Journal of cell science.
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Fig. 4. SCAR RNAi in S2R+ cells inhibits lamellipodial protrusion. (A) Morphometry. Projected area (left) and P/A1/2 cell shape index (right) of control (gray bars, n=24) and dsSCAR-treated for 4 days (black bars, n=26) S2R+ cells. Higher values of the index correspond to more irregular cell shape (see text for details). (B,C). Kinetics of lamellipodial protrusion on day 3 (B) and day 4 (C) of SCAR RNAi. Time series of enlarged boxed regions are shown on the right. Time in sec. (B) Cell is able to form lamellipodia, but protrusion is not coordinated along the edge. Formation of individual protuberances is indicated by arrowheads. (C) Cell is not able to form lamellipodia; it forms long narrow curved processes, which express dynamics at their tips and along their length (arrowheads). (D,E). Structure of actin cytoskeleton on day 3 (D) and day 4 (E) of SCAR RNAi (EM). (D) Lamellipodia-like protrusions contain very sparse dendritic actin filament network. (E) Long processes contain branched filament network, but not bundles of long filaments. Bars, 10 µm (B,C) and 0.2 µm (D,E).
- During this time, cells became more compact and developed a highly irregular shape, displaying long narrow processes (Fig. 3A and Fig. 4B,C).
- To quantify the reduction in cell spreading, we compared the projected area of control and SCAR-depleted cells (Fig. 4A, left).
- The irregularity of cell shape was evaluated using a dimension-less index – namely, the ratio of perimeter (P) over the square root of projected area (A) (Fig. 4A, right).
- At 3 days of treatment, cells still were able to protrude lamellipodia, although their activity was more erratic compared with untreated cells, and lamellipodia protruded with discrete protuberances instead of a smooth regular outline (Fig. 4B).
- At 4-5 days treatment, cells were unable to form lamellipodia; instead, they protruded long narrow processes, which expressed dynamics at their tips, as well as along their length (Fig. 4C).
- Therefore, we analyzed actin organization by phalloidin staining (Fig. 3D) and EM (Fig. 4D,E).
- By EM, residual lamellipodia of SCAR-depleted cells contained very sparse filament networks that retained dendritic features (Fig. 4D).
- In the narrow processes, actin filaments were also organized into a branched network of rather short actin filaments (Fig. 4E), but not into parallel bundles as is characteristic of filopodia.
Journal of andrology.
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Figure 5. Schematic diagram that emphasizes the importance of gap junctions to penile erection. As illustrated, intercellular communication provides the anatomic substrate for the establishment of corporal smooth muscle cell networks. These cellular networks are critical to rapid and syncytial contraction and relaxation responses required for detumescence and erection, respectively. In addition, the presence of gap junctions ensures that phenotypic cellular heterogeneity (ie, expression of distinct receptor/effector mechanism on corporal smooth muscle cells) can be tolerated. Thus, the presence of gap junctions guarantees that not all corporal smooth muscle cells need to be directly activated/affected by any given stimulus or therapy; this has important implications to gene therapy of erectile dysfunction (see text).
- The presence of these aqueous intercellular channels provides partial cytoplasmic continuity between coupled smooth muscle cells and ensures the intercellular transit of most of the known second-messenger molecules/ions that regulate corporal smooth muscle cell tone (Figure 5).
- The fact that relatively low-level hSlo transfection rates produced dramatic changes in erectile capacity (ie, intracavernous pressure) was presumed related to the presence and physiological relevance of the intercellular pathway provided by the connexin43-derived gap junction channels (see Figure 5).
Development (Cambridge, England)
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Fig. 5. Presomitic mesoderm explants form VEGF-dependent vascular networks. 8.5 dpc Flt1+/–, eGFP+/– mouse presomitic mesoderm explants were embedded in collagen gels and cultured for 72 hours. Explants were stained for ß-gal activity (A,C,E), and the same explants were visualized for eGFP expression (B,D,F). Explants were incubated with basal medium alone (A,B), or with added 30 ng/ml VEGFA (C,D) or 25 ng/ml bFGF (E,F). n=" BORDER="0">7 for all three conditions. The arrows in C indicate the vascular plexus, and the arrowheads in A and E indicate ß-gal positive (blue) presumptive vascular cells. Scale bar: 200 µm for all panels.
- To further dissect the molecular composition of the neural tube vascular patterning signal, we developed a collagen gel explant model (Fig. 5).
- After 72 hours in basal growth conditions, only a few ß-gal-positive cells were seen (Fig. 5A, arrow), although vigorous expression of eGFP showed that the explants had grown considerably and were healthy (Fig. 5B).
- However, a robust primitive vascular network was seen when the explants were incubated with added VEGFA (Fig. 5C,D).
- By contrast, bFGF addition produced only a few small clumps of ß-gal-positive cells (Fig. 5E,F), and no plexus formed even in a range of bFGF concentrations (5-100 ng/ml, data not shown), indicating that the vascular network was specific to VEGFA.
- This plexus was similar in complexity to the presomitic mesoderm-derived vascular plexus formed in the presence of VEGFA (compare Fig. 5C with Fig. 6G).
- Moreover, the complete lack of vascular cells in the presence of the inhibitors suggested to us that our basal conditions may include trace amounts of VEGF (Fig. 5A).
Protein engineering, design & selection : PEDS.
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Fig. 4. Neural network score distributions. Known secretory proteins of the non-classical pathway have similar neural network output scores to the positive training examples (truncated secretory proteins of the classical pathway without the signal peptide), easily distinguishable from the negative training examples. The data set extracted from the IPI database display a scoring pattern similar to the negative training set, indicating that the majority of the IPI set is non-secreted. All score distributions were normalized and smoothed by a Gaussian kernel density estimation.
- The score distributions shown in Figure 4 indicate clearly that this view may not be entirely correct.
- To analyze this, the neural network score distribution for the set of 13 such proteins was compared with the score distributions obtained for positive and negative training examples (see Figure 4).
- However, this was clearly not the case as a very large number of proteins received high scores from the neural networks (see Figure 4).
Genome Biology
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Edge-removal criteria. (a) Pseudocode of the algorithm including positive and negative regulation. Acc(i) and Adj(i) indicate the accessibility and adjacency lists for gene i, respectively, and Acc(i,j) indicates the value (+1 or -1) of the edge from i to j. (b) The original algorithm will pare away any edge connecting two nodes that already have a pathway between them. (c) Algorithm taking positive and negative regulation into account will only pare away an edge if its sign is equal to the product of the signs of the remaining edges in the pathway.
- Inspection reveals that any indirect regulatory pathway will have a value equal to the product of the intermediate edges, so the extended algorithm only prunes an edge, by converting the matrix element to zero, if this condition is met (Figure 2a, lines 15-19).
- For example, if the two intermediate edges both have a positive sign, the original algorithm will remove the shortcut regardless of its sign (Figure 2b), but the extended algorithm will only prune the edge if it is also positive (Figure 2c).
- These 'pseudocycles' interfere with the search phase of the algorithm (Figure 2a, lines 4-14) and often result in reconstructions of poor quality.
- This suggests that antagonistic feed-forward pathways ("incoherent feedforward loops" in the terminology of Shen-Orr et al. [27]) (Figure 2b, bottom) are common in real biological regulatory networks.
- Such pathways are also encountered in experimental data (data not shown) and the algorithm can get caught in these loops during the search phase (Figure 2a, lines 4-14).
BMC Cancer
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Networks of genes commonly regulated after tipifarnib treatment. (A) Twenty-three genes that were down-regulated in patient leukemic cells and AML cell lines were analyzed by the Ingenuity Pathway Analysis tool. The shown major network that was found to be significantly down-regulated by tipifarnib was associated with proliferation (p = 10-10). (B) Twenty-nine genes that were up-regulated were also analyzed for associated networks that were significantly affected by tipifarnib. The shown network was significantly associated with apoptosis (p = 10-10) and immunity (p = 10-7). Shaded genes are the genes identified by microarray analysis and others are those associated with the regulated genes based on the pathway analysis. The meaning of the node shapes is also indicated. Asterisks indicate genes that were identified multiple times.
- For example, the network of up-regulated genes (Fig 5A) includes the lamin B gene, which is indeed a direct target of FTIs.
- Also, the PIK3R2 gene, which regulates AKT and is a known target of FTIs [3], can be found in the down-regulated network of genes (Fig. 5B).
Lipids in Health and Disease
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Pathway analysis. Figures 3–7 represent signaling pathways for 58 focus genes selected from Table 3 by Ingenuity Systems software (Redwood City, CA. The following signaling cascades are shown: JUN, TNF, and CDKN2A signaling cascade affecting: DNA replication; recombination and repair; immune response; and cell cycle (Fig. 3); TGFB1 signaling cascade affecting: cell morphology; cancer; and tumor morphology (Fig. 4); CTNB1 signaling cascade affecting: cell signaling; gene expression; and cell cycle (Fig. 5); INS1/hRAS signaling cascade affecting: carbohydrate metabolism; endocrine disorders; and metabolic disease (Fig. 6); and MYC signaling cascade affecting: viral function; gene expression; and cell Cycle (Fig. 7). Differentiating groups (per Table 3) are overlaid onto the signaling diagrams, and abbreviated: FU, fungal; FI, fish oil; CO, combination diet. When CO was the differentiating group, absolute differences between FU and FI are indicated. Intracellular location of focus genes (subscripts) are annotated: C, cytoplasm; E, extracellular; N, nucleus; P, plasma membrane; U, unknown. Major canonical functional/signaling categories associated with genes in the figures identified by the software, are shown in yellow boxes.
- Pathway analysis (Figs. 3, 4, 5, 6, 7) was performed on selected transcripts differentiating the groups (Table 3; described in next section).
- JUN/TGIF/TNF/CDKN2A signaling (Fig. 3)
- Herein, we focus on genes implicated in pathway analysis (Figs 3, 4, 5, 6, 7) and linkable to specific functions (Tables 2, 3).
- Changes to these transcripts up- and down stream of HSPs were not found but the upstream activator of HSPs, junD, was decreased with FISH (Fig. 3).
- Pathway analysis (Figs. 3, 4, 5, 6, 7)
- Network diagrams including these 58 focus genes and some connection genes were redrawn for simplicity (Figs. 3, 4, 5, 6, 7).
- The 5 networks were independent from one another, except for an indirect connection via JUN linking Figs. 3, 4.
Diabetes.
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FIG. 4. PlGF and PlGF/VEGF promote and prolong in vitro angiogenesis. A: BRECs were sandwiched between two layers of type I collagen in serum-deprived medium in the presence of VEGF (100 ng/ml), PlGF (100 ng/ml), or PlGF/VEGF (100 ng/ml) for up to 14 days. Images of representative microscope fields (x10): I–II, Control; IV–VI, VEGF; VII–IX, PlGF; and X–XII, PlGF/VEGF. B: Total tube length was measured in each treatment at indicated time points as mm/mm2. VEGF induced a rapid increase in capillary networks that reached a maximum at day 5; however, PlGF had no effect on tube formation until day 5, while PlGF/VEGF sustained in vitro angiogenesis over the entire 14 days. Data are means ± SD or representative of three independent experiments. **P < 0.01, ***P < 0.01 vs. control.
- All three growth factors induced angiogenesis, but only PlGF-containing ligands sustained tube formation over this period (Fig. 4A).
- VEGF, PlGF, and PlGF/VEGF induced in vitro angiogenesis as determined by total tube lengths in a time-dependent manner (Fig. 4B).
The Journal of biological chemistry.
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Fig. 5. NRK cells transfected with Rab2 (Q65L) protein or with anti-GAPDH antibody display altered microtubule organization. NRK cells (1 × 104/10-cm dish) were plated overnight on coverslips, then each coverslip was transfected with 1 µg of recombinant Rab2 (Q65L) protein or 5 µg of affinity-purified anti-GAPDH polyclonal antibody using Chariot protein transfection reagent as described under "Experimental Procedures." The cells were incubated for 4 h at 37 °C in a 5% CO2 incubator, then fixed in 3% formaldehyde and immunostained with affinity-purified anti-GAPDH polyclonal antibody (control and Rab2Q65L) and anti-{beta}-tubulin monoclonal antibody, as described under "Experimental Procedures." Rab2 (Q65L)-transfected cells displayed prominent {beta}-tubulin-labeled structures that resembled MT bundles. These structures codistributed with elements that immunolabeled with anti-GAPDH polyclonal antibody. In contrast, cells transfected with anti-GAPDH antibody lacked MT networks.
- In control cells, MTs are concentrated near the nucleus and radiate out toward the cell periphery from a centrally located microtubule-organizing center, whereas GAPDH is distributed diffusely throughout the cytoplasm (Fig. 5).
- When these cells were costained for GAPDH, the anti-GAPDH antibody labeled the filamentous structures that overlapped with the anti-tubulin-labeled elements (Fig. 5).