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.     

T and B cell recovery in arthritis adoptively transferred to SCID mice: antigen-specific activation is required for restoration of autopathogenic CD4+ Th1 cells in a syngeneic system

T cell homeostasis is a physiological function of the immune system that maintains a balance in the numbers and ratios of T cells at the periphery. A self-MHC/self-peptide ligand can induce weak (covert) signals via the TCR, thus providing an extended lifespan for naive T cells. A similar mechanism is responsible for the restoration of immune homeostasis in severe lymphopenic conditions such as those following irradiation or chemotherapy, or upon transfer of lymphocytes to nu/nu or SCID mice. To date, the genetic backgrounds of donor and recipient SCID mice were unmatched in all autoimmune arthritis transfer experiments, and the recovery of lymphoid cells in the host has not been followed. In this study, we present the adoptive transfer of proteoglycan (PG)-induced arthritis using unseparated and T or B cell-depleted lymphocytes from arthritic BALB/c donors to genetically matched syngeneic SCID recipient mice. We demonstrate that selectively recovered lymphoid subsets determine the clinical and immunological status of the recipient. We found that when T cells were depleted (>98% depleted), B cells did not produce PG-specific anti-mouse (auto) Abs unless SCID mice received a second Ag (PG) injection, which promoted the recovery of Ag-specific CD4(+) Th1 cells. Reciprocally, as a result of B cell recovery, high levels of serum anti-PG Abs were found in SCID mice that received B cell-depleted (>99% depleted) T lymphocytes. Our results indicate a selective and highly effective cooperation between CD4(+) T cells and B lymphocytes that is required for the restoration of pathological homeostasis and development of autoimmune arthritis in SCID mice.     

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.     

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.     

Significant differences in capillary electrophoretic patterns of follicular fluids and sera from women pre-treated for in vitro fertilization

Human ovarian follicular fluids and sera obtained from women pre-treated for in vitro fertilization (IVF) were investigated by capillary zone electrophoresis. Comparison of the matching physiological liquids showed substantial differences in the electrophoretic patterns. Significant decrease in the alpha(1)- and gamma-fractions of follicular fluids of every woman were observed, whereas other fractions of the samples did not show such alterations. Since follicular fluid is a product of both, secretion by granulosa cells and diffusion from the theca capillaries, we can assume that the forced production of follicular fluid upon hormone stimulation (with gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH) and corionic gonadotroph hormone (hCG)) may play role in the uneven presence of the proteins.     

In vitro induction of bilirubin conjugation in primary rat hepatocyte culture

UDP-glucuronosyltransferase (UGT1A1) is a critical enzyme in the elimination of bilirubin. The aim of our study was to investigate bilirubin conjugation in primary rat hepatocyte culture and the in vitro inducibility of this isoenzyme by inducing compounds of different classes: dexamethasone, clofibrate, rifampicin, and methylcholanthrene. Hepatocytes exhibited a marked decline in UGT1A1 activity in the first 4 h of culturing (10% of initial activity) and the recovery took 72 h. Immunoblot analysis proved that the loss of enzyme activity was associated with the decrease of protein concentration. Marked induction was detected in the cases of dexamethasone, clofibrate, and rifampicin treatments for 96 h both in enzyme activity (178, 176, and 168%) and in UGT1A1 protein level (362, 328, and 250%). The effects of dexamethasone and clofibrate were additive (210%). Methylcholanthrene had no influence on bilirubin conjugation in our system.