SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale

Cytoskeletal dynamics during cell behaviours ranging from endocytosis and exocytosis to cell division and movement is controlled by a complex network of signalling pathways, the full details of which are as yet unresolved. Here we show that SILAC-based proteomic methods can be used to characterize the rapid chemoattractant-induced dynamic changes in the actin–myosin cytoskeleton and regulatory elements on a proteome-wide scale with a second to minute timescale resolution. This approach provides novel insights in the ensemble kinetics of key cytoskeletal constituents and association of known and novel identified binding proteins. We validate the proteomic data by detailed microscopy-based analysis of in vivo translocation dynamics for key signalling factors. This rapid large-scale proteomic approach may be applied to other situations where highly dynamic changes in complex cellular compartments are expected to play a key role

Environmental enrichment has no effect on the development of dopaminergic and GABAergic fibers during methylphenidate treatment of early traumatized gerbils

It is widely believed, that environmental factors play a crucial role in the etiology and outcome of psychiatric diseases such as Attention-Deficit/Hyperactivity Disorder (ADHD). A former study from our laboratory has shown that both methylphenidate (MP) and handling have a positive effect on the dopaminergic fiber density in the prefrontal cortex (PFC) of early traumatized gerbils (Meriones unguiculatus). The current study was performed to investigate if enriched environment during MP application has an additional influence on the dopaminergic and GABAergic fiber densities in the PFC and amygdala in this animal model

Gene Expression Profiles of the NCI-60 Human Tumor Cell Lines Define Molecular Interaction Networks Governing Cell Migration Processes

Although there is extensive information on gene expression and molecular interactions in various cell types, integrating those data in a functionally coherent manner remains challenging. This study explores the premise that genes whose expression at the mRNA level is correlated over diverse cell lines are likely to function together in a network of molecular interactions. We previously derived expression-correlated gene clusters from the database of the NCI-60 human tumor cell lines and associated each cluster with function categories of the Gene Ontology (GO) database. From a cluster rich in genes associated with GO categories related to cell migration, we extracted 15 genes that were highly cross-correlated; prominent among them were RRAS, AXL, ADAM9, FN14, and integrin-beta1. We then used those 15 genes as bait to identify other correlated genes in the NCI-60 database. A survey of current literature disclosed, not only that many of the expression-correlated genes engaged in molecular interactions related to migration, invasion, and metastasis, but that highly cross-correlated subsets of those genes engaged in specific cell migration processes. We assembled this information in molecular interaction maps (MIMs) that depict networks governing 3 cell migration processes: degradation of extracellular matrix, production of transient focal complexes at the leading edge of the cell, and retraction of the rear part of the cell. Also depicted are interactions controlling the release and effects of calcium ions, which may regulate migration in a spaciotemporal manner in the cell. The MIMs and associated text comprise a detailed and integrated summary of what is currently known or surmised about the role of the expression cross-correlated genes in molecular networks governing those processes

<span style="font-size: 21.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"">Electromagnetic exposure effects the hippocampal dentate cell proliferation in gerbils <i>(Meriones unguiculatus)</i> </span>

1220-1226<span style="font-size: 16.0pt;mso-bidi-font-size:9.0pt;font-family:" times="" new="" roman","serif""="">The chronic effect on hippocampal neurogenesis after exposure (30 min/day for 14 days) to a high frequency (35,53 kHz) electromagnetic field, double modulated at extremely low frequencies (ELF; 1, 8, 12, 29 and 50 Hz), was studied in young adult gerbils. Immediately after the last exposure proliferation of dentate granule cells was identified by in vivo labeling with 5-bromo-2-desoxyuridine (BrdU). Exposure to 1, 29 and 50 <span style="font-size:16.0pt; mso-bidi-font-size:9.0pt;font-family:" times="" new="" roman","serif""="">Hz resulted in a statistically significant reduction of cell proliferation rates, but only the 50Hz-group manifested the effect highly significantly (-29,3 %). On the other hand, gerbils exposed to 8 and 12 Hz showed no significant change of postmitotic cell proliferation as compared with the sham treated controls. The results suggest that the effects of ELF on the granule cell proliferation are mediated by neurotmnsmitters and hormones which regulate hippocampal neurogenesis. </span

Contralateral prefrontal projections in gerbils mature abnormally after early methamphetamine trauma and isolated rearing

As previously shown, a miswiring of ipsilateral prefrontal projections after methamphetamine (MA) intoxication and/or isolated rearing (IR) may serve as a model of so-called “dysconnection” in human schizophrenia. We here find that deep prefrontal projections to contralateral targets were drastically reduced by both MA and IR alone, but remained equally dense if both impairments cumulated. Projections from superficial layers were not altered by MA and/or IR. These findings confirm that the normal intercortical integration of information is compromised in this animal model of schizophrenia

Electromagnetic exposure effects the hippocampal dentate cell proliferation in gerbils (Meriones unguiculatus)

Hoffmann K, Bagorda F, Stevenson AF, Teuchert-Noodt G. Electromagnetic exposure effects the hippocampal dentate cell proliferation in gerbils (Meriones unguiculatus). Indian J Exp Biol. 2001;39(12):1220-1226.The chronic effect on hippocampal neurogenesis after exposure (30 min/day for 14 days) to a high frequency (35,53 kHz) electromagnetic field, double modulated at extremely low frequencies (ELF; 1, 8, 12, 29 and 50 Hz), was studied in young adult gerbils. Immediately after the last exposure proliferation of dentate granule cells was identified by in vivo labeling with 5-bromo-2-desoxyuridine (BrdU). Exposure to 1, 29 and 50 Hz resulted in a statistically significant reduction of cell proliferation rates, but only the 50 Hz-group manifested the effect highly significantly (-29,3 %). On the other hand, gerbils exposed to 8 and 12 Hz showed no significant change of postmitotic cell proliferation as compared with the sham treated controls. The results suggest that the effects of ELF on the granule cell proliferation are mediated by neurotransmitters and hormones which regulate hippocampal neurogenesis

Naloxone inhibits A6 cell Na+/H+ exchange by activating protein kinase C via the mobilization of intracellular calcium

The opioid receptor antagonist, naloxone, has been shown to have beneficial effects in the kidney and to be implicated in renal salt and water balance. In the present study the signal transduction pathways utilized by naloxone were studied in an epithelial cell line model of the cortical collecting duct, A6 cells. We found that naloxone has a dual effect depending on the concentration used: at a low concentration (10(-6) M) it antagonized the beta-endorphin-dependent increase in cytoplasmic calcium [Ca(2+)](i), while at higher concentrations (>10(-5) M) it increased [Ca(2+)](i) and intracellular inositol phosphate levels. While naloxone-induced increases in [Ca(2+)](i) occurred in the absence of external calcium, it was significantly stimulated by increasing the external calcium concentration

NHE3 inhibits PKA-dependent functional expression of CFTR by NHERF2 PDZ interactions

It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is "sequestered" by interacting with NHE3 it can no longer participate in CFTR functional expression. (c) 2006 Elsevier Inc. All rights reserved.X1116sciescopu

NHE3 inhibits PKA-dependent functional expression of CFTR by NHERF2 PDZ interactions

It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is &quot;sequestered&quot; by interacting with NHE3 it can no longer participate in CFTR functional expression.close151