Epigenetic asymmetry of imprinted genes in plant gametes

Plant imprinted genes show parent-of-origin expression in seed endosperm, but little is known about the nature of parental imprints in gametes before fertilization. We show here that single differentially methylated regions (DMRs) correlate with allele-specific expression of two maternally expressed genes in the seed and that one DMR is differentially methylated between gametes. Thus, plants seem to have developed similar strategies as mammals to epigenetically mark imprinted genes.     

Radioprotectors from pyrodextrins

Cornstarch heated in the range of 230–280°C depolymerized into pyrodextrins characterized by two-component EPR signals of relatively stable free radicals. These thermally generated radicals could serve as efficient scavengers for free radicals generated from pyrodextrins with the 200 Gy dose of γ-radiation. The most efficient traps/scavengers were produced from cornstarch at 250–280°C. IR data indicated incorporation of the OH groups to the pyrodextrins. These groups most probably originated from the OH· radicals formed by the radiolysis of water. EPR spectra provided evidence for trapping free radicals generated by γ-irradiation and for their subsequent annihilation on contact with pyrodextrins. Water affected radical processes occurring in pyrodextrins caused by γ-irradiation.     

Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.     

Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome

To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation.     

Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-alpha receptor (PDGFR-alpha) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-alpha associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-alpha was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-alpha blocking antibody (IMC-3G3) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-alpha was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-alpha, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-alpha phosphorylation. Taken together, these data indicate that PDGFR-alpha is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.     

Role of kinetin in the dormancy ofCercis siliquastrum seeds

Summary Studies conducted onCercis siliquastrum seeds treated with kinetin confirm that this hormone does not interrupt dormancy in either whole seeds or those decoated at the radical pole. Seeds totally decoated or decoated at the cotyledon pole only demonstrated atypical germinations linked to cotyledon growth, permitting the embryo to escape the inhibitory action present in the endosperm; this does not occur when the cotyledon surface is experimentally reduced.This investigation was supported by the Italian National Research Council (C.N.R.) Project Biology of Reproduction.     

Prediction of central nervous system embryonal tumour outcome based on gene expression.

Embryonal tumours of the central nervous system (CNS) represent a heterogeneous group of tumours about which little is known biologically, and whose diagnosis, on the basis of morphologic appearance alone, is controversial. Medulloblastomas, for example, are the most common malignant brain tumour of childhood, but their pathogenesis is unknown, their relationship to other embryonal CNS tumours is debated, and patients' response to therapy is difficult to predict. We approached these problems by developing a classification system based on DNA microarray gene expression data derived from 99 patient samples. Here we demonstrate that medulloblastomas are molecularly distinct from other brain tumours including primitive neuroectodermal tumours (PNETs), atypical teratoid/rhabdoid tumours (AT/RTs) and malignant gliomas. Previously unrecognized evidence supporting the derivation of medulloblastomas from cerebellar granule cells through activation of the Sonic Hedgehog (SHH) pathway was also revealed. We show further that the clinical outcome of children with medulloblastomas is highly predictable on the basis of the gene expression profiles of their tumours at diagnosis.     

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).     

Low density lipoprotein receptor-related protein 1 couples β1 integrin activation to degradation

Low density lipoprotein receptor-related protein (LRP) 1 modulates cell adhesion and motility under normal and pathological conditions. Previous studies documented that LRP1 binds several integrin receptors and mediates their trafficking to the cell surface and endocytosis. However, the mechanism by which LRP1 may regulate integrin activation remains unknown. Here we report that LRP1 promotes the activation and subsequent degradation of β1 integrin and thus supports cell adhesion, spreading, migration and integrin signaling on fibronectin. LRP1 interacts with surface β1 integrin, binds the integrin activator kindlin2 and stimulates β1 integrin–kindlin2 complex formation. Specifically, serine 76 in the LRP1 cytoplasmic tail is crucial for the interaction with kindlin2, β1 integrin activation and cell adhesion. Interestingly, a loss of LRP1 induces the accumulation of several integrin receptors on the cell surface. Following internalization, intracellular trafficking of integrins is driven by LRP1 in a protein kinase C- and class II myosin-dependent manner. Ultimately, LRP1 dictates the fate of endocytosed β1 integrin by directing it down the pathway of lysosomal and proteasomal degradation. We propose that LRP1 mediates cell adhesion by orchestrating a multi-protein pathway to activate, traffic and degrade integrins. Thus, LRP1 may serve as a focal point in the integrin quality control system to ensure a firm connection to the extracellular matrix.