Prominent Narp expression in projection pathways and terminal fields

Narp (neuronal activity regulated pentraxin) is a secreted immediate early gene product that is induced by synaptic activity. Recent studies have indicated that Narp may be an extracellular aggregating factor for AMPA receptors. Immunohistochemical studies have revealed prominent expression of Narp in the mossy fiber pathway of the dentate gyrus, suggesting it may be released pre-synaptically. However, in vitro studies using recombinant Narp indicate that Narp may act when expressed by either pre- or post-synaptic elements. To help define Narp's mode of action, we have examined its localization in the habenula-interpeduncular pathway which also displays robust Narp expression. Focusing on this pathway as well as hippocampal and cortical Narp expression, we found prominent Narp staining in projection pathways and terminal fields. In contrast, Narp expression in dendrites was minimal in these neuronal populations. These findings indicate that, under physiological conditions, Narp is targeted to the synapse from pre- rather than post-synaptic elements. Our results also suggest that future studies focusing on these projection pathways that express high levels of Narp, in vivo, may help to understand the regulation and function of endogenous Narp.     

Ionworks HT: a new high-throughput electrophysiology measurement platform

To address the throughput restrictions of classical patch clamp electrophysiology, Essen Instruments has developed a plate-based electrophysiology measurement platform. The instrument is an integrated platform that consists of computer-controlled fluid handling, recording electronics, and processing tools capable of voltage clamp whole-cell recordings from thousands of individual cells per day. To establish a recording, the system uses a planar, multiwell substrate (a PatchPlate). The system effectively positions 1 cell into a hole separating 2 fluid compartments in each well of the substrate. Voltage control and current recordings from the cell membrane are made subsequent to gaining access to the cell interior by applying a permeabilizing agent to the intracellular side. Based on the multiwell design of the PatchPlate, voltage clamp recordings of up to 384 individual cells can be made in minutes and are comparable to measurements made using traditional electrophysiology techniques. An integrated pipetting system allows for up to 2 additions of modulation agents. Typical throughput, measurement fidelity, stability, and comparative pharmacology of a recombinant voltage-dependent sodium channel (hNav1.3) and a voltage-gated potassium channel (hKv1.5) exogenously expressed in CHO cells are presented. The IonWorks HT device can be used in biophysical and pharmacological profiling of ion channels in an environment compatible with high-capacity screening.     

Cellular activation of proMMP-13 by MT1-MMP depends on the C-terminal domain of MMP-13

Procollagenase-3 (proMMP-13) can be activated by soluble or cell associated membrane type matrix metalloproteinase 1 (MT1-MMP). In this study we show that the cell based activation of proMMP-13 by MT1-MMP was dependent on the C-terminal domain, as delta(249-451) proMMP-13, which lacks the haemopexin domain, and a chimaera from N-terminal MMP-13 and C-terminal MMP-19 (proMMP-13/19) were not processed by MT1-MMP expressing cells. Only the initial cleavage at Gly(35)-Ile(36) was dependent on MT1-MMP activity, as conversion to the active enzyme (Tyr(85) N-terminus) required a functional MMP-13 active site. Unlike proMMP-2 activation, this process was independent of tissue inhibitor of metalloproteinase-2 (TIMP-2) as MT1-MMP expressing cells from the TIMP-2-/- mouse efficiently activated proMMP-13.     

Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin

To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.     

Floral Display in Narcissus: Variation in Flower Size and Number at the Species, Population, and Individual Levels

Floral display (the size, number, and arrangement of open flowers) influences pollinator visitation to animal-pollinated plants and should be an important determinant of reproductive success. We examined variation in the size and number of open flowers in wild daffodils (Narcissus). Our analysis of published data on 45 taxa showed that flower number varied negatively with flower diameter among Narcissus species, which supports the widespread assumption that there is a trade-off between these traits. In contrast, field measurements indicated a positive relation between flower number and diameter within two populations of Narcissus dubius, and no relation was evident after we controlled for variation in bulb size. The discrepancy between inter- and intraspecific patterns may have occurred because variable resource levels obscure trade-offs when variation in flower size is low (e.g., within species). Size-related increases in floral tube length were half as great as corresponding increases in flower diameter, a result that is consistent with stronger stabilizing selection on tube length. Staggered flowering within N. dubius inflorescences limited the mean number of open flowers to <66% of total flower number, and slow expansion by later opening flowers resulted in significant differences in flower size throughout flowering. Although pollinators preferred large flowers, experimental reductions in flower diameter did not affect seed production. Our results illustrate how the relative importance of the factors influencing floral display can vary among levels of biological organization. Interspecific variation in flower size and number appeared to be constrained by allocation trade-offs, but intraspecific variation in both traits was more greatly influenced by plant resource status. Within plants, the size and number of open flowers reflected the relative age of individual flowers and floral longevity.     

Developmental regulation of GDNF response and receptor expression in the enteric nervous system

The development of the enteric nervous system is dependent upon the actions of glial cell line-derived neurotrophic factor (GDNF) on neural crest-derived precursor cells in the embryonic gut. GDNF treatment of cultured enteric precursor cells leads to an increase in the number of neurons that develop and/or survive. Here we demonstrate that, although GDNF promoted an increase in neuron number at all embryonic ages examined, there was a developmental shift from a mitogenic to a trophic response by the developing enteric neurons. The timing of this shift corresponded to developmental changes in gut expression of GFR alpha-1, a co-receptor in the GDNF-Ret signaling complex. GFR alpha-1 was broadly expressed in the gut at early developmental stages, at which times soluble GFR alpha-1 was released into the medium by cultured gut cells. At later times, GFR alpha-1 became restricted to neural crest-derived cells. GFR alpha-1 could participate in GDNF signaling when expressed in cis on the surface of enteric precursor cells, or as a soluble protein. The GDNF-mediated response was greater when cell surface, compared with soluble, GFR alpha-1 was present, with the maximal response seen the presence of both cis and trans forms of GFR alpha-1. In addition to contributing to GDNF signaling, cell-surface GFR alpha-1 modulated the specificity of interactions between GDNF and soluble GFR alphas. These experiments demonstrate that complex, developmentally regulated, signaling interactions contribute to the GDNF-dependent development of enteric neurons.     

Salmonella SsrB activates a global regulon of horizontally acquired genes

Salmonella enterica is a bacterial pathogen of humans that can proliferate within epithelial cells as well as professional phagocytes of the immune system. This ability requires an S. enterica specific locus termed Salmonella pathogenicity island 2 (SPI-2). SPI-2 encodes a type III secretion system that injects effectors encoded within the island into host cell cytosol to promote virulence. SsrAB is a two-component regulator encoded within SPI-2 that was assumed to activate SPI-2 genes exclusively. Here, it is shown that SsrB in fact activates a global regulon. At least 10 genes outside SPI-2 are SsrB regulated within epithelial and macrophage cells. Nine of these 10 SsrB-regulated genes outside SPI-2 reside within previously undescribed regions of the Salmonella genome. Most share no sequence homology with current database entries. However, one is remarkably homologous to human glucosyl ceramidase, an enzyme involved in the ceramide signalling pathway. The SsrB regulon is modulated by the two-component regulatory systems PhoP/PhoQ and OmpR/EnvZ, and is upregulated in the intracellular microenvironment.