Structure of the Cdt1 C‐terminal domain: Conservation of the winged helix fold in replication licensing factors

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2‐7 complex onto the origin of chromosome. The C‐terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2‐7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X‐ray crystallography and solution NMR spectroscopy, respectively. While the N‐terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.     

From lead to preclinical candidate: Optimization of β-homophenylalanine based inhibitors of dipeptidyl peptidase IV

A series of highly potent and selective inhibitors of DPP-4 was optimized for ADMET properties. The effort resulted in the discovery of inhibitor 1g, that exhibits excellent efficacy in an oral glucose tolerance test and an attractive pharmacokinetic profile.     

LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase

Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.     

Potential activity of methane and ammonia oxidation by methanotropic communities from soda lakes of the southern Transbaikal

Radioisotopic measurements of the methane consumption by mud samples taken from nine Southern Transbaikal soda lakes (pH 9.5-10.6) showed an intense oxidation of methane in the muds of lakes Khuzhirta, Bumalai Nur, Gorbunka, and Suduntuiskii Torom, with the maximum oxidation rate in the mud of lakes Khuzhirta (33.2 nmol/(ml day)). The incorporation rate of the radioactive label from 14CH4 into 14CO2 was higher than into acid-stable metabolites. Optimum pH values for methane oxidation in water samples were 7-8, whereas mud samples exhibited two peaks of methane oxidation activity (at pH 8.15-9.4 and 5.8-7.0). The majority of samples could oxidize ammonium to nitrites; the oxidation was inhibited by methane. The PCR amplification analysis of samples revealed the presence of genes encoding soluble and particulate methane monooxygenase and methanol dehydrogenase. Three alkaliphilic methanotrophic bacteria of morphotype I were isolated from mud samples in pure cultures, one of which, B5, was able to oxidize ammonium to nitrites at pH 7-11. The data obtained suggest that methanotrophs are widely spread in the soda lakes of Southern Transbaikal, where they actively oxidize methane and ammonium.     

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Dynamics and thermodynamic properties of CXCL7 chemokine

Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5‐Cys31 and Cys7‐Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer. Proteins 2015; 83:1987–2007. © 2015 Wiley Periodicals, Inc.     

The average local ionization energy as a tool for identifying reactive sites on defect-containing model graphene systems

In a continuing effort to further explore the use of the average local ionization energy $ \overline{\mathrm{I}}\left( \mathbf{r} \right) $ as a computational tool, we have investigated how well $ \overline{\mathrm{I}}\left( \mathbf{r} \right) $ computed on molecular surfaces serves as a predictive tool for identifying the sites of the more reactive electrons in several nonplanar defect-containing model graphene systems, each containing one or more pentagons. They include corannulene (C₂₀H₁₀), two inverse Stone-Thrower-Wales defect-containing structures C₂₆H₁₂ and C₄₂H₁₆, and a nanotube cap model C₂₂H₆, whose end is formed by three fused pentagons. Coronene (C₂₄H₁₂) has been included as a reference planar defect-free graphene model. We have optimized the structures of these systems as well as several monohydrogenated derivatives at the B3PW91/6-31G* level, and have computed their $ \overline{\mathrm{I}}\left( \mathbf{r} \right) $ on molecular surfaces corresponding to the 0.001 au, 0.003 au and 0.005 au contours of the electronic density. We find that (1) the convex sides of the interior carbons of the nonplanar models are more reactive than the concave sides, and (2) the magnitudes of the lowest $ \overline{\mathrm{I}}\left( \mathbf{r} \right) $ surface minima (the $ {{\overline{\mathrm{I}}}_{{\mathrm{S}\text{,}\min }}} $ ) correlate well with the interaction energies for hydrogenation at these sites. These $ {{\overline{\mathrm{I}}}_{{\mathrm{S}\text{,}\min }}} $ values decrease in magnitude as the nonplanarity of the site increases, consistent with earlier studies. A practical benefit of the use of $ \overline{\mathrm{I}}\left( \mathbf{r} \right) $ is that a single calculation suffices to characterize the numerous sites on a large molecular system, such as graphene and defect-containing graphene models.

The first report on the induction of hairy roots in Trapa natans,a unique aquatic plant with photosynthesizing roots

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation.