Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.     

Nicotinic Acetylcholine Receptors Containing the α7-Like Subunit Mediate Contractions of Muscles Responsible for Space Positioning of the Snail,Helix pomatia L. Tentacle

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na⁺ and Ca²⁺ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.     

ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.     

Crystal Structure of human pyridoxal kinase: structural basis of M(+) and M(2+) activation

Pyridoxal kinase catalyzes the transfer of a phosphate group from ATP to the 5' alcohol of pyridoxine, pyridoxamine, and pyridoxal. In this work, kinetic studies were conducted to examine monovalent cation dependence of human pyridoxal kinase kinetic parameters. The results show that hPLK affinity for ATP and PL is increased manyfold in the presence of K(+) when compared to Na(+); however, the maximal activity of the Na(+) form of the enzyme is more than double the activity in the presence of K(+). Other monovalent cations, Li(+), Cs(+), and Rb(+) do not show significant activity. We have determined the crystal structure of hPLK in the unliganded form, and in complex with MgATP to 2.0 and 2.2 A resolution, respectively. Overall, the two structures show similar open conformation, and likely represent the catalytically idle state. The crystal structure of the MgATP complex also reveals Mg(2+) and Na(+) acting in tandem to anchor the ATP at the active site. Interestingly, the active site of hPLK acts as a sink to bind several molecules of MPD. The features of monovalent and divalent metal cation binding, active site structure, and vitamin B6 specificity are discussed in terms of the kinetic and structural studies, and are compared with those of the sheep and Escherichia coli enzymes.     

Arabidopsis PPP family of serine/threonine phosphatases

Serine/threonine-specific phosphoprotein phosphatases (PPPs) are ubiquitous enzymes in all eukaryotes, but their regulatory functions are largely unknown in higher plants. The Arabidopsis genome encodes 26 PPP catalytic subunits related to type 1, type 2A and so-called novel phosphatases, including four plant-specific enzymes carrying large N-terminal kelch-domains, but no apparent homologue of the PP2B family. The catalytic subunits of PPPs associate with regulatory protein partners that target them to well defined cellular locations and modulate their activity. Recent studies of phosphatase partners and their interactions have directed attention again to functional dissection of plant PPP families, and highlight their intriguing roles in the regulation of metabolism, cell cycle and development, as well as their roles in light, stress and hormonal signalling.     

Polymerization of hemoglobins in Arctic fish: Lycodes reticulatus and Gadus morhua

In vitro, and possibly in vivo, hemoglobin polymerization and red blood cell sickling appear to be widespread in codfish. In this article, we show that the hemoglobins of the two Arctic fish Lycodes reticulatus and Gadus morhua also have the tendency to polymerize, as monitored by dynamic light scattering experiments. The elucidation of the primary structure of the single hemoglobin of the zoarcid L. reticulatus shows the presence of a large number of cysteyl residues in α and β chains. Their role in eliciting the ability to produce polymers was also addressed by MALDI-TOF and TOF-TOF mass spectrometry. The G.morhua globins are also rich in Cys, but unlike in L. reticulatus, polymerization does not seem to be disulfide driven. The widespread occurrence of the polymerization phenomenon displayed by hemoglobins of Arctic fish supports the hypothesis that this feature may bea response to stressful environmental conditions.     

The antiviral efficacy of HIV-specific CD8⁺ T-cells to a conserved epitope is heavily dependent on the infecting HIV-1 isolate

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef_90–97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A–H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.     

Roadside Reclamation Outside the Revegetation Season: Management Options under Schedule Pressure

Roadside reclamation involves standard revegetation practices that often fail under the adverse conditions imposed by subordination to the infrastructure construction schedule. We experimentally tested for seed and microsite limitations on roadslopes by assessing the effects of seed addition and habitat suitability upon plant cover and species richness. The relative contributions of topsoil seed bank, seed rain, and hydroseeding with standard or native seed mixtures were analyzed in relation to soil texture, fertility, and stability. In order to increase applicability, this research was fitted into the actual construction design and schedule of a highway in central Spain, which resulted in topsoil of varying quality, steep roadcuts and embankments (34°), and out‐of‐season hydroseedings. During the first 2 years following roadslope construction, there was an uneven but sustained increase in plant cover and species richness. Topsoil spread on embankments led to greater plant cover in a shorter time and to lower sedimentation rates at slope bases. The topsoil seed bank was extremely poor. Hydroseeding invariably failed, regardless of seed mixture and roadslope type. The seed rain provided seven times more seeds than hydroseedings, and was correlated with the distance to vegetation patches. Recruitment, however, was limited by microsite suitability, as the initial soil content in nitrate, total nitrogen, and organic matter explained up to 80% of variation in plant cover. In conclusion, when revegetation was performed outside the optimal season due to schedule constraints, measures aimed at overcoming microsite limitation were more cost‐effective and enhanced roadside carrying capacity for local species.