Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.     

Partial molar volumes of mRNA 5' cap analogues

Partial molar volumes in aqueous solution of eleven selected 7-methylguanine cap-analogues and their guanine counterparts were determined by means of density measurements. Hydrophobicity of the investigated compounds regarding their structural features was analysed within the framework of the solute-solvent interaction model, based on the relative density of the molecular solvation shell.     

Rtg2 protein links metabolism and genome stability in yeast longevity

Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromosomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relaying the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.     

Aggregation and structural changes of α(S1)-, β- and κ-caseins induced by homocysteinylation

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.     

Expansion of effector memory regulatory T cells represents a novel prognostic factor in lower risk myelodysplastic syndrome

Myelodysplastic syndromes are premalignant diseases characterized by cytopenias, myeloid dysplasia, immune dysregulation with association to autoimmunity, and variable risk for acute myeloid leukemia transformation. Studies of FOXP3(+) regulatory T cells (Tregs) indicate that the number and/or activation state may influence cancer progression in these patients. Focusing on patients with a lower risk for leukemia transformation, 18 (34.6%) of 52 patients studied displayed an altered Treg compartment compared with age-matched controls. Delineation of unique Treg subsets revealed that an increase in the absolute number of CD4(+)FOXP3(+)CD25(+)CD127(low)CD45RA(-)CD27(-) Tregs (effector memory Tregs [Treg(EM)]) was significantly associated with anemia (p = 0.046), reduced hemoglobin (p = 0.038), and blast counts ≥5% (p = 0.006). In healthy donors, this Treg(EM) population constitutes only 2% of all Tregs (one to six Tregs per microliter) in peripheral blood but, when isolated, exhibit greater suppressive activity in vitro. With a median follow-up of 3.1 y (range 2.7-4.9 y) from sample acquisition, increased numbers of Treg(EM) cells proved to have independent prognostic importance in survival estimates, suggesting that enumeration of this Treg subset may be a more reliable indicator of immunological escape than FOXP3(+) T cells as a whole. Based on multivariate analyses, Treg(EM) impacted survival independently from myeloblast characteristics, cytopenias, karyotype, and comorbidities. Based on these findings, Treg(EM) cell expansion may be synonymous with human Treg activation and indicate microenvironmental changes conducive to transformation in myelodysplastic syndromes.     

Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme

The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.     

Uncovering hidden coral diversity: a new cryptic lobophylliid scleractinian from the Indian Ocean

Extant biodiversity can easily be underestimated owing to the presence of cryptic taxa, even among commonly observed species. Scleractinian corals are challenging to identify because of their ecophenotypic variation and morphological plasticity. In addition, molecular analyses have revealed the occurrence of cryptic speciation. Here, we describe a new cryptic lobophylliid genus and species Paraechinophyllia variabilis gen. nov., sp. nov., which is morphologically similar to Echinophyllia aspera and E. orpheensis. The new taxon occurs in Mayotte Island, Madagascar, the Gulf of Aden and the Red Sea. Six molecular markers (COI, 12S, ATP6-NAD4, NAD3-NAD5, histone H3 and ITS) and 46 morphological characters at three different levels (macromorphology, micromorphology and microstructure) were examined. The resulting molecular phylogenetic reconstruction showed that Paraechinophyllia gen. nov. represents a distinct group within the Lobophylliidae that diverged from the lineage leading to Echinophyllia and Oxypora in the Early Miocene, approximately 21.5 Ma. The morphological phylogenetic reconstruction clustered Paraechinophyllia gen. nov., Echinophyllia and Oxypora together in a single clade. A sole morphological character, calice relief, discriminated Paraechinophyllia gen. nov. from the latter two genera, suggesting that limited morphological variation has occurred over a long period. These results highlight the importance of cryptic taxa in reef corals, with implications for population genetics, ecological studies and conservation.     

Seeing the forest through the trees: comprehensive inference on individual mating patterns in a mixed stand of Quercus robur and Q. petraea

Background and Aims

Sexual reproduction is one of the most important moments in a life cycle, determining the genetic composition of individual offspring. Controlled pollination experiments often show high variation in the mating system at the individual level, suggesting a persistence of individual variation in natural populations. Individual variation in mating patterns may have significant adaptive implications for a population and for the entire species. Nevertheless, field data rarely address individual differences in mating patterns, focusing rather on averages. This study aimed to quantify individual variation in the different components of mating patterns.

Methods

Microsatellite data were used from 421 adult trees and 1911 seeds, structured in 72 half-sib families collected in a single mixed stand of Quercus robur and Q. petraea in northern Poland. Using a Bayesian approach, mating patterns were investigated, taking into account pollen dispersal, male fecundity, possible hybridization and heterogeneity in immigrant pollen pools.

Key Results

Pollen dispersal followed a heavy-tailed distribution (283 m on average). In spite of high pollen mobility, immigrant pollen pools showed strong genetic structuring among mothers. At the individual level, immigrant pollen pools showed highly variable divergence rates, revealing that sources of immigrant pollen can vary greatly among particular trees. Within the stand, the distribution of male fecundity appeared highly skewed, with a small number of dominant males, resulting in a ratio of census to effective density of pollen donors of 5·3. Male fecundity was not correlated with tree diameter but showed strong cline-like spatial variation. This pattern can be attributed to environmental variation. Quercus petraea revealed a greater preference (74 %) towards intraspecific mating than Q. robur (36 %), although mating preferences varied among trees.

Conclusions

Mating patterns can reveal great variation among individuals, even within a single even-age stand. The results show that trees can mate assortatively, with little respect for spatial proximity. Such selective mating may be a result of variable combining compatibility among trees due to genetic and/or environmental factors.     

Mycobacterium tuberculosis AtsG (Rv0296c), GlmU (Rv1018c) and SahH (Rv3248c) Proteins Function as the Human IL-8-Binding Effectors and Contribute to Pathogen Entry into Human Neutrophils

Mycobacterium tuberculosis is an extremely successful intracellular pathogen that has evolved a broad spectrum of pathogenic mechanisms that enable its manipulation of host defense elements and its survival in the hostile environment inside phagocytes. Cellular influx into the site of mycobacterial entry is mediated by a variety of chemokines, including interleukin-8 (IL-8), and the innate cytokine network is critical for the development of an adaptive immune response and infection control. Using affinity chromatography, liquid chromatography electrospray ionization tandem mass spectrometry and surface plasmon resonance techniques, we identified M. tuberculosis AtsG arylsulphatase, bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyl transferase (GlmU) and S-adenosyl-L-homocysteine hydrolase (SahH) as the pathogen proteins that bind to human IL-8. The interactions of all of the identified proteins (AtsG, GlmU and SahH) with IL-8 were characterized by high binding affinity with K_D values of 6.83x10^-6 M, 5.24x10^-6 M and 7.14x10^-10 M, respectively. Furthermore, the construction of Mtb mutant strains overproducing AtsG, GlmU or SahH allowed determination of the contribution of these proteins to mycobacterial entry into human neutrophils. The significantly increased number of intracellularly located bacilli of the overproducing M. tuberculosis mutant strains compared with those of “wild-type” M. tuberculosis and the binding interaction of AtsG, GlmU and SahH proteins with human IL-8 may indicate that these proteins participate in the modulation of the early events of infection with tubercle bacilli and could affect pathogen attachment to target cells.