RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.     

Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy (DMD). However, new studies evidence that all three NOS isoforms-NOS1, NOS2, and NOS3-are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.     

Bacterial Contamination of Drinking Water Supplies in a Modern Rural Neighborhood

On six occasions during a 15-month period, the private well and spring water supplies in a modern rural neighborhood of 78 households were examined for total coliforms, fecal coliforms, Staphylococcus aureus, and standard plate count bacteria. More than one-third of the water supplies were unsatisfactory on at least one occasion as judged by standard plate counts over 10³/ml and the presence of coliforms, fecal coliforms, and/or S. aureus. Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli were the most frequently isolated total coliforms. At least 12 other genera of bacteria were identified from standard plate count agar. Coliform contamination was found to be higher after periods of rainfall, and high standard plate counts were more prevalent during warmer weather. These observations probably reflect leakage of surface water into improperly sealed wells or aquifer contamination during winter and the lack of chlorination to control microbial regrowth during the warm season. An inverse correlation was found between the presence of high standard plate counts and incidence of coliforms. Consumer education and at least a twice yearly monitoring of private water supplies (winter and summer) are suggested methods to signal that treatment may be necessary to reduce the risk of waterborne disease.     

Fine-tuning the space, time, and host distribution of mycobacteria in wildlife

Background  

We describe the diversity of two kinds of mycobacteria isolates, environmental mycobacteria and Mycobacterium bovis collected from wild boar, fallow deer, red deer and cattle in Do?ana National Park (DNP, Spain), analyzing their association with temporal, spatial and environmental factors.     

Effect of interleukin-1β on spinal cord nociceptive transmission of normal and monoarthritic rats after disruption of glial function

Introduction  

Cytokines produced by spinal cord glia after peripheral injuries have a relevant role in the maintenance of pain states. Thus, while IL-1β is overexpressed in the spinal cords of animals submitted to experimental arthritis and other chronic pain models, intrathecal administration of IL-1β to healthy animals induces hyperalgesia and allodynia and enhances wind-up activity in dorsal horn neurons.     

Frecuencia de envejecimiento exitoso y fragilidad. Factores de riesgo asociados

Objective

To determine the frequency of successful aging (SA) and its relationship with frailty in an elderly population.

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine intestinal function

The effects of deficiencies in the antioxidant nutrients, vitamin E and selenium, on the host response to gastrointestinal nematode infection are unknown. The aim of the study was to determine the effect of antioxidant deficiencies on nematode-induced alterations in intestinal function in mice. BALB/c mice were fed control diets or diets deficient in selenium or vitamin E and the response to a secondary challenge inoculation with Heligmosomoides polygyrus was determined. Egg and worm counts were assessed to determine host resistance. Sections of jejunum were mounted in Ussing chambers to measure changes in permeability, absorption, and secretion, or suspended in organ baths to determine smooth muscle contraction. Both selenium and vitamin E deficient diets reduced resistance to helminth infection. Vitamin E, but not selenium, deficiency prevented nematode-induced decreases in glucose absorption and hyper-contractility of smooth muscle. Thus, vitamin E status is an important factor in the physiological response to intestinal nematode infection and may contribute to antioxidant-dependent protective mechanisms in the small intestine.     

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/delta + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked dimer, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/delta + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (2 out of 20) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/delta lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-2, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-2-dependent proliferative response of cultured NK and TCR-gamma/delta + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')2 fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-2 to its surface receptors. Altogether the data point out that the Kp43 dimer is involved in the regulation of the IL-2-dependent proliferative response of NK cells and a subset of TCR-gamma/delta + T lymphocytes.