The hydrophobic substituent in aminophospholipids affects the formation kinetics of their Schiff bases

Schiff bases (SBs) are the initial products of non-enzymatic glycation reactions, which are associated to some diabetes-related diseases. In this work, we used physiological pH and temperature conditions to study the formation kinetics of the SBs of 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DPHE) and 1,2-dihexanoyl-sn-glycero-3-phospho-l-serine (DHPS) with various glycating compounds and with pyridoxal 5’-phosphate (an effective glycation inhibitor). Based on the obtained results, the hydrophobic environment simultaneously decreases the nucleophilic character of the amino group (k₁) and increases its pK_a, thereby increasing the formation rate of SB (k_obs). Therefore, the presence of hydrophobic chains in aminophospholipids facilitates the formation and stabilization of SBs, and also, in a biological environment, their glycation. Additionally, the results confirm the inhibitory action of B₆ vitamers on aminophospholipid glycation.     

The impact of transposable elements in environmental adaptation

Transposable elements (TEs) play an important role in the responsive capacity of their hosts in the face of environmental challenges. The variety of mechanisms by which TEs influence the capacity of adaptation of the host is as large as the variety of TEs and host genomes. For example, TEs might directly affect the function of individual genes, provide a mechanism for rapidly acquiring new genetic material and disseminate regulatory elements that can lead to the creation of stress‐inducible regulatory networks. In this review, we summarize recent examples that are part of an increasing body of evidence suggesting a significant role of TEs in the host response to an ever‐changing environment, both in prokaryote and in eukaryote organisms. We argue that in the near future, the increasing availability of genome sequences and the development of new tools to discover and analyse TE insertions will further show the relevant role of TEs in environmental adaptation.     

Repeated jumps from Northwest Africa to the European continent: The case of peripheral populations of an annual plant

Peripheral populations (i.e., those occurring on the edge of a species’ distribution range) can have different origins and genetic characteristics, and they may be critical for the conservation of genetic diversity. We investigated European peripheral populations of Scrophularia arguta, a widespread, annual plant distributed from Arabia to Northwest Africa and Macaronesia. Only two small disjunct population groups of this species occur in Europe, specifically in West‐Central and Southeast Iberia. To disclose the origin of these populations and determine their importance for the conservation of S. arguta genetic diversity, we analyzed DNA sequences from two nuclear and two plastid regions and amplified fragment length polymorphism markers in populations sampled mainly across the western distribution range of the species, and modeled the species distribution under present and late Quaternary conditions. The analyses revealed the presence of three distinct lineages of S. arguta in Europe, as a result of multiple colonization waves at different times in the Quaternary. Two of these lineages, occurring in Southeast Iberia, are the result of more or less recent dispersal from Northwest Africa. In contrast, West‐Central Iberian populations are strongly differentiated from the remaining range of S. arguta and can be considered as peripheral relict populations. Our study is the first to demonstrate the occurrence of at least three colonizations of the European continent from Africa by a native plant species. The diverse histories and genetic makeup of the resulting populations confirm the importance of peripheral populations, and particularly of ancient relict populations, for the conservation of global genetic diversity in widespread species.     

Metal ion binding patterns of acyclovir: molecular recognition between this antiviral agent and copper(II) chelates with iminodiacetate or glycylglycinate

In order to deepen on metal-binding patterns of acyclovir (acv), {[Cu(IDA)(acv)]·2MeOH}_n (1) and [Cu(glygly)(acv)]·H₂O (2) compounds have been synthesized and investigated by X-ray crystallography as well as spectral and thermal methods. These compounds have been chosen upon the assumption that iminodiacetate (IDA) and glycylglycinate (glygly) chelating ligands would bind copper(II) with mer-tridentate conformation, supplying two terminal H-acceptor carboxylate groups (IDA) or one H-acceptor carboxylate and one H-donor primary amino group (glygly). The main aim of this work was to clarify if the amino group of glygly can build an intra-molecular interligand H-bonding interaction to reinforce the Cu―N7(acv) bond. Our results are discussed in the context of an up-to-date critical look regarding the related structural information. From the viewpoint of molecular recognition, the structure of 1 shows that the chelate-nucleoside recognition only involves the Cu―N7(acv) coordination bond. In contrast, the molecular complex of 2 exhibits the Cu―N7(acv) coordination bond reinforced by an intra-molecular (glygly)N-H···O6(acv) interaction (2.961(3) Å, 140.5°).     

Evaluation of the Use of Selective PCR Amplification of LPS Biosynthesis Genes for Molecular Typing of <Emphasis Type="Italic">Leptospira</Emphasis> at the Serovar Level

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.     

Gene sequencing and tissue expression of unknown isoforms of an angiotensin II type 2 receptor interacting protein, ATIP, in the rat

To investigate the expression of the unknown angiotensin II type 2 receptor interacting protein (ATIP) isoforms in the rat we used the known sequences of human and mouse ATIP to design sequencing primers to enable us to sequence rat ATIP3 and ATIP4. Exon 4, which is present in human but not mouse ATIP, was not identified in the coding region of rat ATIP. The expression levels of these genes in a range of rat tissues were examined, and we concluded that there is little similarity in the relative tissue distribution of the various ATIP isoforms in rat and human.     

Modulation of microbial predator-prey dynamics by phosphorus availability: growth patterns and survival strategies of bacterial phylogenetic clades

We simultaneously studied the impact of top-down (protistan grazing) and bottom-up (phosphorus availability) factors on the numbers and biomasses of bacteria from various phylogenetic lineages, and on their growth and activity parameters in the oligo-mesotrophic Piburger See, Austria. Enhanced grazing resulted in decreased proportions of bacteria with high nucleic acid content (high-NA bacteria) and lower detection rates by FISH. There was a change in the composition of the bacterial assemblage, whereby Betaproteobacteria were heavily grazed while Alphaproteobacteria and Cytophaga-Flavobacterium-Bacteroides were less affected by predators. Changes in bacterial assemblage composition were also apparent in the treatments enriched with phosphorus, and even more pronounced in the incubations in dialysis tubes (allowing relatively free nutrient exchange). Here, Betaproteobacteria became dominant and appeared to act as successful opportunistic competitors for nutrients. In contrast, Actinobacteria did not respond to surplus phosphorus by population growth, and, moreover, maintained their small size, which resulted in a very low biomass contribution. In addition, significant relationships between high-NA bacteria and several bacterial phylogenetic clades were found, indicating an enhanced activity status. By combining several single-cell methods, new insight is gained into the competitive abilities of freshwater bacteria from a variety of phylogenetic lineages under contrasting sets of bottom-up and top-down constraints.     

Effects of ethanol on the remodeling of neutral lipids and phospholipids in brain mitochondria and microsomes

We have analyzed the effects of ethanol in vitro on the remodeling of neutral lipids and phospholipids in mitochondria and microsomes isolated from chick brain. We used three different fatty acyl-CoAs of similar chain lengths but different degrees of unsaturation. Our results demonstrate the existence of active mechanisms for acyl-CoA transfer into neutral lipids and phospholipids in both mitochondria and microsomes. The profile of fatty acid incorporation was clearly different according to the membrane and lipid fraction in question. Thus, in mitochondrial lipids, the remodeling processes showed a clear preference for the saturated fatty acid whilst the polyunsaturated one was the preferred substrate for microsomal lipid acylation. With regard to the effects of ethanol in vitro, we were able to demonstrate that exposure of the membrane to ethanol led to an increase in the incorporation of polyunsaturated fatty acid into triacylglycerol (TG) in both mitochondria and microsomes, indicating that it directly stimulates the acylation of diacylglycerol (DG) to give TG. This effect may then contribute to the widely reported stimulation of TG biosynthesis in cases of both acute and chronic ethanol ingestion. It is noteworthy that the exposure of microsomes to ethanol in vitro also stimulated the incorporation of oleoyl-CoA into the aminophospholipids phosphatidylethanolamine (PE) and phosphatidylserine (PS). We also demonstrate that both mitochondria and microsomes synthesize fatty acid ethyl esters (FAEEs) from fatty acyl-CoA, although there is a clear difference in preference for the fatty acid used as substrate in the esterification of the alcohol. Thus, mitochondria were capable of forming FAEEs from the polyunsaturated fatty acid whilst in microsomes the saturated fatty acid was the preferred substrate. In both types of membrane, FAEE production was lowest with the monounsaturated fatty acyl-CoA.     

The structure of the Shiga toxin 2a A‐subunit dictates the interactions of the toxin with blood components

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)‐producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A‐subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re‐evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS‐pathogenesis and to develop therapeutic approaches.     

Intact cell mass spectrometry as a progress tracking tool for batch and fed-batch fermentation processes

Penicillin production during a fermentation process using industrial strains of Penicillium chrysogenum is a research topic permanently discussed since the accidental discovery of the antibiotic. Intact cell mass spectrometry (ICMS) can be a fast and novel monitoring tool for the fermentation progress during penicillin V production in a nearly real-time fashion. This method is already used for the characterization of microorganisms and the differentiation of fungal strains; therefore, the application of ICMS to samples directly harvested from a fermenter is a promising possibility to get fast information about the progress of fungal growth. After the optimization of the ICMS method to penicillin V fermentation broth samples, the obtained ICMS data were evaluated by hierarchical cluster analysis or an in-house software solution written especially for ICMS data comparison. Growth stages of a batch and fed-batch fermentation of Penicillium chrysogenum are differentiated by one of those statistical approaches. The application of two matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) instruments in the linear positive ion mode from different vendors demonstrated the universal applicability of the developed ICMS method. The base for a fast and easy-to-use method for monitoring the fermentation progress of P. chrysogenum is created with this ICMS method developed especially for fermentation broth samples.