The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Canine heartworm in the domestic and wild canids of southeastern Nebraska

The prevalence of canine heartworm (Dirofilaria immitis) was examined in the domestic dog, coyote (Canis latrans), and red fox (Vulpes fulva) populations of southeastern Nebraska. Microfilariae were detected in 21.4% (22 of 103) of the domestic dogs. The average age of infection for dogs was 5.8 yr. Nine of the 22 infected dogs also were positive for Dipetalonema reconditum. Thirty-nine of 443 (8.9%) coyotes were found to have adult heartworms. The average number of male and female worms per heart was 3.7 and 3.9, respectively. The mean age of infected coyotes was 3.6 yr. Red foxes had an infection rate of 4.8% (1 of 21). This fox heart had only 1 immature female worm. A mail survey of 6 veterinary clinics was also conducted. Veterinarians reported infection rates of 0% to 18.8% in domestic dogs for their localities.     

Incidence of resistance to neonicotinoid insecticides in greenhouse populations of the whitefly, Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) from Greece

The greenhouse whitefly, Trialeurodes vaporariorum Westwood, is an important pest of field and greenhouse crops of horticultural and ornamental plants. In integrated pest management programs its control is mainly based on the release of biological control agents and application of chemical insecticides. Neonicotinoids are relatively new chemicals currently applied for the chemical control of T. vaporariorum. However, cases of development of insecticide resistance to neonicotinoids have already been reported. The state of resistance to neonicotinoid insecticides for populations of the greenhouse whitefly in Greece is currently unknown. The objective of our study was to screen a number of whitefly populations for resistance to the neonicotinoids imidacloprid and thiacloprid. Seven whitefly populations were collected from tomato greenhouse crops from different areas of central and northern Greece. LC₅₀ values were estimated for all populations following the method proposed by the Insecticide Resistance Action Committee (IRAC). The development of resistance to both neonicotinoids was confirmed for all tested populations with resistance ratios ranging from 1.5 to 4.4-fold and from 1.4 to 12.2-fold for imidacloprid and thiacloprid, respectively. We discuss our results with regard to the development of neonicotinoid resistance in T. vaporariorum populations and its implications for whitefly control.     

QUANTITATIVE METHOD FOR DETERMINING A REPRESENTATIVE ALGAL SAMPLE COUNT1

A method for determining a representative count of a sample dependent on number of species is presented for application to various algal communities. Constant species curves are calculated as efficiency = (number of individuals–number of species)/number of individuals and diagrammed on a plot of efficiency versus number of individuals counted. Efficiency is defined as the probability that a new species encountered is minimal. That is, as the ratio of number of species to number of individuals approaches 1, more individuals will need to be counted in order to achieve a representative count. Data and calculations of efficiency from two algal communities are presented for illustration.     

Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System

Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

AN ELECTRON MICROSCOPIC STUDY OF PINOCYTOSIS IN AMEBA : I. The Surface Attachment Phase

The attachment to the surface of the ameba (Chaos chaos L. (Pelomyxa carolinensis, Wilson)) of two proteins, ribonuclease and ferritin, and two colloidal suspensions, thorium dioxide and gold, was studied in the electron microscope. The initial step in the pinocytosis of ferritin and thorium dioxide particles by amebas is shown to be the attachment of these substances to the "hairlike" extensions of the plasmalemma. Ribonuclease caused alterations in the structure of the plasmalemma, but on account of its relative lack of density, it could not be definitely localized. Colloidal gold did not appear to be active with respect to pinocytosis in amebas. Since molecules in solution and particles in suspension are taken up by the same mechanism, the first step of which is their attachment to the cell surface, it is suggested that a single mechanism underlies phagocytosis, pinocytosis, ropheocytosis, cytopempsis, and potocytosis.     

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.