Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

Anti-influenza activity of diazaadamantanes combined with monoterpene moieties

The antiviral activity of several diaza-adamantanes containing monoterpenoid moieties against a rimantadine-resistant strain of the influenza A/Puerto Rico/8/34 (H1N1) virus was studied. Hetero-adamantanes containing monoterpene moieties at the aminal position of the heterocycle were found to exhibit lower activity compared to compounds with a diaza-adamantane fragment and a monoterpene moiety linked via an amino group at the 6-position of the hetero-adamantane ring. The highest selectivity index (a ratio of the 50% cytotoxic concentration to the 50% inhibitory concentration) out of 30 was observed for compound 8d, which contains a citronellal monoterpenoid moiety. Diaza-adamantane 8d was superior to its adamantane-containing analog 5 both in its anti-influenza activity and selectivity. Furthermore, 8d has more balanced physicochemical properties than 5, making the former a more promising drug candidate. Modelling these compounds against an influenza virus M2 ion channel predicted plausible binding modes to both the wild-type and the mutant (S31N).     

Complex biogeographic history of a Holarctic passerine

Our analysis of the ND2 sequences revealed six clades within winter wrens (Troglodytes troglodytes). These clades corresponded to six geographical regions: western Nearctic, eastern Nearctic, eastern Asia, Nepal, Caucasus and Europe, and differed by 3-8.8% of sequence divergence. Differences among regions explained 96% of the sequence variation in winter wren. Differences among individuals within localities explained 3% of the sequence variation, and differences among localities within regions explained 1%. Grouping sequences into subspecies instead of localities did not change these proportions. Proliferation of the six clades coincided with Early and Middle Pleistocene glaciations. The distribution of winter wren clades can be explained by a series of five consecutive vicariant events. Western Nearctic wrens diverged from the Holarctic ancestor 1.6 Myr before the present time (MYBP). Eastern Nearctic and Palaearctic wrens diverged 1 MYBP. Eastern and western Palaearctic birds diverged 0.83 MYBP. Nepalese and east Asian wrens diverged 0.67 MYBP, and Caucasian birds diverged from European wrens 0.54 MYBP. The winter wren has a much greater degree of inter- and intracontinental differentiation than the three other Holarctic birds studied to date--dunlin (Calidris alpina), common raven (Corvus corax) and three-toed woodpecker (Picoides trydactylus)--and represents an example of cryptic speciation that has been overlooked.     

Molecular evolution of the Est-6 gene in Drosophila melanogaster: contrasting patterns of DNA variability in adjacent functional regions

We have investigated nucleotide polymorphism at the esterase 6 gene (Est-6) gene, including the complete coding region (1686 bp), as well as the 5'-flanking (1183 bp) and 3'-flanking (193 bp) regions of the gene, in 30 strains of Drosophila melanogaster and in one strain of Drosophila simulans. The level of silent variation is similar in the coding and in the 3'-flanking region, but smaller in the 5'-flanking region. Strong linkage disequilibrium occurs within each region; and also, although less pronounced, between the 5'-flanking region and the rest of the gene, including the 3'-flanking region. We suggest that the pattern of nucleotide polymorphism of Est-6 may be shaped by: (1) directional and balancing selection acting on the promoter and the coding region; and (2) interactions between the two regions that involve variable degrees of hitchhiking. The patterns of linkage disequilibrium, as well as the statistics Z(nS) (Genetics 146 (1997)1197) and B and Q (Genet. Res. 74 (1999) 65), may be interpreted as there being multiple targets of selection within the gene. The previously reported Est-6 allozyme latitudinal clines may be accounted for by the interaction between selective processes in the promoter and coding regions.     

Inhibition of APCCdh1 activity by Cdh1/Acm1/Bmh1 ternary complex formation

The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing proteolysis of key regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G(1) phase, whereas phosphorylation of Cdh1 at the end of G(1) phase by cyclin-dependent kinases assists in the inactivation of APC(Cdh1). Here, we suggest additional components are involved in the inactivation of APC(Cdh1) independent of Cdh1 phosphorylation. We have identified proteins known as Acm1 and Bmh1, which bind and form a ternary complex with Cdh1. The presence of phosphorylated Acm1 is critical for the ternary complex formation, and Acm1 is predominantly expressed in S phase when APC(Cdh1) is inactive. The assembly of the ternary complex inhibits ubiquitination of Clb2 in vitro by blocking the interaction of Cdh1 with Clb2. In vivo, lethality caused by overexpression of constitutively active Cdh1 is rescued by overexpression of Acm1. Partially phosphorylated Cdh1 in the absence of ACM1 still binds to and activates the APC. However, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Taken together, our results suggest an additional inactivation mechanism exists for APC(Cdh1) that is independent of Cdh1 phosphorylation.     

Description of a new diatom genus Dorofeyukea gen. nov. with remarks on phylogeny of the family Stauroneidaceae

Type material of Navicula kotschyi was studied, and this species was transferred to Dorofeyukea gen. nov. as D. kotschyi comb. nov. Dorofeyukea was described on the basis of DNA sequence and morphological data. Additional species assigned to this genus that were previously included in Navicula include: D. ancisa comb. nov., D. grimmei comb. nov., D. ivatoensis comb. nov., D. orangiana comb. nov., D. rostellata comb. nov. & stat. nov., D. savannahiana comb. nov., D. tenuipunctata comb. nov., and D. texana comb. nov. All Dorofeyukea species share the same morphological features, including having a narrow stauroid fascia surrounded by 1–3 irregularly shortened striae, uniseriate, and weakly radiate striae, circular, or rectangular puncta in the striae that are covered internally by dome‐shaped hymenes, presence of a pseudoseptum at each apex and absence of septa. Partial DNA sequences of SSU and rbcL loci show Dorofeuykae belongs to the clade of stauroneioid diatoms together with Stauroneis, Prestauroneis, Craticula, Karayevia, Madinithidium, Fistulifera, Parlibellus, and, possibly, Schizostauron. A new species from the monoraphid genus Madinithidium, M. vietnamica sp. nov., was described based on valve and chloroplast morphology as well as DNA sequence data.     

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR,a light-driven proton pump from Exiguobacterium sibiricum

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H⁺ transfer.     

Adaptive radiation of barbs of the genus Labeobarbus (Cyprinidae) in an East African river

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Effects of Asymmetric Nuclear Introgression,Introgressive Mitochondrial Sweep,and Purifying Selection on Phylogenetic Reconstruction and Divergence Estimates in the Pacific Clade of Locustella Warblers

When isolated but reproductively compatible populations expand geographically and meet, simulations predict asymmetric introgression of neutral loci from a local to invading taxon. Genetic introgression may affect phylogenetic reconstruction by obscuring topology and divergence estimates. We combined phylogenetic analysis of sequences from one mtDNA and 12 nuDNA loci with analysis of gene flow among 5 species of Pacific Locustella warblers to test for presence of genetic introgression and its effects on tree topology and divergence estimates. Our data showed that nuDNA introgression was substantial and asymmetrical among all members of superspecies groups whereas mtDNA showed no introgression except a single species pair where the invader''s mtDNA was swept by mtDNA of the local species. This introgressive sweep of mtDNA had the opposite direction of the nuDNA introgression and resulted in the paraphyly of the local species'' mtDNA haplotypes with respect to those of the invader. Тhe multilocus nuDNA species tree resolved all inter- and intraspecific relationships despite substantial introgression. However, the node ages on the species tree may be underestimated as suggested by the differences in node age estimates based on non-introgressing mtDNA and introgressing nuDNA. In turn, the introgressive sweep and strong purifying selection appear to elongate internal branches in the mtDNA gene tree.