LTD₄ induces HB-EGF-dependent CXCL8 release through EGFR activation in human bronchial epithelial cells

Airway epithelial cells release proinflammatory mediators that may contribute to airway remodeling and leukocyte recruitment. We explored the hypothesis that leukotriene D? (LTD?) may trigger the release of proremodeling factors through activation of the EGF receptor (EGFR). We particularly focused on the effects of LTD? on release of heparin-binding EGF-like factor (HB-EGF) and IL-8 (CXCL8), a potent neutrophil chemoattractant that may be released downstream of EGFR activation. To address this hypothesis, both primary (NHBE) and transformed bronchial human epithelial cells (BEAS-2B) were grown on an air-liquid interface and stimulated with LTD?. HB-EGF and CXCL8 were evaluated by ELISA in cell culture supernatants. To explore the EGFR signaling pathway, we used a broad-spectrum matrix metalloproteinase (MMP) inhibitor, GM-6001, two selective EGFR tyrosine kinase inhibitors, AG-1478 and PD-153035, an HB-EGF neutralizing antibody, and a specific small interfering RNA (siRNA) against the EGFR. Expression of the CysLT? cysteinyl leukotriene receptor was demonstrated by RT-PCR and immunocytochemistry in both BEAS-2B and NHBE cells. Four hours after stimulation with LTD?, HB-EGF and CXCL8 were significantly increased in cell culture supernatant. GM-6001 and montelukast, a specific CysLT? receptor antagonist, blocked the LTD?-induced increase in HB-EGF. All inhibitors/antagonists decreased LTD?-induced CXCL8 release. siRNA against EGFR abrogated CXCL8 release following stimulation with LTD? and exogenous HB-EGF. These findings suggest LTD? induced EGFR transactivation through the release of HB-EGF in human bronchial epithelial cells with downstream release of CXCL8. These effects may contribute to epithelial-mediated airway remodeling in asthma and other conditions associated with cysteinyl leukotriene release.     

A fluorescence-based,high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.     

Intersection of endocytic and exocytic systems in Toxoplasma gondii

Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome‐like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant‐like features suggest T. gondii endocytic trafficking involves transit through early and late endosome‐like compartments (ELCs) and potentially the trans‐Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin‐related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post‐invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host‐derived proteins, suggesting that DrpB is not required for parasite endocytosis.      

Impact on disease development, genomic location and biological function of copy number alterations in non-small cell lung cancer

Lung cancer, of which more than 80% is non-small cell, is the leading cause of cancer-related death in the United States. Copy number alterations (CNAs) in lung cancer have been shown to be positionally clustered in certain genomic regions. However, it remains unclear whether genes with copy number changes are functionally clustered. Using a dense single nucleotide polymorphism array, we performed genome-wide copy number analyses of a large collection of non-small cell lung tumors (n = 301). We proposed a formal statistical test for CNAs between different groups (e.g., non-involved lung vs. tumors, early vs. late stage tumors). We also customized the gene set enrichment analysis (GSEA) algorithm to investigate the overrepresentation of genes with CNAs in predefined biological pathways and gene sets (i.e., functional clustering). We found that CNAs events increase substantially from germline, early stage to late stage tumor. In addition to genomic position, CNAs tend to occur away from the gene locations, especially in germline, non-involved tissue and early stage tumors. Such tendency decreases from germline to early stage and then to late stage tumors, suggesting a relaxation of selection during tumor progression. Furthermore, genes with CNAs in non-small cell lung tumors were enriched in certain gene sets and biological pathways that play crucial roles in oncogenesis and cancer progression, demonstrating the functional aspect of CNAs in the context of biological pathways that were overlooked previously. We conclude that CNAs increase with disease progression and CNAs are both positionally and functionally clustered. The potential functional capabilities acquired via CNAs may be sufficient for normal cells to transform into malignant cells.     

Cardiovascular responses of eggs, embryos and alevins of Atlantic salmon and rainbow trout to nitric oxide donors, sodium nitroprusside and isosorbide dinitrate, and inhibitors of nitric oxide synthase, N-nitro-l-arginine methyl ester and aminoguanidine, following short- and long-term exposure

Atlantic salmon embryos and alevins Salmo salar that had been exposed to isosorbide dinitrate (ISDN) for 4 weeks, on transfer to fresh water, showed an increase in heart rate. Unexposed embryos and alevins showed a decrease in heart rate following transfer to 100 μmol l⁻¹ ISDN for 4 h. This is in contrast to adult rainbow trout and higher vertebrates where tachycardia occurred in response to nitric oxide (NO) donors. The decreased heart rate in response to ISDN was inhibited by 2 mg 1⁻¹ methylene blue, indicating that NO activates cardiovascular events via guanylyl cyclase and cyclic guanidine monophosphate. Heart rate of rainbow trout alevins Oncorhynchus mykiss exposed to 100 μmol l⁻¹ aminoguanidine responded with a slowly developed but significant bradycardia over 10 min as did those reared in aminoguanidine for 4 weeks then transferred to fresh water. A potentiated increase in heart rate on exposure to the NO donor sodium nitroprusside (SNP), occurred within 1 min in salmon alevins reared in l -nitro-arginine methyl ester ( l -NAME) for 4 weeks, indicating up-regulation of NO receptors. The evidence for down-regulation of SNP-reared alevins exposed to l -NAME was less well defined. The results suggest that both salmonid embryos and alevins have a functional l -arginine-NO pathway and that NO has a physiological role in control of cardiovascular events.     

Early steps of Bacillus subtilis primosome assembly

Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.