SUR-dependent modulation of KATP channels by an N-terminal KIR6.2 peptide. Defining intersubunit gating interactions

Ntp and Ctp, synthetic peptides based on the N- and C-terminal sequences of K(IR)6.0, respectively, were used to probe gating of K(IR)6.0/SUR K(ATP) channels. Micromolar Ntp dose-dependently increased the mean open channel probability in ligand-free solution (P(O(max))) and attenuated the ATP inhibition of K(IR)6.2/SUR1, but had no effect on homomeric K(IR)6.2 channels. Ntp (up to approximately 10(-4) m) did not affect significantly the mean open or "fast," K(+) driving force-dependent, intraburst closed times, verifying that Ntp selectively modulates the ratio of mean burst to interburst times. Ctp and Rnp, a randomized Ntp, had no effect, indicating that the effects of Ntp are structure specific. Ntp opened K(IR)6.1/SUR1 channels normally silent in the absence of stimulatory Mg(-) nucleotide(s) and attenuated the coupling of high-affinity sulfonylurea binding with K(ATP) pore closure. These effects resemble those seen with N-terminal deletions (DeltaN) of K(IR)6.0, and application of Ntp to DeltaNK(ATP) channels decreased their P(O(max)) and apparent IC(50) for ATP in the absence of Mg(2+). The results are consistent with a competition between Ntp and the endogenous N terminus for a site of interaction on the cytoplasmic face of the channel or with partial replacement of the deleted N terminus by Ntp, respectively. The K(IR) N terminus and the TMD0-L0 segment of SUR1 are known to control the P(O(max)). The L0 linker has been reported to be required for glibenclamide binding, and DeltaNK(IR)6.2/SUR1 channels exhibit reduced labeling of K(IR) with (125)I-azidoglibenclamide, implying that the K(IR) N terminus and L0 of SUR1 are in proximity. We hypothesize that L0 interacts with the K(IR) N terminus in ligand-inhibited K(ATP) channels and put forward a model, based on the architecture of BtuCD, MsbA, and the KcsA channel, in which TMD0-L0 links the MDR-like core of SUR with the K(IR) pore.     

Isolation and identification of the enzyme complex in Streptomyces recifensis var. lyticus 2435 with beta-lactamase activity

Ability of an enzyme complex from Streptomyces recifensis var. lyticus 2435 to inactivate beta-lactam antibiotics was shown. Two lytic endopeptidases with beta-lactamase activity were isolated and identified as beta-lactamases of classes II and V according to the Richmond and Sykes classification system. The ability of the endopeptidases to hydrolyze the beta-lactam ring confirmed the absence of strict substrate specificity in them. Correlation between the capacity of the lytic endopeptidases for lysing staphylococcal cells and their capacity for inactivating beta-lactam antibiotics was observed.     

Chiral superstructures of insulin amyloid fibrils

Hydrodynamic forces are capable of inducing structural order in dispersed solid phases, and of causing symmetry-breaking when chiral crystals precipitate from an achiral liquid phase. Until it was observed upon vortex-assisted fibrillation of insulin, such behavior had been thought to be confined to few unbiological systems. In this paper we are discussing chiroptical properties of two chiral variants of insulin amyloid, termed +ICD and -ICD, which form during the process of chiral bifurcation in vortexed solutions of aggregating insulin. As conventional measurements of circular dichroism of solid, anisotropic substances are particularly vulnerable to overlapping influences of linear birefringence and linear dichroism, we have employed complementary tools including dedicated universal chiroptical spectrophotometer to rule out such artifacts. We propose that the strong chiroptical properties of +ICD and -ICD insulin fibrils are an aspect of genuine superstructural chirality of amyloid fibrils and of powerful excitonic couplings taking place within them. A comparison of thioflavin T complexes with fibrils formed by insulin and polyglutamic acid suggests that the extrinsic Cotton effect stemming from the level of single twisted dye molecules is weaker, although diagnostically useful, and cannot account for the overall magnitude of ICD of the dye bound to ±ICD insulin amyloid.     

Evolutionary conservation suggests a regulatory function of AUG triplets in 5'-UTRs of eukaryotic genes

By comparing sequences of human, mouse and rat orthologous genes, we show that in 5′-untranslated regions (5′-UTRs) of mammalian cDNAs but not in 3′-UTRs or coding sequences, AUG is conserved to a significantly greater extent than any of the other 63 nt triplets. This effect is likely to reflect, primarily, bona fide evolutionary conservation, rather than cDNA annotation artifacts, because the excess of conserved upstream AUGs (uAUGs) is seen in 5′-UTRs containing stop codons in-frame with the start AUG and many of the conserved AUGs are found in different frames, consistent with the location in authentic non-coding sequences. Altogether, conserved uAUGs are present in at least 20–30% of mammalian genes. Qualitatively similar results were obtained by comparison of orthologous genes from different species of the yeast genus Saccharomyces. Together with the observation that mammalian and yeast 5′-UTRs are significantly depleted in overall AUG content, these findings suggest that AUG triplets in 5′-UTRs are subject to the pressure of purifying selection in two opposite directions: the uAUGs that have no specific function tend to be deleterious and get eliminated during evolution, whereas those uAUGs that do serve a function are conserved. Most probably, the principal role of the conserved uAUGs is attenuation of translation at the initiation stage, which is often additionally regulated by alternative splicing in the mammalian 5′-UTRs. Consistent with this hypothesis, we found that open reading frames starting from conserved uAUGs are significantly shorter than those starting from non-conserved uAUGs, possibly, owing to selection for optimization of the level of attenuation.     

Conservation versus parallel gains in intron evolution

Orthologous genes from distant eukaryotic species, e.g. animals and plants, share up to 25–30% intron positions. However, the relative contributions of evolutionary conservation and parallel gain of new introns into this pattern remain unknown. Here, the extent of independent insertion of introns in the same sites (parallel gain) in orthologous genes from phylogenetically distant eukaryotes is assessed within the framework of the protosplice site model. It is shown that protosplice sites are no more conserved during evolution of eukaryotic gene sequences than random sites. Simulation of intron insertion into protosplice sites with the observed protosplice site frequencies and intron densities shows that parallel gain can account but for a small fraction (5–10%) of shared intron positions in distantly related species. Thus, the presence of numerous introns in the same positions in orthologous genes from distant eukaryotes, such as animals, fungi and plants, appears to reflect mostly bona fide evolutionary conservation.     

The springtail (Hexapoda,Collembola) fauna of Wrangel Island

Analysis of the collembolan fauna of Wrangel Island based on published data and recent sampling was performed. The species richness of springtails on the island (89 species from 41 genera and 12 families) exceeds the levels characteristic of most other insular territories of the Arctic Ocean, except for Greenland and some small islands located within the southern tundra belt. The specificity of the species composition of the springtail fauna on the island depends on a high proportion of species with amphi-Pacific ranges, reflecting its formation within the Beringian Arctic sector. At the same time, the structure of the springtail fauna of the island is rather similar to those of the known tundra faunas of the Siberian sector, with no anomalies being detected.     

Molecular genetic analysis of the intratumoral clonal heterogeneity of colorectal adenocarcinomas

Molecular genetic analysis of allelic deletions from the loci containing the tumor suppressor genes p16, p15, p19 (9p21), RB1 (13p14), PTEN (10q23), and TP53 (17p13); microsatellite instability; and activating mutations of K-RAS (codons 12 and 13) was performed in four different segments of sporadic colorectal cancer (CRC) in 11 patients. Intratumoral genetic heterogenity was detected in 9 out of 11 (81%) colorectal adenocarcinomas and was morphologically validated. Analysis of different segments of one tumor reported that not only intratumoral heterogeneity, but also the order of the appearance and distribution of molecular anomalies during tumorigenesis in sporadic CRC. K-RAS point mutations and anomalies of the p16-RB1-cyclin D pathway were assumed to occur prior to microsatellite instability and PTEN deletions during tumor progression.     

Springtails (Hexapoda, Collembola) in karst landscapes of the Pinega State Reserve

The collembolan fauna of the Pinega State Reserve (Arkhangelsk Province) was examined. The study area is highly specific due to its karst topography. In all, 126 Collembola species were recorded in the reserve, a significant fraction of their diversity being restricted to karst habitats. Several species that had never been recorded in plain forest biotopes were of special interest. Such forms with distribution ranges mainly limited to the tundra zone comprised about 10% of the local fauna. Regarding the longitudinal sizes of the ranges, this group was not homogeneous and included both widespread circumpolar and east-Palaearctic (Siberian) species. Several arctoalpine forms were also recorded. Most of these arctic species were not numerous over the area studied and in general, the collembolan assemblages at the coldest sites of the reserve did not differ significantly from those typical of the forest zone, with the exception of two species, Desoria tshernovi and D. inupikella, which often predominated in the karst communities. The origin and appropriateness of the term relict for the revealed “insular” populations of arctic species are discussed.