Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.     

Major histocompatibility complex controls susceptibility and dominant inheritance,but not the severity of the disease in mouse models of rheumatoid arthritis

Collagen-induced arthritis (CIA) in DBA/1 and proteoglycan-induced arthritis (PGIA) in BALB/c mice are the most frequently used mouse models for studying clinical, immunological and genetic factors contributing to rheumatoid arthritis. DBA/1 ( H2(q)) mice are susceptible to CIA but resistant to PGIA, whereas BALB/c mice ( H2 (d)) are susceptible to PGIA and resistant to CIA. To gain insight into the mechanisms of how the major clinical (disease susceptibility, severity and onset of arthritis) and immunological traits (antigen-specific T- and B-cell responses) are influenced by the major histocompatibility complex (MHC), we have generated a unique intercross of BALB/c and DBA/1 parent strains, and the F1 and F2 hybrids were immunized for either CIA or PGIA. The major clinical and immunological traits were identified as either binary (qualitative) or quantitative traits on Chromosome 17 with a peak at MHC when the entire population was analyzed. In contrast, when only arthritic (susceptible) mice were selected and analyzed, the major clinical traits (severity and onset) 'lost' the linkage to MHC. Thus, MHC dictates disease susceptibility, but not the severity of arthritis. This was even more evident in the case of the H2(q) allele, which was clearly responsible for the dominant inheritance of arthritis in F2 hybrids (either CIA or PGIA). In conclusion, while certain MHC alleles strongly affect disease susceptibility and determine the mode of inheritance of a polygenic autoimmune disease, neither the type of inheritance (dominant vs recessive) nor other MHC components have evident effects upon the clinical symptoms of arthritis.     

Structure of variation in enzyme activity in natural Drosophila melanogaster populations

Enzyme activity variation was assessed in several isofemale lines originating from two Hungarian Drosophila melanogaster populations. Samples from each population were taken from from two villages; 8-9 isofemale lines were established from each village. The activities of ADH, alphaGPDH, IDH and 6PGDH were determined in the adults (in the F1 generation) and in the larvae (in the F3 generation) as well. Enzyme activities were measured on starch gel after electrophoresis. The activity of the enzyme was detected in a single individual and it was also possible to determine its genotype. The results showed that most of the variation occurred within sites for all four enzymes. This within site variation was more or less equally partitioned into within and between isofemale line (family) components. A smaller portion of variation was attributable to the differences between the populations. Nevertheless, adult alphaGPDH, and larval IDH and 6PGDH activities exhibited significant differences between the two populations. Variation in larval activities of all enzymes was higher than that of the adults, but 6PGDH had considerably higher variation in the adults. The greater variation in larval activities probably reflected the greater environmental variation in the microhabitat of the larvae compared to that of the adults. Larval activities of the investigated enzymes showed much stronger correlation than adult activities. The correlation pattern in the adults differed greatly between the two populations.     

Clathrin-mediated endocytosis and recycling of the neuron-specific Na+/H+ exchanger NHE5 isoform. Regulation by phosphatidylinositol 3'-kinase and the actin cytoskeleton

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.     

Central representation of internal and external sensory information in the CNS of Helix pomatia L. and Lymnaea stagnalis L

The central representation of intero- and exteroreceptors located in visceral organs and the osphradium were compared in the CNS of Helix pomatia L. (Gastropoda, Stylommatophora) and Lymnaea stagnalis L. (Gastropoda, Basommatophora), two pulmonate snail species inhabiting a terrestrial and anaquatic environment, respectively. Semi-intact preparations were used comprising the CNS connected by the corresponding nerves either to the cardio-renal, respiratory and genital systems or to the osphradium. Spike discharges of central neurons and the nerves were recorded simultaneously. The central representation of intero- and exteroreceptors was found to be distributed throughout the CNS and involved about 300 neurons. The majority of the neurons received sensory information from all the studied visceral organs and the osphradium. Among the neurons responding to intero- and exteroreceptors a multimodal reaction to tactile, chemical and osmotic stimuli prevailed while in the osphradium specific reactions also were demonstrated. Central neurons receiving sensory information from visceral organs and the osphradium form overlapping and reorganizing neural circuits using the same neurons in the regulation of heart activity, respiration or reproduction producing the appropriate behaviour. In the selection of sensory information the firing pattern appears to be the main determining factor as bursting neurons do not receive sensory information. The central representation of intero- and exteroreceptors and its variability can be a model system for cellular studies of motivational state and self-perception.     

Extracellular mRNA induces dendritic cell activation by stimulating tumor necrosis factor-alpha secretion and signaling through a nucleotide receptor

We previously demonstrated that dendritic cell (DC) pulsing with antigen-encoded mRNA resulted in the loading of both major histocompatibility complex class I and II antigen presentation pathways and the delivery of an activation signal. Coculture of mRNA-pulsed DC with T cells led to the induction of a potent primary immune response. DC, in addition to recognizing foreign antigens through pattern recognition receptors, also must respond to altered self, transformed, or intracellularly infected cells. This occurs through cell surface receptors that recognize products of inflammation and cell death. In this report, we characterize two signaling pathways utilized by extracellular mRNA to activate DC. In addition, a novel ligand, poly(A), is identified that mediates signaling through a receptor that can be inhibited by pertussis toxin and suramin and can be desensitized by ATP and ADP, suggesting a P2Y type nucleotide receptor. The role of this signaling activity in vaccine design and the potential effect of mRNA released by damaged cells in the induction of immune responsiveness is discussed.