CDK targeting of NBS1 promotes DNA-end resection, replication restart and homologous recombination

The conserved MRE11–RAD50–NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.     

Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.     

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.     

Synthesis and antiviral activity of certain second generation methylenecyclopropane nucleosides

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers.     

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.     

Molecular pathogenesis of sporadic prion diseases in man

The yeast, fungal and mammalian prions determine heritable and infectious traits that are encoded in alternative conformations of proteins. They cause lethal sporadic, familial and infectious neurodegenerative conditions in man, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), kuru, sporadic fatal insomnia (SFI) and likely variable protease-sensitive prionopathy (VPSPr). The most prevalent of human prion diseases is sporadic (s)CJD. Recent advances in amplification and detection of prions led to considerable optimism that early and possibly preclinical diagnosis and therapy might become a reality. Although several drugs have already been tested in small numbers of sCJD patients, there is no clear evidence of any agent’s efficacy. Therefore, it remains crucial to determine the full spectrum of sCJD prion strains and the conformational features in the pathogenic human prion protein governing replication of sCJD prions. Research in this direction is essential for the rational development of diagnostic as well as therapeutic strategies. Moreover, there is growing recognition that fundamental processes involved in human prion propagation – intercellular induction of protein misfolding and seeded aggregation of misfolded host proteins – are of far wider significance. This insight leads to new avenues of research in the ever-widening spectrum of age-related human neurodegenerative diseases that are caused by protein misfolding and that pose a major challenge for healthcare.     

Low beta diversity of ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) in lowland rainforests of Papua New Guinea

We assessed the effect of geographical distance on insect species turnover in a situation where other major environmental factors, including host plant species, altitude, and climate, were constant. We sampled ambrosia beetles (Coleoptera, Curculionidae: Scolytinae and Platypodinae) from four tree species: Artocarpus altilis , Ficus nodosa , Leea indica and Nauclea orientalis , at three sites forming a 1000 km transect in lowland rainforests of northern Papua New Guinea. A standardized volume of wood from trunk, branches and twigs was sampled for ambrosia beetles from three individuals of the four tree species at each site. Each tree was killed standing and left exposed to beetle colonization for 20 days prior to sampling. We obtained 12 751 individuals from 84 morphospecies of ambrosia beetles. We surveyed most of the local species richness at each site, predicted by Chao 2 species richness estimates. The similarity of ambrosia beetle communities, estimated by Chao-Sorensen index, was not correlated with their geographical distance. Likelihood analysis and Q-mode analysis using Monte Carlo-generated null distribution of beetles among sites supported the hypothesis that the assemblages of ambrosia beetles at different sites are drawn from the same species pool, regardless of their geographical distance. Tree part (trunk, branch, or twig) was more important predictor of the composition of ambrosia beetle communities than was the host species or geographical location. All three variables, however, explained only a small portion of variability in ambrosia assemblages. The distribution of ambrosia beetles among tree parts, tree species and study sites was mostly random, suggesting limited importance of host specificity or dispersal limitation.     

Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.