production (514 mg liter-1) was achieved at a suboptimal growth temperature (20 degrees C) and pH 6.0. Continuous cultures showed that EPS A, synthesized mainly at low dilution rates, is clearly dependent on the growth rate, whereas EPS B synthesis was hardly affected. EPS production was also detected in supplemented skimmed milk, but no increase on the viscosity of the fermented milk was recorded. This could be linked to the high proportion of the low-Mw polysaccharide produced in these conditions in contrast to that observed in culture media. Overall, the present study shows that culture conditions have a clear impact on the type and concentration of EPS produced by strain LPS26, and consequently, these conditions should be carefully selected for optimization and application studies. Finally, it should be noted that this is, to our knowledge, the first report on EPS production by L. pentosus.     

Glutathione-Dependent Conversion of N-Ethylmaleimide to the Maleamic Acid by Escherichia coli: an Intracellular Detoxification Process

The electrophile N-ethylmaleimide (NEM) elicits rapid K⁺ efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K⁺ efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-Ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD⁺-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K⁺ efflux systems.     

Determination of age and age classes of Akodon olivaceus in Southern Chile

Age and age classes in Akodon olivaceus brachiotis were determined by means of a modification of the method used by Sluiter (1961) to quantify tooth wear. Ten age categories were obtained. From April to July the population was mainly composed of young animals. From November to March the population was heterogeneous due to the coexistence of new‐born animals and animals born in the previous reproductive season.     

Assessment of cell line competence for studies of pharmacological GPR30 modulation

Context/objective: Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30.

Materials and methods: The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals.

Results: None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50–55?kDa previously identified in cell lines that respond to 17β-estradiol and G-1.

Discussion and conclusion: Cells that do not express the 44 and 50–55?kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.