The relationship between the in situ reduction level of the cytochrome c pool of Azorhizobium caulinodans growing in a chemostat with NH4+ or N2 as the N source and the total activity of cytochrome c oxidases

Abstract The in situ method for determination of reduction levels of cytochromes b and c pools during steady-state growth (Pronk et al., Anal. Biochem. 214, 149–155, 1993) was applied to chemostat cultures of the wild-type, a cytochrome aa₃ single mutant and a cytochrome aa₃/d double mutant of Azorhizobium caulinodans . For growth with NH₄⁺ as the N source, the results indicate that (i) the aa₃ mutant strains growing at a dissolved O₂ tension of 0.5% possess an active alternative cytochrome c oxidase, which is hardly present during fully aerobic growth, and assuming that (i) also pertains to the wild-type, (ii) the wild-type uses cytochrome aa₃ under fully aerobic conditions. For growth with N₂ as the N source, it was found that the aa₃ mutant strains growing at dissolved O₂ tensions ranging from 0.5 to 3.0% also contain an active alternative cytochrome c oxidase.     

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.     

A two-entropies analysis to identify functional positions in the transmembrane region of class A G protein-coupled receptors

Residues in the transmembrane region of G protein-coupled receptors (GPCRs) are important for ligand binding and activation, but the function of individual positions is poorly understood. Using a sequence alignment of class A GPCRs (grouped in subfamilies), we propose a so-called "two-entropies analysis" to determine the potential role of individual positions in the transmembrane region of class A GPCRs. In our approach, such positions appear scattered, while largely clustered according to their biological function. Our method appears superior when compared to other bioinformatics approaches, such as the evolutionary trace method, entropy-variability plot, and correlated mutation analysis, both qualitatively and quantitatively.     

Structural and biological evaluation of some loloatin C analogues

Loloatin C is a cyclic cationic antimicrobial peptide which is active against Gram positive as well as certain Gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure–activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.     

Unraveling the diversification and systematic puzzle of the highly polymorphic Psammobates tentorius (Bell, 1828) complex (Reptilia: Testudinidae) through phylogenetic analyses and species delimitation approaches

The high level of phenotypic diversity in southern African tent tortoises (Psammobates tentorius complex) has for decades prevented systematists from developing a stable taxonomy for the group. Here, we used a comprehensive DNA sequence dataset (mtDNA: Cytb, ND4, ND4 adjacent tRNA-His, and tRNA-Ser, 12S, 16S; and nDNA: PRLR gene) of 455 specimens, and the latest phylogenetic and species delimitation analytical procedures, to unravel the long-standing P. tentorius complex systematic puzzle. Our results for mtDNA and nDNA were incongruent, with the poorly supported nDNA phylogeny differentiating the three recognized subspecies, and showing potential hybridization in some regions. In contrast, the concatenated mtDNA phylogeny identified seven operational taxonomic units, with strong support. Clades 1, 4, 5, and 7 corresponded to tortoises identified as P. t. tentorius, clade 3 to P. t. trimeni, and clades 2 and 6 to P. t. verroxii. Our analyses showed conflicting topologies for the placement of C6 (P. t. verroxii north of the Orange River), with stronger support for it being sister to C2 + C3 than to the other clades. Clades 1, 2, and 6 had significantly higher genetic diversity than clades 3, 4, 5, and 7, perhaps because these clades inhabit substantially larger areas. The potential for future cladogenic radiations seems high in C1 and C6, particularly in C6 for which the within-clade diversification level was highest. Further research involving microsatellite DNA, phylogeographic evaluations, and morphological variation among clades is crucial for understanding the adaptive radiation of the P. tentorius complex and for modifying their taxonomy.     

A comparative analysis of testicular sperm morphology in fossorial and surface-living skinks in South Africa

We described for the first time the spermatozoan ultrastructure of the fully pentadactyl surface-living skink Trachylepis punctatissima, and limbless fossorial skink Acontias meleagris. The spermatozoa of both species follow the general patterns observed within the Squamata. However, several important differences were detected between the two species in the head region (shape of the anterior acrosome, size of acrosome and nucleus) and especially in the midpiece (size of the midpiece, the presence of regular rows of dense bodies, size and number of mitochondria and beginning of the fibrous sheath). Both species shared more characters with the Sphenomorphus + Egernia group than with the Eugongylus group proposed by Jamieson, Oliver, and Scheltinga (Acta Zoologica, 77, 85). Differences in the spermatozoan ultrastructure between T. punctatissima and A. meleagris could be due to distinct ecological and physiological requirements for fertilization, but additional research on these genera and within the Scincidae is required to disentangle this hypothesis, and to disentangle the phylogenetic relationships among skinks.     

Rapid reversal of human intestinal ischemia-reperfusion induced damage by shedding of injured enterocytes and reepithelialisation

Background

Intestinal ischemia-reperfusion (IR) is a phenomenon related to physiological conditions (e.g. exercise, stress) and to pathophysiological events (e.g. acute mesenteric ischemia, aortic surgery). Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce. Therefore, an experimental harmless model for human intestinal IR was developed, enabling us to clarify the sequelae of human intestinal IR for the first time.

Methods and Findings

In 30 patients undergoing pancreatico-duodenectomy we took advantage of the fact that in this procedure a variable length of jejunum is removed. Isolated jejunum (5 cm) was subjected to 30 minutes ischemia followed by reperfusion. Intestinal Fatty Acid Binding Protein (I-FABP) arteriovenous concentration differences across the bowel segment were measured before and after ischemia to assess epithelial cell damage. Tissue sections were collected after ischemia and at 25, 60 and 120 minutes reperfusion and stained with H&E, and for I-FABP and the apoptosis marker M30. Bonferroni''s test was used to compare I-FABP differences. Mean (SEM) arteriovenous concentration gradients of I-FABP across the jejunum revealed rapidly developing epithelial cell damage. I-FABP release significantly increased from 290 (46) pg/ml before ischemia towards 3,997 (554) pg/ml immediately after ischemia (p<0.001) and declined gradually to 1,143 (237) pg/ml within 1 hour reperfusion (p<0.001). Directly after ischemia the intestinal epithelial lining was microscopically normal, while subepithelial spaces appeared at the villus tip. However, after 25 minutes reperfusion, enterocyte M30 immunostaining was observed at the villus tip accompanied by shedding of mature enterocytes into the lumen and loss of I-FABP staining. Interestingly, within 60 minutes reperfusion the epithelial barrier resealed, while debris of apoptotic, shedded epithelial cells was observed in the lumen. At the same time, M30 immunoreactivity was absent in intact epithelial lining.

Conclusions

This is the first human study to clarify intestinal IR induced cell damage and repair and its direct consequences. It reveals a unique, endogenous clearing mechanism for injured enterocytes: rapid detachment of damaged apoptotic enterocytes into the lumen. This process is followed by repair of the epithelial continuity within an hour, resulting in a normal epithelial lining.     

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A_2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A_2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.     

Multiple paternity analysis in the thornback ray Raja clavata L

Skates (Rajidae) are characterized by slow growth rate, low fecundity, and late maturity and are thus considered to be vulnerable to exploitation. Although understanding mating systems and behavior are important for long-term conservation and fisheries management, this aspect of life history is poorly understood in skates. Using 5 highly polymorphic microsatellite loci, we analyzed egg clutches collected from 4 female Raja clavata captured in the wild to test for multiple paternity. Using the reconstructed multilocus genotypes method to explain the progeny genotype array, we showed that all 4 clutches were sired by a minimum of 4-6 fathers and, thus, female thornback rays are polyandrous. Whether polyandry in R. clavata is natural or a consequence of overexploitation remains uncertain. This is the first report of multiple paternity in a rajiform species and any oviparous elasmobranch.