Reexamination of the recognition preference of the specificity pocket of the Abl SH3 domain

Src homology-3 (SH3) domains mediate important protein-protein interactions in a variety of normal and pathological cellular processes, thus providing an attractive target for the selective interference of SH3-dependent signaling events that govern these processes. Most SH3 domains recognize proline-rich peptides with low affinity and poor selectivity, and the goal to design potent and specific ligands for various SH3 domains remains elusive. Better understanding of the molecular basis for SH3 domain recognition is needed in order to design such ligands with potency and specificity. In this report, we seek to define a clear recognition preference of the specificity pocket of the Abl SH3 domain using targeted synthetic peptide libraries. High-resolution affinity panning coupled with mass spectrometric readout allows for quick identification of Trp as the preferred fourth residue in the decapeptide ligand APTWSPPPPP, which binds to Abl SH3 four times stronger than does the decapeptide containing Tyr or Phe in the fourth position. This finding is in contrast to several reports that Tyr is the only residue selected from phage displayed peptide libraries that interacts with the specificity pocket of Abl SH3. This simple, unbiased approach can fine-tune the affinity and selectivity of both natural and unnatural SH3 ligands whose consensus binding sequence has been pre-defined by combinatorial library methods.     

In vivo studies on the nuclear behavior of the arbuscular mycorrhizal fungusGigaspora rosea grown under axenic conditions

Summary The distribution and fate of nuclei of the arbuscular-my-corrhizal fungusGigaspora rosea during late stages of axenic cultures were studied in fixed cultures by transmitted light, conventional and confocal laser scanning microscopy, and in live cultures with two-photon fluorescence microscopy. Mature specimens not yet showing apical septation displayed oval-shaped nuclei localized in lateral positions of the hypha all along the germ-tube length. Beside these, round-shaped nuclei were found to migrate along the central germ-tube core. Some (rare) germ-tube areas, delimited by septa and containing irregularly shaped, much brighter fluorescent nuclei were also found. Specimens that had just initiated the septation process after germ-tube growth arrest displayed round or oval-shaped nuclei in several portions of the germ tubes. These hyphal areas often alternated with other septa-delimited cytoplasmic clusters which contained distorted, brightly fluorescent nuclei. Completely septated specimens mostly lacked nuclei along their germ tubes. However, highly fluorescent chromatin masses appeared within remnants of cytoplasmic material, often compressed between close septa. Our results provide a first clear picture of the in vivo distribution of nuclei along arbuscular mycorrhizal fungal germ tubes issued from resting spores, and suggest that selective areas of their coenocytic hyphae are under specific, single nuclear control. They indicate as well that random autolytic processes occur along senescingG. rosea germ tubes, probably as a consequence of the absence of a host root signal for mycorrhizal formation. Finally, the data presented here allow us to envisage the fate of nuclei released by the germinating spore after nonsymbiotic fungal growth arrest.Abbreviations AM fungi arbuscular-mycorrhizal fungi - DAPI 4, 6-diamidino-2-phenylindole - FM fluorescence microscopy - CLSM confocal laser scanning microscopy - 2PM two-photon microscopy - PI propidium iodide - PMT photomultiplier tube     

Oligomeric state study of prokaryotic rhomboid proteases

Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases.     

Niche partitioning of the genetic lineages of the oak powdery mildew complex

Recent molecular studies have shown that four genetic lineages, probably cryptic species, cause oak powdery mildew in Europe. We tested the hypothesis of niche differentiation between the two most frequent species (Erysiphe alphitoides and Erysiphe quercicola), by determining their relative prevalences at various times during the growing season. E. quercicola was strictly associated with the flag-shoot symptom in a 2-yr sampling campaign of 35 natural oak seedling populations. Additional sampling during Aug.–Sep. in the same stands showed that E. alphitoides predominated in most (70 %) cases. Time-course monitoring of powdery mildew in two natural regenerating oak populations confirmed the inverse patterns of relative abundance for these two species in early and late season. The coexistence of these two closely related species may be due to the use of different strategies, resulting from a trade-off between overwintering (flag-shoots vs. chasmothecia) and late-season performance.     

Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study

Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45dimCD34+VEGFR-2+ and CD45dimCD133−CD34+CD144+ respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill’s colonies). At baseline TG had lower cEPCs, PACs, CFU-Hills’ colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill’s colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill’s colonies with cECs. Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill’s colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132     

Arabidopsis poly(A) polymerase PAPS1 limits founder‐cell recruitment to organ primordia and suppresses the salicylic acid‐independent immune response downstream of EDS1/PAD4

Polyadenylation of pre‐mRNAs by poly(A) polymerase (PAPS) is a critical process in eukaryotic gene expression. As found in vertebrates, plant genomes encode several isoforms of canonical nuclear PAPS enzymes. In Arabidopsis thaliana these isoforms are functionally specialized, with PAPS1 affecting both organ growth and immune response, at least in part by the preferential polyadenylation of subsets of pre‐mRNAs. Here, we demonstrate that the opposite effects of PAPS1 on leaf and flower growth reflect the different identities of these organs, and identify a role for PAPS1 in the elusive connection between organ identity and growth patterns. The overgrowth of paps1 mutant petals is due to increased recruitment of founder cells into early organ primordia, and suggests that PAPS1 activity plays unique roles in influencing organ growth. By contrast, the leaf phenotype of paps1 mutants is dominated by a constitutive immune response that leads to increased resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis and reflects activation of the salicylic acid‐independent signalling pathway downstream of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)/PHYTOALEXIN DEFICIENT4 (PAD4). These findings provide an insight into the developmental and physiological basis of the functional specialization amongst plant PAPS isoforms.     

Skeletal Muscle Insulin Resistance and Absence of Inflammation Characterize Insulin-Resistant Grade I Obese Women

ContextObesity is associated with insulin-resistance (IR), the key feature of type 2 diabetes. Although chronic low-grade inflammation has been identified as a central effector of IR development, it has never been investigated simultaneously at systemic level and locally in skeletal muscle and adipose tissue in obese humans characterized for their insulin sensitivity.ObjectivesWe compared metabolic parameters and inflammation at systemic and tissue levels in normal-weight and obese subjects with different insulin sensitivity to better understand the mechanisms involved in IR development.Methods30 post-menopausal women were classified as normal-weight insulin-sensitive (controls, CT) and obese (grade I) insulin-sensitive (OIS) or insulin-resistant (OIR) according to their body mass index and homeostasis model assessment of IR index. They underwent a hyperinsulinemic-euglycemic clamp, blood sampling, skeletal muscle and subcutaneous adipose tissue biopsies, an activity questionnaire and a self-administrated dietary recall. We analyzed insulin sensitivity, inflammation and IR-related parameters at the systemic level. In tissues, insulin response was assessed by P-Akt/Akt expression and inflammation by macrophage infiltration as well as cytokines and IκBα expression.ResultsSystemic levels of lipids, adipokines, inflammatory cytokines, and lipopolysaccharides were equivalent between OIS and OIR subjects. In subcutaneous adipose tissue, the number of anti-inflammatory macrophages was higher in OIR than in CT and OIS and was associated with higher IL-6 level. Insulin induced Akt phosphorylation to the same extent in CT, OIS and OIR. In skeletal muscle, we could not detect any inflammation even though IκBα expression was lower in OIR compared to CT. However, while P-Akt/Akt level increased following insulin stimulation in CT and OIS, it remained unchanged in OIR.ConclusionOur results show that systemic IR occurs without any change in systemic and tissues inflammation. We identified a muscle defect in insulin response as an early mechanism of IR development in grade I obese post-menopausal women.     

NK cell-mediated targeting of human cancer and possibilities for new means of immunotherapy

Insights into the molecular basis for natural killer (NK) cell recognition of human cancer have been obtained in recent years. Here, we review current knowledge on the molecular specificity and function of human NK cells. Evidence for NK cell targeting of human tumors is provided and new strategies for NK cell-based immunotherapy against human cancer are discussed. Based on current knowledge, we foresee a development where more cancers may be subject to treatment with drugs or other immunomodulatory agents affecting NK cells, either directly or indirectly. We also envisage a possibility that certain forms of cancers may be subject to treatment with adoptively transferred NK cells, either alone or in combination with other therapeutic interventions.     

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.