A functional link between Polo-like kinase 1 and the mammalian Target-Of-Rapamycin pathway?

Polo like kinase-1 is a key effector of cell division and its over-expression in several cancers is often linked with negative prognostic. We recently described that Plk1 is over-expressed in acute myeloid leukemia, and that its inhibition selectively reduces the proliferation of leukemic cells. Here, we report that Plk1 inhibition or depletion using pharmacological and siRNA approaches decreased the phosphorylation of two mTOR substrates in AML cells. In HCT116 cells, inducible expression of a constitutively active form of Plk1 leads to activation of mTOR, as shown by increased phosphorylation of its 4E-BP1 and RPS6 down-stream targets. In addition, HCT116 cells over-expressing the active form of Plk1 were characterized by abnormal growth that could be reversed by rapamycin, a specific inhibitor of the TORC1 complex. Altogether these data suggest the existence of a molecular and functional link between the Plk1 mitotic kinase and the mTOR pathway. Given the different established functions of Plk1 and mTOR during the cell cycle, we will discuss the possible meaning of this functional relationship.     

Kringle 4 from human plasminogen:1H-nuclear magnetic resonance study of the interactions between ω-amino acid ligands and aromatic residues at the lysine-binding site

The interactions of theω-amino acid ligandsε-aminocaproic acid andp-benzylaminesulphonic acid with the isolated kringle 4 domain from human plasminogen have been investigated by¹H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Overall, the data indicate that binding either ligand does not cause the kringle to undergo significant conformational changes. When p-benzylaminesulphonic acid is in excess relative to the kringles, progressive exchange-broadening and high field chemical shifts are observed for the proton resonances of the ligand. The largest effect is seen at the amino end of the molecule, which indicates that the — NH ₃ ⁺ group of the ligand penetrates deeper into the binding site than does the — SO ₃ ^- . Ligand-binding causes signals from the ring-current shifted Leu⁴⁶ CH ₃ ^δ .δ groups and from a number of aromatic side-chains to shift. Depending on the ligand, the latter include Tyr-II (Tyr⁵⁰), Tyr-V (an immobile ring), His-II and His-III imidazole groups and the three Trp indole groups present in kringle 4. In particular,p-benzylaminesulphonic acid-binding induces large high field shifts on the Trp-II H6 triplet and the Trp-III (Trp⁷²) H2 singlet. On the other hand,ε-aminocaproic acid bound to kringle 4 exhibits large chemical shifts of its CH₂ proton resonances, which indicates that the lysine-binding site is rich in aromatic side chains. Overhauser experiments centered on thep-benzylaminesulphonic acid H2,6 and H3,5 aromatic transitions as well as on the shifted Trp-II and Trp-III signals reveal efficient cross-relaxation between these two indole side chains and thep-benzylaminesulphonic acid ring. These experiments also show that the side chains from Phe⁶⁴, Tyr-II (Tyr⁵⁰), Tyr-IV, and His-II (His³¹) interact with the ligand. In combination with reported chemical modification experiments that show requirement of Asp⁵⁷, Arg⁷¹ and Trp⁷² integrity for ligand-binding, our study underscores the relevance of the Cys⁵¹-Cys⁷⁵ loop in defining the kringles’ lysine-binding site. Furthermore, the Cys²²-Cys⁶³ loop is folded so as to place His³¹, His³³, Tyr⁴¹ and Leu⁴⁶ in proximity to the binding site. The involvement of residues within the Cys⁵¹-Cys⁷⁵ loop in ligand-binding suggests that Trp-II and Tyr-IV may correspond to Trp⁶² and Tyr⁷⁴, respectively. As shown by Overhauser experiments, these two residues are in close contact with each other. From these studies and from the shielding and deshielding effects caused byp-benzylaminesulphonic acid, we suggest that the ligand is sandwiched between the indole rings of Trp-II and Trp-III, which form part of the hydrophobic binding site.     

Do large effect QTL fractionate? A case study at the maize domestication QTL teosinte branched1

Quantitative trait loci (QTL) mapping is a valuable tool for studying the genetic architecture of trait variation. Despite the large number of QTL studies reported in the literature, the identified QTL are rarely mapped to the underlying genes and it is usually unclear whether a QTL corresponds to one or multiple linked genes. Similarly, when QTL for several traits colocalize, it is usually unclear whether this is due to the pleiotropic action of a single gene or multiple linked genes, each affecting one trait. The domestication gene teosinte branched1 (tb1) was previously identified as a major domestication QTL with large effects on the differences in plant and ear architecture between maize and teosinte. Here we present the results of two experiments that were performed to determine whether the single gene tb1 explains all trait variation for its genomic region or whether the domestication QTL at tb1 fractionates into multiple linked QTL. For traits measuring plant architecture, we detected only one QTL per trait and these QTL all mapped to tb1. These results indicate that tb1 is the sole gene for plant architecture traits that segregates in our QTL mapping populations. For most traits related to ear morphology, we detected multiple QTL per trait in the tb1 genomic region, including a large effect QTL at tb1 itself plus one or two additional linked QTL. tb1 is epistatic to two of these additional QTL for ear traits. Overall, these results provide examples for both a major QTL that maps to a single gene, as well as a case in which a QTL fractionates into multiple linked QTL.     

Is the association between optimistic cardiovascular risk perceptions and lower rates of cardiovascular disease mortality explained by biomarkers of systemic inflammation or endothelial function? A case-cohort study

Background  

More optimistic perceptions of cardiovascular disease risk are associated with substantively lower rates of cardiovascular death among men. It remains unknown whether this association represents causality (i.e. perception leads to actions/conditions that influence cardiovascular disease occurrence) or residual confounding by unmeasured factors that associate with risk perceptions and with physiological processes that promote cardiovascular disease (i.e. inflammation or endothelial dysfunction).     

Methylation of progesterone receptor isoform A promoter in normal breast tissue: An epigenetic link between early age at menarche and risk of breast cancer?

Emerging evidence indicates that some altered patterns of methylation that occur in breast tumors may also be found in breast tissue of healthy women in relation to the breast cancer (BC) risk factors. Progesterone receptor (PR) isoform α is a crucial regulator of breast hormone responsiveness and its hypermethylation plays an important role in the initiation and development of breast tumors. However, such a methylation change in healthy women and its link with the different risk factors has not yet been investigated. In the present study, we aimed to examine the relationship of possible methylation changes within a critical region in the promoter CpG island of PGR-α (progesterone receptor α) gene in the healthy women with a set of reproductive and nonreproductive BC risk factors. The breast tissues were collected from 120 cancer-free women who had undergone cosmetic mammoplasty. The genomic DNA was extracted from the breast tissues and the methylation level of PGR-α promoter CpG island was determined by using MeDIP-qPCR assay. Using regression analysis, we found that increasing menarche age is inversely associated with the high methylation of PGR-α promoter ( β = −0.790, SE = 0.362; P = 0.031). Although lactating women had more methylation than nonlactating women (P = 0.026, the t test), this result was not confirmed by regression models. Such an observation may be helpful in better understanding of the underlying mechanisms by which early age at menarche increases the risk of BC. However, this perspective requires further validations in larger studies of more subjects as well as the inclusion of other related genes.     

Macrophage inflammatory protein-1α: A link between innate immunity and familial mediterranean fever?

The aim of this study is to investigate the relationship between chemokines and the inflammation in Familial Mediterranean Fever (FMF). Forty-nine patients with FMF (41 in remission and 8 in acute attack period) and 20 healthy controls were included in the study. Serum levels of macrophage inflammatory protein-1alpha (MIP-1alpha) were assessed in the patients and the controls, along with other parameters of disease activity, i.e., fibrinogen, C-reactive protein and erythrocyte sedimentation rate. Serum MIP-1alpha levels of the patients with FMF in acute attack period were significantly higher than the patients in remission and healthy controls (p=0.02 and p=0.038, respectively). MIP-1alpha levels were weakly correlated with CRP (r=0.32, p=0.032) levels. MIP-1alpha may have a role in the pathogenesis of FMF attacks. MIP-1alpha and other chemokines may constitute a link between the innate immune system and FMF.     

A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

An integrative approach combining ion mobility mass spectrometry,X-ray crystallography,and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1-antitrypsin upon ligand binding

Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α₁-antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α₁-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α₁-antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α₁-antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.PDB Code(s): 4PYW     

A DFT study on the mechanisms for the cycloaddition reactions between 1−aza-2-azoniaallene cations and acetylenes

The mechanisms of cycloaddition reactions between 1-aza-2-azoniaallene cations 1 and acetylenes 2 have been investigated using the global electrophilicity and nucleophilicity of the corresponding reactants as global reactivity indexes defined within the conceptual density functional theory. The reactivity and regioselectivity of these reactions were predicted by analysis of the energies, geometries, and electronic nature of the transition state structures. The theoretical results revealed that the reaction features a tandem process: an ionic 1,3-dipolar cycloaddition to produce the cycloadducts 3?H-pyrazolium salts 3 followed by a [1,2]-shift affording the thermodynamically more stable adducts 4 or 5. The mechanism of the cycloaddition reactions can be described as an asynchronous concerted pathway with reverse electron demand. The model reaction has also been investigated at the QCISD/6-31++G(d,p) and CCSD(T)/6-31++G(d,p)//B3LYP/6-31++G(d,p) levels as well as by the DFT. The polarizable continuum model, at the B3LYP/6-31++G(d,p) level of theory, was used to study solvent effects on all the studied reactions. In solvent dichloromethane, all the initial cycloadducts 3 were obtained via direct ionic process as the result of the solvent effect. The consecutive [1,2]-shift reaction, in which intermediates 3 are rearranged to the five-membered heterocycles 4/5, is proved to be a kinetically controlled reaction, and the regioselectivity can be modulated by varying the migrant. The LOL function and RDG function based on localized electron analysis were used to analysis the covalent bond and noncovalent interactions in order to unravel the mechanism of the title reactions.     

A combined nuclear magnetic resonance and molecular dynamics study of the two structural motifs for mixed-linkage β-glucans: methyl β-cellobioside and methyl β-laminarabioside

The conformational and hydration properties of the two disaccharides methyl β-cellobioside and methyl β-laminarabioside were investigated by NMR spectroscopy and explicit solvation molecular dynamics simulations using the carbohydrate solution force field (CSFF). Adiabatic maps produced with this force field displayed 4 minima A: (Φ = 300°, Ψ = 280°), B: (Φ = 280°, Ψ = 210°), C: (Φ = 260°, Ψ = 60°), and D: (Φ = 60°, Ψ = 260°) for methyl β-cellobioside and 3 minima A: (Φ = 290°, Ψ = 130°), B: (Φ = 270°, Ψ = 290°), and C: (Φ = 60°, Ψ = 120°) for methyl β-laminarabioside. Molecular dynamics simulations were initiated from all minima. For each disaccharide, the simulation started from the A minimum was conducted for 50 ns, while the other minima were explored for 10 ns. The simulations revealed two stable minima for both compounds. For methyl β-cellobioside, the simulation minima in aqueous solution were shifted from their adiabatic map counterparts, while the simulation minima for methyl β-laminarabioside coincided with the corresponding adiabatic map minima. To validate the simulation results, NMR-derived NOEs and coupling constants across the glycoside linkage, ³J_HC and ³J_CH, were compared with values calculated from the MD trajectories. For each disaccharide, the best agreement was obtained for the simulations started at the A minimum. For both compounds, inter-ring water bridges in combination with the direct hydrogen bonds between the same groups were found to be determining factors for the overall solution structure of the disaccharides which differed from solid-state structures. Comparison with helical parameters showed that the preferred glycosidic dihedral configurations in the methyl β-cellobioside simulation were not highly compatible with the structure of cellulose, but that curdlan helix structures agreed relatively well with the methyl β-laminarabioside simulation. Polymers generated using glycosidic dihedral angles from the simulations revealed secondary structure motifs that that may help to elucidate polymer associations and small-molecule binding.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Matrix metalloproteinases in the adult brain physiology: a link between c-Fos,AP-1 and remodeling of neuronal connections?

Matrix metalloproteinases (MMPs), together with their endogenous inhibitors (TIMPs) form an enzymatic system that plays an important role in a variety of physiological and pathological conditions. These proteins are also expressed in the brain, especially under pathological conditions, in which glia as well as invading inflammatory cells provide the major source of the MMP activity. Surprisingly little is known about the MMP function(s) in adult neuronal physiology. This review describes available data on this topic, which is presented in a context of knowledge about the MMP/TIMP system in other organs as well as in brain disorders. An analysis of the MMP and TIMP expression patterns in the brain, along with a consideration of their regulatory mechanisms and substrates, leads to the proposal of possible roles of the MMP system in the brain. This analysis suggests that MMPs may play an important role in the neuronal physiology, especially in neuronal plasticity, including their direct participation in the remodeling of synaptic connections-a mechanism pivotal for learning and memory.     

Do climatically similar regions contain similar alien floras? A comparison between the mediterranean areas of central Chile and California

Aim Taxonomic comparisons of alien floras across climatically similar regions have been proposed as a powerful approach for increasing our understanding of plant invasions across scales. However, detailed comparisons between the alien biotas of climatically similar regions are scarce. This study aims to compare the taxonomic patterns of alien species richness in mediterranean‐type climate areas of central Chile and California, in order to better understand how climatically similar regions converge or diverge in terms of their alien flora. Location Central Chile and California, United States. Methods We compared the alien floras of the state of California in the United States and central Chile, considering within‐region variation and taxonomic composition up to the species level. To test for within‐region variation, administrative units and counties were grouped within seven latitudinal bands for each region. We tested for differences in the relative contributions of the various origins of the naturalized species to each region. We used a family naturalization index to establish which families had relatively higher numbers of naturalized species in each region. We evaluated the similarity, using cluster analyses with Jaccard’s similarity index, of alien taxa between regions and latitudinal bands using presence–absence matrices at the species, genus and family levels. We used principal components analysis to determine the presence of a compositional gradient including all latitudinal bands. Results We recorded 1212 alien plant species in California and 593 in central Chile, of which 491 are shared between the two regions. These figures include 25 species that are native to California and 37 that are native to Chile. A comparison between the alien floras of central Chile and California reveals three major trends: (1) higher naturalized species diversity for California than for Chile, at all taxonomic levels; (2) differences in the proportion of species according to origin, with America, Africa, Asia and Australia providing a larger number of species in California than in Chile; (3) segregation between regions in terms of taxonomic composition of their alien flora, and a rather weak differentiation within regions; and (4) a trend towards higher similarity between the alien floras of latitudinal bands associated with higher levels of human disturbances. Main conclusions The alien floras of central Chile and California are significantly different, but this difference diminishes in highly disturbed areas. Thus, the current high levels of species movement caused by globalization, together with increasing levels of anthropogenic disturbances, should reduce the differentiation of the alien floras in these regions, increasing overall biotic homogenization.     

Crosstalk between the M1 muscarinic acetylcholine receptor and the endocannabinoid system: A relevance for Alzheimer's disease?

Alzheimer's disease (AD) is a neurodegenerative disorder which accounts for 60–70% of the 50 million worldwide cases of dementia and is characterised by cognitive impairments, many of which have long been associated with dysfunction of the cholinergic system. Although the M₁ muscarinic acetylcholine receptor (mAChR) is considered a promising drug target for AD, ligands targeting this receptor have so far been unsuccessful in clinical trials. As modulatory receptors to cholinergic transmission, the endocannabinoid system may be a promising drug target to allow fine tuning of the cholinergic system. Furthermore, disease-related changes have been found in the endocannabinoid system during AD progression and indeed targeting the endocannabinoid system at specific disease stages alleviates cognitive symptoms in numerous mouse models of AD. Here we review the role of the endocannabinoid system in AD, and its crosstalk with mAChRs as a potential drug target for cholinergic dysfunction.     

Is the association between TNF-alpha-308 A allele and DMT1 independent of HLA-DRB1, DQB1 alleles?

The aim of the study was to assess chosen factors of genetic susceptibility to DMT1: DRB1, DQB1, and TNF-alpha polymorphisms-308 (G/A) in children with DMT1 and their up-to-now healthy siblings. Then we tested whether the association between TNF-alpha genes and DMT1 is independent of HLA. 87 diabetic children, their 78 siblings, and 85 persons from healthy control group were followed up. The highest risk of DMT1 was connected with alleles: DRB1*0401 (OR = 3.39; CI: 1.55-7.41), DRB1*0301 (OR = 2.72; CI: 1.48-5.01), DQB1*0201 (OR = 4.04; CI: 2.17-7.52), DQB1*0302 (OR = 5.08; CI: 2.54-10.14), and TNF-alpha-308 A allele (OR = 2.59; CI: 1.23-5.44). Moreover linkage disequilibrium for TNF-alpha-308 A allele with DRB1*0301 and DQB1*0201 was observed in both diabetic children and their siblings. Diabetic children and their siblings present similar genetic risk factors for DMT1. The association between TNF-alpha-308 A allele and DMT1 is dependent of HLA-DRB1 and DQB1 alleles.     

Backbone 1H, 13C and 15N resonance assignments of the 39?kDa staphylococcal hemoglobin receptor IsdH

During infections Stahpylococcus aureus preferentially uses heme as an iron source, which it captures from human hemoglobin using the Iron regulated surface determinant (Isd) system. On the cell surface two related staphylococcal surface receptors called IsdH and IsdB bind to hemoglobin and extract its heme. Both receptors contain multiple NEAr iron Transporter (NEAT) domains that either bind to hemoglobin, or to heme. All previous structural studies have investigated individual NEAT domains and have not explored how the domains might interact with one another to synergistically extract heme from hemoglobin. Here, we report the near complete (1)H, (13)C and (15)N backbone resonance assignments of a bi-domain unit from IsdH that contains the N2 and N3 NEAT domains, which bind to hemoglobin and heme, respectively (IsdH(N2N3), residues 326-660, 39 kDa). The assigned backbone resonances lay the foundation for future NMR studies that will explore the molecular basis of IsdH function.