Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues – 2′-deoxyguanosine-5′-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5′-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3′-5′)-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3′-5′)-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.     

Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-d-aspartate receptors

The synthesis and structure–activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modulated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and α₁-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood–brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency.     

DNA Polymerase ζ-Dependent Lesion Bypass in Saccharomyces cerevisiae Is Accompanied by Error-Prone Copying of Long Stretches of Adjacent DNA

Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.     

Human Adipose-Tissue Derived Stromal Cells in Combination with Hypoxia Effectively Support Ex Vivo Expansion of Cord Blood Haematopoietic Progenitors

The optimisation of haematopoietic stem and progenitor cell expansion is on demand in modern cell therapy. In this work, haematopoietic stem/progenitor cells (HSPCs) have been selected from unmanipulated cord blood mononuclear cells (cbMNCs) due to adhesion to human adipose-tissue derived stromal cells (ASCs) under standard (20%) and tissue-related (5%) oxygen. ASCs efficiently maintained viability and supported further HSPC expansion at 20% and 5% O₂. During co-culture with ASCs, a new floating population of differently committed HSPCs (HSPCs-1) grew. This suspension was enriched with СD34⁺ cells up to 6 (20% O₂) and 8 (5% O₂) times. Functional analysis of HSPCs-1 revealed cobble-stone area forming cells (CAFCs) and lineage-restricted colony-forming cells (CFCs). The number of CFCs was 1.6 times higher at tissue-related O₂, than in standard cultivation (20% O₂). This increase was related to a rise in the number of multipotent precursors - BFU-E, CFU-GEMM and CFU-GM. These changes were at least partly ensured by the increased concentration of MCP-1 and IL-8 at 5% O₂. In summary, our data demonstrated that human ASCs enables the selection of functionally active HSPCs from unfractionated cbMNCs, the further expansion of which without exogenous cytokines provides enrichment with CD34⁺ cells. ASCs efficiently support the viability and proliferation of cord blood haematopoietic progenitors of different commitment at standard and tissue-related O₂ levels at the expense of direct and paracrine cell-to-cell interactions.     

Plant volatiles providing additional evidences to the occurence of a wild-growing population of Calamintha vardarensis (Greuter et Burdet) Šilić outside of its natural habitat

The essential‐oil profile of a Calamintha species, wild‐growing in the urban settings of the city of Ni? (South Serbia) and botanically tentatively identified as C. vardarensis (endemic species native to FYR Macedonia and East Serbia), has been statistically (multivariate statistical analyses) compared with those of other Calamintha species, including two previously investigated C. vardarensis populations, as a means of corroboration of the surprising occurence of this Calamintha population outside of its natural distributional range. Agglomerative hierarchical clustering reveals a close link of C. vardarensis from Ni? (with neo‐menthol (40.0%), menthone (21.8%), and pulegone (27.2%) as its major oil contributors) and C. vardarensis from FYR Macedonia.     

The epithelial potassium channel Kir7.1 is stimulated by progesterone

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present.     

Differential ATP binding and intrinsic ATP hydrolysis by amino-terminal domains of the yeast Mlh1 and Pms1 proteins.

MutL homologs belong to a family of proteins that share a conserved ATP binding site. We demonstrate that amino-terminal domains of the yeast MutL homologs Mlh1 and Pms1 required for DNA mismatch repair both possess independent, intrinsic ATPase activities. Amino acid substitutions in the conserved ATP binding sites concomitantly reduce ATP binding, ATP hydrolysis, and DNA mismatch repair in vivo. The ATPase activities are weak, consistent with the hypothesis that ATP binding is primarily responsible for modulating interactions with other MMR components. Three approaches, ATP hydrolysis assays, limited proteolysis protection, and equilibrium dialysis, provide evidence that the amino-terminal domain of Mlh1 binds ATP with >10-fold higher affinity than does the amino-terminal domain of Pms1. This is consistent with a model wherein ATP may first bind to Mlh1, resulting in events that permit ATP binding to Pms1 and later steps in DNA mismatch repair.