Dissecting the complete lipoprotein biogenesis pathway in Streptomyces scabies

Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.     

Requirements for mammalian carboxylesterase inhibition by substituted ethane-1,2-diones

Carboxylesterases (CE) are ubiquitous enzymes found in both human and animal tissues and are responsible for the metabolism of xenobiotics. This includes numerous natural products, as well as a many clinically used drugs. Hence, the activity of these agents is likely dependent upon the levels and location of CE expression. We have recently identified benzil is a potent inhibitor of mammalian CEs, and in this study, we have assessed the ability of analogues of this compound to inhibit these enzymes. Three different classes of molecules were assayed: one containing different atoms vicinal to the carbonyl carbon atom and the benzene ring [PhXC(O)C(O)XPh, where X=CH?, CHBr, N, S, or O]; a second containing a panel of alkyl 1,2-diones demonstrating increasing alkyl chain length; and a third consisting of a series of 1-phenyl-2-alkyl-1,2-diones. In general, with the former series of molecules, heteroatoms resulted in either loss of inhibitory potency (when X=N), or conversion of the compounds into substrates for the enzymes (when X=S or O). However, the inclusion of a brominated methylene atom resulted in potent CE inhibition. Subsequent analysis with the alkyl diones [RC(O)C(O)R, where R ranged from CH? to C?H??] and 1-phenyl-2-alkyl-1,2-diones [PhC(O)C(O)R where R ranged from CH? to C?H??], demonstrated that the potency of enzyme inhibition directly correlated with the hydrophobicity (clogP) of the molecules. We conclude from these studies that that the inhibitory power of these 1,2-dione derivatives depends primarily upon the hydrophobicity of the R group, but also on the electrophilicity of the carbonyl group.     

Advancing cervical cancer prevention initiatives in resource-constrained settings: insights from the Cervical Cancer Prevention Program in Zambia

Groesbeck Parham and colleagues describe their Cervical Cancer Prevention Program in Zambia, which has provided services to over 58,000 women over the past five years, and share lessons learned from the program's implementation and integration with existing HIV/AIDS programs.     

Hypoxic alligator embryos: chronic hypoxia, catecholamine levels and autonomic responses of in ovo alligators

Hypoxia is a naturally occurring environmental challenge for embryonic reptiles, and this is the first study to investigate the impact of chronic hypoxia on the in ovo development of autonomic cardiovascular regulation and circulating catecholamine levels in a reptile. We measured heart rate (f(H)) and chorioallantoic arterial blood pressure (MAP) in normoxic ('N21') and hypoxic-incubated ('H10'; 10% O(2)) American alligator embryos (Alligator mississippiensis) at 70, 80 and 90% of development. Embryonic alligator responses to adrenergic blockade with propranolol and phentolamine were very similar to previously reported responses of embryonic chicken, and demonstrated that embryonic alligator has α and β-adrenergic tone over the final third of development. However, adrenergic tone originates entirely from circulating catecholamines and is not altered by chronic hypoxic incubation, as neither cholinergic blockade with atropine nor ganglionic blockade with hexamethonium altered baseline cardiovascular variables in N21 or H10 embryos. In addition, both atropine and hexamethonium injection did not alter the generally depressive effects of acute hypoxia - bradycardia and hypotension. However, H10 embryos showed significantly higher levels of noradrenaline and adrenaline at 70% of development, as well as higher noradrenaline at 80% of development, suggesting that circulating catecholamines reach maximal levels earlier in incubation for H10 embryos, compared to N21 embryos. Chronically elevated levels of catecholamines may alter the normal balance between α and β-adrenoreceptors in H10 alligator embryos, causing chronic bradycardia and hypotension of H10 embryos measured in normoxia.     

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

Background  

Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.     

Copy number variation across European populations

Genome analysis provides a powerful approach to test for evidence of genetic variation within and between geographical regions and local populations. Copy number variants which comprise insertions, deletions and duplications of genomic sequence provide one such convenient and informative source. Here, we investigate copy number variants from genome wide scans of single nucleotide polymorphisms in three European population isolates, the island of Vis in Croatia, the islands of Orkney in Scotland and the South Tyrol in Italy. We show that whereas the overall copy number variant frequencies are similar between populations, their distribution is highly specific to the population of origin, a finding which is supported by evidence for increased kinship correlation for specific copy number variants within populations.     

The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.