Metal allergens nickel and cobalt facilitate TLR4 homodimerization independently of MD2

Development of contact allergy requires cooperation of adaptive and innate immunity. Ni²⁺ stimulates innate immunity via TLR4/MD2, the bacterial LPS receptor. This likely involves receptor dimerization, but direct proof is pending and it is unclear if related haptens share this mechanism. We reveal Co²⁺ as second metal stimulating TLR4 and confirm necessity of H456/H458 therein. Experiments with a new TLR4 dimerization mutant established dimerization as a mechanism of metal‐ and LPS‐induced TLR4 activation. Yet, in interaction studies only LPS‐ but not metal‐induced dimerization required MD2. Consistently, soluble TLR4 expressed without MD2 inhibited metal‐ but not LPS‐induced responses, opening new therapeutic perspectives.     

Resveratrol improves cardiac contractility following trauma-hemorrhage by modulating Sirt1

Mitochondria play a critical role in metabolic homeostasis of a cell. Our recent studies, based on the reported interrelationship between c-Myc and Sirt1 (mammalian orthologue of yeast sir2 [silent information regulator 2]) expression and their role in mitochondrial biogenesis and function, demonstrated a significant downregulation of Sirt1 protein expression and an upregulation of c-Myc following trauma-hemorrhage (T-H). Activators of Sirt1 are known to improve mitochondrial function and the naturally occurring polyphenol resveratrol (RSV) has been shown to significantly increase Sirt1 activity by increasing its affinity to both NAD+ and the acetylated substrate. In this study we tested the salutary effect of RSV following T-H and its influence on Sirt1 expression. Rats were subjected to T-H or sham operation. RSV (8 mg/kg body weight, intravenously) or vehicle was administered 10 min after the onset of resuscitation, and the rats were killed 2 h following resuscitation. Sirtinol, a Sirt1 inhibitor, was administered 5 min prior to RSV administration. Cardiac contractility (±dP/dt) was measured and heart tissue was tested for Sirt1, Pgc-1α, c-Myc, cytosolic cytochrome C expression and ATP level. Left ventricular function, after T-H, was improved (P < 0.05) following RSV treatment, with significantly elevated expression of Sirt1 (P < 0.05) and Pgc-1α (P < 0.05), and decreased c-Myc (P < 0.05). We also observed significantly higher cardiac ATP content, declined cytosolic cytochrome C and decreased plasma tumor necrosis factor-α in the T-H-RSV group. The salutary effect due to RSV was abolished by sirtinol, indicating a Sirt1-mediated effect. We conclude that RSV may be a useful adjunct to resuscitation fluid following T-H.     

Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R(2) = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.     

Biological monitoring among benzene-exposed workers in Bangalore city, India.

Environmental and biological monitoring was carried out in the winter season of 2004 for 30 gasoline station workers (study subjects) and 30 office workers (controls) of Bangalore city, India. Personal air sampling was carried out in the breathing zone of workers using an Anasorb CSC sorbent tube (SKC 226-01) fitted to the low-flow personal samplers (PCXR4 and pocket pump Model No. 210-1002) at a flow rate of 200 ml min(-1) during the shift work of 8 h. The benzene content adsorbed in the sorbent tube (SKC 226-01) was desorbed with 1 ml of benzene-free carbon disulfide on a developing vibrator and later analysed by Trace GC fitted with MXT-624 column and flame ionization detector. The mean time weighted average benzene concentration found among study and controls was 1.10+/-1.08 and 0.070+/-0.035 mg m(-3), respectively. Biological monitoring for benzene exposure was performed by measuring trans,trans muconic acid (t,t-MA) in the end shift urine samples using HPLC-UV technique. End-shift urine samples (1 ml) were adjusted to pH 7-9 with phosphate buffer pH 7.4 passed through the preconditioned Q-SAX anion-exchange cartridge and the (t,t-MA) is extracted with 10% acetic acid and later analysed by HPLC-UV detection The mean t,t-MA found among study and controls were 563.16+/-281.81 and 266.88+/-110.65 microg g(-1) creatinine. About 50% of the study subjects (15) have higher t,t-MA values than the biological exposure index of the American Conference of Government Industrial Hygienist (ACGIH). Correlation is significant at 5% level (p<0.05) between personal air benzene concentration and urinary t,t-MA in the study group. Based on these findings, the t,t-MA can be used as a biomarker for benzene exposure.     

Transmembrane mechanisms in the assembly of the postsynaptic apparatus at the neuromuscular junction

The vertebrate neuromuscular junction (NMJ) is marked by molecular specializations that include postsynaptic clusters of acetylcholine receptor (AChR) and acetylcholinesterase (AChE). Whereas AChRs are aggregated in the postsynaptic muscle membrane to a density of 10,000/mum(2), AChE is concentrated, also to a high density, in the synaptic basement membrane (BM). In recent years considerable progress has been made in understanding the cellular and molecular mechanisms of AChR clustering. It is known that during the early stages of motoneuron-muscle interaction, the nerve-secreted proteoglycan agrin activates the muscle-specific kinase MuSK, which leads to the formation of a postsynaptic cytoskeletal scaffold that immobilizes and concentrates AChRs through a process generally accepted to involve diffusion-mediated trapping of the receptors. We have recently tested this diffusion-trap model at the single molecule level for the first time by using quantum-dot labeling to track individual AChRs during NMJ development. Our results showed that single AChRs exhibit Brownian-type movement, with diffusion coefficients of 10(-11) to 10(-9)cm(2)/s, until they become immobilized at "traps" assembled in response to synaptogenic stimuli. Thus, free diffusion of AChRs is an integral part of their clustering mechanism. What is the mechanism for AChE clustering? We previously showed that the A(12) asymmetric form of AChE binds to perlecan, a heparan-sulfate proteoglycan which in turn interacts with the transmembrane dystroglycan complex. Through this linkage AChE becomes bound to the muscle membrane and, like AChRs, may exhibit lateral mobility along the membrane. Consistent with this idea, pre-existent AChE at the cell surface becomes clustered together with AChRs following synaptogenic stimulation. Future studies testing diffusion-mediated trapping of AChE should provide insights into the synaptic localization of BM-bound molecules at the NMJ.     

Human cytomegalovirus regulates bioactive sphingolipids

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.     

Involvement of p120 catenin in myopodial assembly and nerve-muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve-secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin-association of p120ctn in cell extracts. Whereas ectopic expression of wild-type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant-negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve- and agrin-induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin-induced myopodial assembly and suggest that myopodia increase muscle-nerve contacts and muscle's access to neural agrin to promote NMJ formation.     

HGF induction of postsynaptic specializations at the neuromuscular junction

A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development.     

Ecological, practical, and political inputs into selection of weed targets: What makes a good biological control target?

The topic of ecological, practical, and political considerations in the selection of weed targets for biological control has been widely discussed during the past two decades, mostly from the perspective of insect herbivores. For conceptual and practical purposes, plant pathogens have been treated in these discussions as if they are a subset of inoculative biocontrol agents, with little said about the inherent differences between pathogens and insects as biocontrol agents or the selection of weed targets for control by the inundative, bioherbicide strategy. Herein, I attempt to address the question of what makes a good biological control target for plant pathogens used as inoculative as well as inundative agents, basing my analysis on examples from the past three decades. Despite the small number of examples available for this analysis, the following generalizations can be made: (1) Weeds with robust capacity for vegetative regeneration are more difficult to control with pathogens than those that lack this trait. (2) A plant’s growth habit is not a reliable guide for target selection; weeds that have been successfully controlled include annual and biennial herbs, perennial shrubs, perennial vines, and trees, while numerous failures have been reported irrespective of the target’s growth habit or reproductive mode. (3) It is more challenging to control species with genetic heterogeneity and capacity for introgression than genetically homogeneous and reproductively conserved species. (4) Matching the target host’s susceptibility with the candidate pathogen’s virulence is of utmost importance for biocontrol success since host–pathogen interactions at the species and subspecies levels are often governed by single-gene differences (e.g., varietal specificity). (5) Practical and political considerations are central to the selection of targets for control with pathogens. (6) Demand from influential stakeholders for control and/or for a nonchemical or economically sustainable control typically drives the initiative as well as the continuance of biocontrol projects to their completion. (7) In the case of inundative, bioherbicide agents, the continuity and ultimate implementation of a project will be dictated by the prospects of economic returns from developing and using a pathogen. (8) The stakeholders’ perceptions of the effectiveness of a biocontrol program can be unpredictable, leading to conflicting views of “success.” In the final analysis, a good weed target for control by a pathogen is one that has strong stakeholder backing and the list of available pathogens for the target suggests a possibility of acceptable control at a cost that is competitive with those of other control options. While this conclusion is also applicable to target selection for insect biocontrol agents, it is more relevant for pathogens because of limited funding and personnel available for development of pathogens and the added cost and technological complexity of implementing bioherbicides compared to classical biocontrols.