A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases

The current article aims to summarize all possible spectrum of protein–protein interactions for thromboxane A synthase (CYP5A1) and prostacyclin synthase (CYP8A1). These enzymes metabolize the same substrate (prostaglandin H₂) and can participate in cardiovascular, inflammatory, immune processes, and apoptosis modulation, as well as significantly influence the risk of cancers. Binary protein–protein and multiprotein complexes are of great importance in enzyme-regulating and signal-transduction pathways. However, protein partners of CYP5A1 and CYP8A1 are not yet fully identified, although both synthases are considered as prospective drug targets. At least 36 novel protein partners of CYP5A1 and CYP8A1 were revealed from different tissue types using an approach based on affinity isolation and mass spectrometry. Enrichment analysis showed that these proteins have different molecular functions: folding (refolding), unfolded protein and chaperon binding, protein transport (export/import), posttranslational modification, protein domain-specific binding, antioxidant activity, and glutathione homeostasis. A significant part of them, belonging to molecular chaperones, were common partners for CYP5A1 and CYP8A1, while other proteins were unique with the tissue-dependent distribution. New aspects of CYP5A1 and CYP8A1 interactomics and hetero-complex formation with different protein partners, including cytochrome P450s are discussed.     

Mitochondrial DNA Variation and Introgression in Siberian Taimen Hucho taimen

Siberian taimen Hucho taimen is the largest representative of the family Salmonidae inhabiting rivers of northern Eurasia. The species is under intensive aquaculture activity. To monitor natural taimen populations we have sequenced a portion (8,141 bp) of the mitochondrial (mt) genome in 28 specimens of H. taimen from six localities in the Amur River basin. Nucleotide variability is low (π = 0.0010), but structured in two divergent haplotype groups. A comparison of the data with the GenBank H. taimen mt genome ({"type":"entrez-nucleotide","attrs":{"text":"HQ897271","term_id":"325127000","term_text":"HQ897271"}}HQ897271) reveals significant differences between them in spite of the fact that the fish specimens come from neighboring geographical areas. The distribution of divergence is non-uniform with two highly pronounced divergent regions centered on two genes, ND3 and ND6. To clarify the pattern of divergence we sequenced the corresponding portion of the mt genome of lenok Brachymystax tumensis and analyzed the GenBank complete mt genomes of related species. We have found that the first and second divergent regions are identical between the GenBank H. taimen and two lenok subspecies, B. lenok and B. lenok tsinlingensis, respectively. Consequently, both divergent regions represent introgressed mtDNA resulting from intergeneric hybridization between the two lenok subspecies and H. taimen. Introgression is, however, not detected in our specimens. This plus the precise identity of the introgressed fragments between the donor and the recipient GenBank sequence suggests that the introgression is local and very recent, probably due to artificial manipulations involving taimen – lenok intergeneric hybridization. Human-mediated hybridization may become a major threat to aquatic biodiversity. Consequently we suggest that due attention needs to be given to this threat by means of responsible breeding program management, so as to prevent a potential spread of hybrid fishes that could jeopardize the resilience of locally adapted gene pools of the native H. taimen populations.     

Breaking the Carboxyl Rule: LYSINE 96 FACILITATES REPROTONATION OF THE SCHIFF BASE IN THE PHOTOCYCLE OF A RETINAL PROTEIN FROM EXIGUOBACTERIUM SIBIRICUM*?

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D₂O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pK_a of 8.5 ± 0.3, which reflects the pK_a of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pK_a of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.     

Long‐term metapopulation study of the Glanville fritillary butterfly (Melitaea cinxia): survey methods,data management,and long‐term population trends

Long‐term observational studies conducted at large (regional) spatial scales contribute to better understanding of landscape effects on population and evolutionary dynamics, including the conditions that affect long‐term viability of species, but large‐scale studies are expensive and logistically challenging to keep running for a long time. Here, we describe the long‐term metapopulation study of the Glanville fritillary butterfly (Melitaea cinxia) that has been conducted since 1991 in a large network of 4000 habitat patches (dry meadows) within a study area of 50 by 70 km in the Åland Islands in Finland. We explain how the landscape structure has been described, including definition, delimitation, and mapping of the habitat patches; methods of field survey, including the logistics, cost, and reliability of the survey; and data management using the EarthCape biodiversity platform. We describe the long‐term metapopulation dynamics of the Glanville fritillary based on the survey. There has been no long‐term change in the overall size of the metapopulation, but the level of spatial synchrony and hence the amplitude of fluctuations in year‐to‐year metapopulation dynamics have increased over the years, possibly due to increasing frequency of exceptional weather conditions. We discuss the added value of large‐scale and long‐term population studies, but also emphasize the need to integrate more targeted experimental studies in the context of long‐term observational studies. For instance, in the case of the Glanville fritillary project, the long‐term study has produced an opportunity to sample individuals for experiments from local populations with a known demographic history. These studies have demonstrated striking differences in dispersal rate and other life‐history traits of individuals from newly established local populations (the offspring of colonizers) versus individuals from old, established local populations. The long‐term observational study has stimulated the development of metapopulation models and provided an opportunity to test model predictions. This combination of empirical studies and modeling has facilitated the study of key phenomena in spatial dynamics, such as extinction threshold and extinction debt.     

Synthesis and cytotoxic potency of novel tris(1-alkylindol-3-yl)methylium salts: Role of N-alkyl substituents

Novel derivatives of tris(indol-3-yl)methane and tris(indol-3-yl)methylium salts with the alkyl substituents at the N-atoms of the indole rings were synthesized. An easy substitution of indole rings in trisindolylmethanes for other indoles under the action of acids is demonstrated, and the mechanism of substitution is discussed. To obtain trisindolylmethylium salts, the environmentally safe method of oxidation of trisindolylmethanes with air oxygen in acidic conditions was developed. Tris(1-alkylindol-3-yl)methanes and tris(1-alkylindol-3-yl)methylium salts represent three-bladed molecular propellers whose physico-chemical and biological properties strongly depend on the N-alkyl substituent. The cytotoxicity of novel compounds increased with the number of C atoms in the alkyl chains, with optimal number n = 3–5 whereas the derivatives with longer side chains were less cytotoxic. The most potent novel compounds killed human tumor cells at nanomolar-to-submicromolar concentrations, being one order of magnitude more potent than the prototype antibiotic turbomycin A [tris(indol-3-yl)methylium salt]. Apoptosis in HCT116 colon carcinoma cell line induced by tris(1-pentyl-1H-indol-3-yl)methylium methanesulfonate was detectable at concentrations tolerable by normal blood lymphocytes. Thus, N-alkyl substituted tris(1-alkylindol-3-yl)methylium salts emerge as perspective anticancer drug candidates.     

Mitochondrial phylogeny of Locustella and related genera

We used maximum likelihood analysis of complete mitochondrial ND2 sequences (1041 bp) to clarify the taxonomy and relationships of various species and genera of grass and bush warblers. The tree revealed two clades of grass and bush warblers. One clade was comprised of all four western Palearctic Locustella and two species of Asian Bradypterus . The other clade included five eastern Palearctic Locustella (including the distinctive Sakhalin warbler Locustella amnicola ) and the marsh grassbird Megalurus pryeri . African Bradypterus and Australian little grassbird Megalurus gramineus were distantly related to their Asian congeners. Therefore, current taxonomy of these genera does not reflect their evolutionary history and needs revision. It is proposed that a phylogenetic analysis of morphology and ecological preferences would show that the current taxonomy of grass and bush warblers reflects species' habitat preferences and morphology related to locomotion and foraging in their habitats, rather than their shared ancestry. Distinct clades were found in grasshopper warbler Locustella naevia and Pallas's grasshopper warbler L. certhiola . Detailed phylogeographic studies are needed to elucidate the species status of the clades within these two species.     

Altered astrocyte glutamate transporter regulation of hypothalamic neurosecretory neurons in heart failure rats

Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-D-aspartate receptor (NMDAR)-mediated inward current (tonic I(NMDA)) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic I(NMDA) activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.     

Atomic Force Microscopy Analysis of the Acinetobacter baumannii Bacteriophage AP22 Lytic Cycle

Background

Acinetobacter baumannii is known for its ability to develop resistance to the major groups of antibiotics, form biofilms, and survive for long periods in hospital environments. The prevalence of infections caused by multidrug-resistant A. baumannii is a significant problem for the modern health care system, and application of lytic bacteriophages for controlling this pathogen may become a solution.

Methodology/Principal Findings

In this study, using atomic force microscopy (AFM) and microbiological assessment we have investigated A. baumannii bacteriophage AP22, which has been recently described. AFM has revealed the morphology of bacteriophage AP22, adsorbed on the surfaces of mica, graphite and host bacterial cells. Besides, morphological changes of bacteriophage AP22-infected A. baumannii cells were characterized at different stages of the lytic cycle, from phage adsorption to the cell lysis. The phage latent period, estimated from AFM was in good agreement with that obtained by microbiological methods (40 min). Bacteriophage AP22, whose head diameter is 62±1 nm and tail length is 88±9 nm, was shown to disperse A. baumannii aggregates and adsorb to the bacterial surface right from the first minute of their mutual incubation at 37°C.

Conclusions/Significance

High rate of bacteriophage AP22 specific adsorption and its ability to disperse bacterial aggregates make this phage very promising for biomedical antimicrobial applications. Complementing microbiological results with AFM data, we demonstrate an effective approach, which allows not only comparing independently obtained characteristics of the lytic cycle but also visualizing the infection process.     

Mitochondrial DNA and plumage evolution in the white wagtail Motacilla alba

We analyzed sequences of two mitochondrial DNA (mtDNA) gene regions (control region and ND2) totaling 1477 base‐pairs from 232 specimens of the white wagtail Motacilla alba obtained from 27 localities throughout Eurasia. Although overall haplotype diversity was relatively low (0.79) and the most common haplotype was shared by 45% of individuals, belonging to six subspecies, a high level of population differentiation was detected. The mtDNA tree revealed three clades: (1) most individuals from Krasnodar (belonging to M. a. alba subspecies), (2) all individuals from Almaty and some from Primor'e (belonging to M. a. personata, M. a. lugens and M. a. leucopsis subspecies), and (3) the remaining individuals (representing all subspecies and all localities except Almaty). We suggest that these three clades represent historically isolated populations that relatively recently came into secondary contact in Krasnodar and Primor'e. None of the six subspecies were reciprocally monophyletic in the mtDNA tree. The Krasnodar population appeared to receive immigrants from other localities, but distinctive haplotypes from this locality did not appear elsewhere, suggesting asymmetric gene flow. Signatures of recent gene flow between northern populations were detected, and there was no evidence of isolation by distance within the northern group of populations. Mismatch distributions for most localities were consistent with population expansions. We also analyzed 12 male plumage characters from 93 study skins sampled from 24 populations. Phylogenetic trees resulting from separate genetic and morphological analyses were incongruent. Plumage evolution seems to be under strong sexual or natural selection, which favors particular phenotypes in various areas irrespective of the mitochondrial background. Dispersal events at different evolutionary times could have obscured the effects of earlier isolation events. The mtDNA data does not support species status for M. a. lugens and M. a. personata, which shared haplotypes with other subspecies of M. alba. We recommend that M. lugens and M. personata are placed as junior synonyms of M. alba.