American journal of physiology. Gastrointestinal and liver physiology.
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Fig. 3. Cartoon depicting the development and plasticity of ICC. Mesenchymal cells at the periphery of the small bowel develop Kit expression by embryonic day 12 (E12). Neuroblasts, in close proximity to the Kit-positive cells, express stem cell factor (Kit ligand) and might provide early signaling; however, functional ICC develop in the absence of neurons. Smooth muscle cells on which Kit-expressing cells develop also express stem cell factor. Cells that are signaled via Kit develop into functional IC-MY, and these cells become pacemaker cells of the intestine. Cells not signaled via Kit develop smooth muscle antigens and may become the longitudinal smooth muscle layer. After development of IC-MY, blocking Kit signaling causes a phenotypic transition toward a smooth muscle-like phenotype. This is accomplished within days in newborn animals but requires weeks in mature animals. Pathophysiological conditions, which are not fully understood, also cause a phenotypic transition that leads to loss of IC-MY and pacemaker activity. Hybrid cells appear that may be similar to the cells that develop when Kit signaling is blocked. An important question is whether the hybrid cells can redifferentiate into functional ICC if normal conditions in the intestinal microenvironment are restored.
- Thus myenteric interstitial cells (IC-MY) and longitudinal muscle cells diverge from a common precursor late in gestation (Fig. 3).
- When the Kit signaling pathway is blocked, ICC appear to differentiate toward a smooth muscle-like phenotype (Fig. 3).
BMC Genomics
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Gene networks showing inter-relationship between differentially expressed genes in lymphoblastoid cell lines from monozygotic twins discordant in severity of autism spectrum disorder and/or language impairment. The over-expressed (red) and under-expressed (green) genes were identified as significant using SAM analysis (FDR = 15.6%) of microarray data across 5 twin pairs. The log2 expression ratio cutoff was set at ± 0.58 and was based upon the mean values for each gene. Differentially expressed genes within this network that have a reported role in nervous system development and function are marked with a "#" symbol and include: ALOX5AP (FLAP), CD44, CHL1, EGR2, F13A1, FLT1, IL6ST, ITGB7, and NAGLU. Gray genes are present but do not meet expression cutoff.
- Once again, pathway analysis of the differentially expressed significant genes revealed an extended network centered on TNF and other cytokines (including IL1B, IL4, and IL6, which was highly expressed in the brain tissues of autistic individuals [34]), connecting a number of neurologically relevant genes (Fig. 2).
- One of the striking results of the pathway analyses is that a relatively large number of the differentially expressed, neurologically relevant genes are linked in networks that are centered on genes involved in inflammation (see Figs. 1 and 2).
BMC Genomics
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Gene networks showing inter-relationship between differentially expressed genes in LCL from 3 discordant autistic twin sets using Ingenuity Pathways Analysis software. The over-expressed (red) and under-expressed (green) genes were identified as significant using SAM analysis (FDR = 26.4%) of microarray data across 3 twin pairs. The log2 expression ratio cutoff was set at ± 0.58 and was based upon the mean values for each gene. Genes within this network that have a reported role in nervous system development and function are marked with a "#" symbol and include: ASS, ALOX5AP (FLAP), DAPK1, F13A1, IL6ST, NAGLU, PTGS2, and ROBO1. Gray genes are present but do not meet expression cutoff.
- in gene expression are generally reproducible when Lowess normalization is used [27], and our own ability to confirm expression changes of at least 1.5-fold by qPCR. Of particular note is the gene network that is derived from pathway analysis of the mean expression values (with log2 ratio ≥ ± 0.58) across 3 sets of discordant twins which shows that the majority of significantly differentially expressed genes are part of an extended network centered on TNF and other inflammatory mediators (Fig. 1).
- One of the striking results of the pathway analyses is that a relatively large number of the differentially expressed, neurologically relevant genes are linked in networks that are centered on genes involved in inflammation (see Figs. 1 and 2).
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.
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Figure 1. Visualization of the pathway of Vectamidine/DNA complex entry into cells by electron microscopy: transmission electron micrographs of BHK21 cells treated with Vectamidine/DNA complexes for 3 hr. Complexes were observed adhering to the cell surface (A) and/or in endosome-like vesicles (B). Arrows denote examples of vesicle structures; arrowheads indicate examples of filament networks. Large arrow in A denotes a complex that is in the process of being endocytosed. Bars = 0.2 µm.
- Vectamidine/DNA complexes were identified on the ultrathin sections by their high electron density and appeared as aggregations of ovoid structures (Figure 1, arrows) and filament networks (Figure 1, arrowheads).
- At 2–3 hr after incubation, these aggregates adhered to the cell surface or were taken up by the cells through an endocytic process (Figure 1A).
- In some instances, aggregates in the process of being engulfed by the cells were observed (an example is indicated by the large arrow in Figure 1A).
- Once in the cytosol, the Vectamidine/DNA aggregates appeared to be contained in large endocytic vesicles that were filled to some extent with the aggregates (Figure 1B).
- Altogether, these results (Figure 1 and Figure 2) clearly demonstrate that the Vectamidine/DNA complexes are internalized by an endocytic process and that they contain the transfected DNA.
- EM data showed that the complexes were organized as spherical and filamentous assemblies (Figure 1 and Figure 2).
Journal of neurophysiology.
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Fig. 2. Synaptic depolarization of TC neurons disrupts spindle synchrony in thalamocortical pathway. Barbiturate anesthesia. Field potential from the depth of area 4 and intracellular activity of thalamocortical (TC) neuron from VL nucleus. Parts marked by horizontal lines are expanded below. At the resting membrane potential, TC neuron produced spindles, almost simultaneously with spindles in the corresponding cortical area. Barrages of excitatory postsynaptic potentials (EPSPs), presumably of cerebellar origin (see Timofeev and Steriade 1997), depolarized the TC neuron and spindle oscillations were no longer synchronous with cortical EEG spindles.
- Short-range synchronization of spindles was observed as relatively large-amplitude field potentials recorded at one site and not accompanied by similar field potentials and/or related cellular activities recorded at remote sites (Fig. 2).
- Detailed examination of recordings showed that the loss of spindle synchrony between TC neurons from VL nucleus of thalamus and cortical field potentials from area 4 occurs when thalamic neurons received barrages of excitatory postsynaptic potential (EPSPs) presumably of cerebellar origin (Timofeev and Steriade 1997; Fig. 2, bottom).
- Note that the amplitude of local depolarizing potentials observed in TC cells during interspindle lull exceeded those typically recorded in vivo (Fig. 2B).
- In in vivo recordings, the frequency of bursting was similar, but, only single bursts or doublets of bursts were recorded (see Fig. 2B).
Molecular & cellular proteomics : MCP.
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FIG. 5. Kinetics of the in vitro kinase reactions are linear with time. Assays were incubated with fixed amounts of HT-29 or HeLa lysates treated with known activators of the pathway of interest (for details, see Fig. 2, A–D). In vitro reactions were allowed to proceed for the indicated times and analyzed as described under "Experimental Procedures." A, Akt assay kinetics with Akt from 500 µg of HT-29 lysate. B, JNK1 assay kinetics with with JNK1 from 200 µg of HT-29 lysate. C, IKK assay kinetics with IKK from 800 µg of HeLa lysate. D, MK2 assay kinetics with MK2 from 200 µg of HT-29 lysate.
- As shown in Fig. 5, A–D, each phosphorylation reaction displayed linear kinetics up to the time of termination, suggesting that neither the [-32P]ATP nor the substrate was significantly depleted over the course of the reaction.
Molecular & cellular proteomics : MCP.
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FIG. 6. Proteins associated with proximal EGF receptor signaling and their modulation by transient exposure to EGF (10 ng/ml 10 min) or to erlotinib (OSI-774; 1 µM for 120 min) and were modeled using present data, Network Explorer (Ingenuity), and literature data.
- Multiple SH2 domain-containing proteins were modulated by erlotinib and/or EGF exposure (Table IV), and these were modeled using the pathway connectivity Network Explorer software, from data obtained by anti-EGFR selection and from data obtained from the literature (Fig. 6).
- Similarly, anti-pY capture of paxillin, p130CAS, and the FAK homolog Pyk2 were all reduced by EGF receptor inhibition (Fig. 7B), while no changes in the phosphorylation of ACK1, -catenin, or Tyk2 (Figs.
Physiological genomics.
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Fig. 2. Two-dimensional hierarchical clustering node of stromal cell-derived factor 1 and related genes. From top to bottom: TM4SF1, transmembrane 4 superfamily member 1; SDF1, chemokine ligand 12 (sdf-1); ANGPTL2, angiopoietin-like 2; MAGED1, melanoma antigen; KIAA0680 protein; EFNB2, ephrin-B2; KIAA0830 protein; CKAP4, cytoskeletal associated protein 4; JAM2, junctional adhesion molecule 2; NOLA2, nucleolar protein family A, member 2; KIAA0193 gene product; GPS2, G protein pathway suppressor 2; GSPT1, G1- to S-phase transition 1; EMCN, endmucin; ANGPTL2, angiopoietin-like 2; FLJ10849; CDH5, cadherin 5, type 2; TM4SF1, transmembrane 4 superfamily member 1; TM4SF1, transmembrane 4 superfamily member 1. Labels across top represent patients in cohort (see Table 1).
- The relevance of this is not known. Figure 2 shows the two-dimensional hierarchical cluster of genes surrounding stromal-derived factor 1, which include angiopoietin-like 2, ephrin-B2, transmembrane 4 superfamily, and cadherin 5.
American journal of physiology. Cell physiology.
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Fig. 11. Insulin signaling events leading to microvillar shape change and formation of submembrane microfilament networks (membrane ruffling). High-affinity binding of actin-binding proteins to D-3 phosphorylated phospholipids (D-3-P lipids) directs a flux of actin monomers to the sites of activated insulin receptors/phosphatidylinositol (PI) 3-kinase. With these monomers and those from the pointed ends of microvillar filaments, a 3-dimensional microfilament network is formed beneath the plasma membrane that exerts an isotropic outwardly directed force (arrows), blowing up this membrane area like a gas-filled balloon (see SEM in Fig. 6B and 12B). The high membrane curvature at the transitional region between the plasma membrane and the microvillar shaft additionally activates the PI 3-kinase by 2 orders of magnitude.
- Downstream of receptor-mediated PI 3-kinase translocation/activation, four mechanistic components are involved in the activation of the microvillar influx pathway (Fig. 11):
- At the least, the reduction of insulin stimulation of transport by cytochalasin D may indicate that PI 3-kinase-induced F-actin reorganization into submembranous networks (membrane ruffling; see Fig. 11) is a necessary mechanistic component for full activation of glucose transport by insulin.