Journal of cell science.
Extra large 
|
|
|
Fig. 2. Rho activity controls the level of tubulin acetylation upstream of HDAC6. (A) Inhibition of Rho by 0.5 µM TAT-C3 for 5 hours induced an accumulation of acetylated microtubules in comparison to 0.5 µM TAT-GFP used as a control. (B) One nucleus per osteoclast was microinjected with either RhoA WT-GFP or a constitutively activated form of Rho, RhoAV14-GFP expression vectors. Cells were fixed 6 hours after microinjection and GFP-expressing cells were detected by GFP fluorescence using a confocal microscope. Acetylated tubulin was detected by indirect immunofluorescence (green) and F-actin by means of phalloidin-RITC (red) and a close-up of each condition is presented. In the presence of RhoA-WT, osteoclasts exhibit the typical podosome belt and dense networks of acetylated microtubules. On the other hand, expression of Rho V14-GFP induced deacetylation of microtubules and disorganisation of podosome belts (arrowhead in close-up area). However, tubulin deacetylation dependent on Rho activation was inhibited after treatment with the HDAC6 inhibitor TSA (3 µM) for 1 hour, showing that this enzyme is downstream of Rho. (C,D) HDAC6 is present and active in osteoclasts. Endogenous HDAC6 was easily detected in osteoclasts by western blotting with a polyclonal anti-HDAC6 antibody (C). The deacetylase activity of HDAC6 was tested with two drugs: TSA, known to inhibit its activity and sodium butyrate, which does not. HDAC6 was indeed active in osteoclasts as confirmed by greatly increased levels of acetylated tubulin in TSA-treated osteoclasts and unchanged levels in the presence of sodium butyrate compared to the control and to the total amount of ß-tubulin (D). Finally, inhibition of Rho by TAT-C3 (for 4 hours) in the presence of TSA had no additional effect on the increase in acetylated tubulin (C). Bar, 20 µm.
- Whereas the Ac-MT level was detectable but low in osteoclasts incubated with TAT-GFP fusion protein, it was increased in osteoclasts maintained in presence of TAT-C3 (Fig. 2A).
- GFP-RhoA WT did not significantly affect levels of Ac-MT whereas V14RhoA promoted a drastic decrease in the amount of Ac-MT together with disruption of the podosome belt (Fig. 2B).
- TSA treatment blocked the V14RhoA-mediated deacetylation of microtubules (Fig. 2B, lower panel), indicating that HDAC6 was downstream of Rho.
- To confirm that microtubule acetylation was increased by HDAC6 or Rho inhibition, we monitored levels of acetylated tubulin in osteoclast lysates treated with TAT-GFP, TAT-C3 or TSA (Fig. 2C).
- The amount of acetylated tubulin did not change in sodium butyrate-treated osteoclasts but did drastically increase when osteoclasts were treated with TSA (Fig. 2D).
Reproductive Biology and Endocrinology
Extra large 
|
|
|
Interactive map (pathway) of 46 genes/proteins created in PathwayAssist® from the list of transcripts up-regulated in the testes of ABP-transgenic mice. AGT, A2M, IL-6, and MYC are "nodes" with multiple connections whereas most other components have only one or two connections. The symbols along the connecting lines indicate the nature of the interaction (Expression: blue square; Regulation: gray square; Molecular transport: gray square with green outline; Protein modification: yellow hexagon; Binding: purple diamond; Promoter binding: green diamond; Molecular synthesis: light blue square with dark blue outline; and Chemical reaction: clear square; stimulation or inhibition is indicated by + or - signs within the symbols).
- All the genes belonging to the up-regulated group were displayed, all were selected, and the "Build Pathway" command was used with the option of "Find only direct interactions between selected nodes" to create a new pathway (Fig. 3).
- This possibility is reinforced by the large number of signal transduction-related genes whose expression is up- or down-regulated in the ABP-TG mice (see Tables 1, 2, 3, 4, and Figs. 3 and 4).
- The interaction networks presented in Figs. 3 and 4 provide additional insights into the changes in testicular gene expression resulting from reduced androgen levels in the testis.
- Figs. 3 and 4 identify 46 and 38 genes (gene products), respectively, that are not only co-regulated in the ABP-TG mice but are also capable of functionally interacting with each other.
- One of only two genes known to be expressed by SC and directly respond to androgens, MYC, has been identified as a critical component of the up-regulated network (Fig. 3, Table 2), whereas CREB1, known to be essential for spermatogenesis, was down-regulated.
The Journal of cell biology.
Extra large 
|
|
|
Figure 4. Kinetic analysis of Golgi membrane reassembly in rat liver cytosol and p115-depleted cytosol. (A–H) MGF isolated through a 0.5-M sucrose cushion were incubated for increasing time at 37°C with either rat liver cytosol (A–D) or p115-depleted cytosol (E–H) with the cytosol concentration set at 10 mg/ml, fixed and processed for EM, and quantitated as described in Materials and Methods. Representative fields are shown. In rat liver cytosol, note that the first intermediate formed is the single cisterna after 5 min (arrows in A). By 15 min, these single cisternae had grown in length, had tubular networks associated with their rims (asterisks in B), and had begun to align and dock to form stacks (arrowheads in B). Many discrete stacks had formed by 45 min (arrowheads in C), which had joined up by 120 min (arrowheads in D). In p115-depleted cytosol, single cisternae were again present after 5 min (arrows in E), and had increased in length by 15 min (arrows in F), but were often blunt-ended (asterisk in F) and were not stacked. At 45 min, these cisternae remained blunt-ended (asterisk in G) and unstacked. By 120 min, some stacks of blunt-ended cisternae had begun to form (arrowhead in H), but many single cisternae remained (arrows in H). Bar, 0.5 µm. (I) Quantitation of the time course. The percentage total membrane present as cisternae ± SEM (n = 3) and stacked regions of cisternae ± SEM (n = 3) are presented for each cytosol at every time point tested. (J) MGF were reassembled in p115-depleted cytosol and supplemented with p115 at different times (time of addition of p115). The reaction was allowed to proceed for a total time of 120 min at 37°C. Samples were then fixed and processed for EM, and the percentage total membrane as stacked regions of cisternae ± SEM (n = 2) are presented for each time point tested.
- Kinetic analysis revealed the reassembly reaction was complete for both cisternal regrowth and stacking after 60 min in rat liver cytosol (10 mg/ml; Figure 4 I).
- The first intermediates that formed quickly during the first 15 min of the incubation were single cisternae (Figure 4A and Figure B), frequently with tubular networks at their rims (asterisks in Figure 4 B).
- By 15 min, these intermediates had begun to dock and align to form the beginnings of stacked Golgi structure (arrowheads in Figure 4 B).
- The lag in the formation of stacked structures (Figure 4 I) therefore may be considered due to the need to form single cisternae first.
- By 45 min, this process was well advanced and Golgi stacks with two or more cisternae per stack were prevalent and these discrete stacks were becoming linked via tubular networks (Figure 4 C).
- In p115-depleted cytosol, single cisternae formed at the start of the reaction, although with a reduced initial rate (Figure 4I and Figure E).
- Once again these cisternae were blunt-ended, indicating the p97 pathway of reassembly may be dominant (asterisks in Figure 4F and Figure G).
- By 15 min, these single cisternae were still well separated (Figure 4 F), and even after 45–60 min, single, blunt-ended cisternae were the major reaction product (Figure 4G and Figure I).
- However, after 120 min, even though no more cisternal regrowth occurred, these single cisternae did begin to align and form stacks (Figure 4H and Figure I).
- Even then, the level of stacking only reached ~50% of that of rat liver cytosol (Figure 4 I), and the intercisternal distance between adjacent cisternae of the stack seemed more variable (compare Figure 4C and Figure H).
- However, if added at 60 min, the p115 only slightly stimulated stacking (Figure 4 J).
- When added at 15 min, the time point when cisternae begin to dock and align (Figure 4 B), the N73pep actually unstacked those stacks that had formed, suggesting that p115 was mediating this event (compare Figure 8A and Figure C).
BMC Bioinformatics
Extra large 
|
|
|
MAPK signaling pathways produced by PathSys in comparison with the canonical KEGG MAPK signaling pathways. a). MAPK signaling pathways in Yeast reproduced by KEGG.b). Pathways generated by PathSys for (b1) Pheromone response, (b2) Cell Wall modeling and (b3) Filamentation of MAPK signaling pathways, with the proteins as red ovals, complexes as blue ovals; processes (binary and multiple) and interaction types as small colored circles, squares, diamonds, etc.; pathways (Cell Cycle pathway) as yellow triangles, compartments as grey boxes. Network graphs are produced by BiologicalNetworks client software.
- An excellent benchmark against which to validate our approach is MAPK pathways involved in pheromone response, filamentous growth, and maintenance cell wall integrity (Figure 4), one of the most thoroughly studied networks in yeast, conserved across all eukaryotes.
- To show the impact of MIG integration in understanding biology, we present a comparison between our results and those obtained from KEGG. First, we start with ALL_HC and extract a subnetwork of genes related to "pheromone response" (Figure 4b1).
- Compared to KEGG (Figure 4a), our results include more members of heterotrimetric G protein complex, including the alpha, beta, and gamma subunits, the GDP-GTP exchange factor, and the GTPase-activating protein (Gpa1p, Ste4p, Ste18p, Cdc24p, and Sst2p, respectively).
- Figure 4b2 shows the result of extracting network for the function "cell wall remodeling" from ALL_HC. It contains both GTPase constituents, Rho1 and Cdc42p, as well as associated GAPs and other interactors, including Rdi1p, Rga1p, and Gic2p.
- The last three proteins are not presented by KEGG (Figure 4a).
- In the result of a query for the filamentation pathway (Figure 4b3), key components of the Ras GTPase are included, such as Cdc25p (the Ras guanine nucleotide factor), Cyr1p (the Ras-associated adenylate cyclase), and Srv2p, which enables the activation of adenylate cyclase by Ras2p.
Reproductive Biology and Endocrinology
Extra large 
|
|
|
Interactive map (pathway) of 38 genes/proteins created in PathwayAssist® from the list of transcripts down-regulated in the testes of ABP-transgenic mice. Major nodes are IL-2, 4, and 10, MAPK8, SOCS1, CD44, and CREB1. For explanation of symbols, see legend to Fig. 3.
- A similar interaction network was created from the list of down-regulated genes (Fig. 4).
- Of the proteins in this gene list, 38 were directly connected to each other through 72 "contols" or interactions (Fig. 4).
- This possibility is reinforced by the large number of signal transduction-related genes whose expression is up- or down-regulated in the ABP-TG mice (see Tables 1, 2, 3, 4, and Figs. 3 and 4).
- The interaction networks presented in Figs. 3 and 4 provide additional insights into the changes in testicular gene expression resulting from reduced androgen levels in the testis.
- Figs. 3 and 4 identify 46 and 38 genes (gene products), respectively, that are not only co-regulated in the ABP-TG mice but are also capable of functionally interacting with each other.
Journal of neurophysiology.
Extra large 
|
|
|
FIG. 8. Proposed interlimb coupling of the locomotor pattern generators, or half-centers. Mutually inhibitory connections between ipsilateral extensors (E) and flexors (F) create alternating flexion and extension of the limb. A and B: parallel descending commands to the left and right limb networks. C: interlimb coupling pathway. If the connections at C have low gain, then ipsilateral muscle coordination patterns would be the same as long as ipsilateral motor command and ipsilateral afferent signals were the same, regardless of contralateral leg movements. An inhibitory connection from E to contralateral F is hypothesized. With only a descending command to the pedaling leg in unilateral pedaling, inhibitory influence from the nonpedaling leg neuronal network is released.
- Because of the flexibility observed in the locomotor pattern, separate locomotor-generating elements for each limb have been hypothesized with interneuronal connections that create coordinated alternation between the limbs [Fig. 8; in humans (Prokop et al.
- One hypothesis compatible with these general concepts of locomotor pattern generation is that the gain of the interneuronal connections between the ipsilateral and contralateral elements is low (Fig. 8, interconnections C).
- In this case, to pedal in the unilateral condition, the descending command would have to excite only the locomotor elements associated with the pedaling leg (Fig. 8, descending command A).
- Perhaps an inhibition of flexor muscles from the sensorimotor control of contralateral limb extension during bilateral pedaling exists (Fig. 8, interconnections C).
- We believe that subjects used the same ipsilateral descending command (Fig. 8, descending command A) to pedal unipedally as bipedally, but the change in sensorimotor state of the nonpedaling leg (e.g., Fig. 1B, right leg) resulted in different motor outputs to the pedaling leg (e.g., Fig. 1B, left leg) because the neuronal circuitry responsible for pedaling is inherently bilateral (cf. "shared bilateral core") (Stein and Smith 1997; Stein et al.
The Journal of experimental medicine.
Extra large 
|
|
|
Figure 5. The structure of HLA-B*4405 alters peptide specificity and increases hydrophobicity in the F pocket. (A) Overview of the structure of B*4405 highlighting Tyr116 and the electron density of the peptide (Dp{{alpha}}46-54, EEFGRAFSF). The structure of the F pocket in HLA-B*4402/EEFGRAFSF (B) (35) and the new structures B*4405/EEFGRAFSF (C) and HLA-B**4403/EEPTVIKKY (D) are shown as ball and stick representations of F pocket amino acids (gray), residue 116 (yellow), and the peptide ligand (green), including the COOH-terminal peptide residue P9 Phe in B and C and P9 Tyr in D. Dotted lines are H bonds. The {{alpha}}2-helix has been removed for clarity. In the B*4402 structure (B), Asp 116 is part of an intricate polar network involving Asp 114, Asp 156, Arg 97, and several conserved water molecules that additionally bridge contacts to the bound peptide. Asp 116 is orientated in the same direction as the aromatic ring of Tyr 116 in the B*4405 structure. A water molecule fills the "cavity" at position 116 of B*4402 such that it superposes closely to Tyr 116 O{{eta}} group in B*4405 and forms a similar role in that it bridges one H bond to Asp 114. In B*4405 (C), the P9 anchor residue Phe projects into a hydrophobic F pocket where it is surrounded by the aromatic rings of Tyr 74, Tyr 116, Tyr123, and Trp 147, as well as making van der Waals contacts with Ile 95 and the aliphatic moiety of Asn77. The main chain of this COOH-terminal residue is tethered by H bonds to Asn77, Tyr84, and Thr143. Tyr 116 forms the base of this pocket, where the aromatic ring points away from the F pocket, such that the Tyr 116 O{{eta}} group points toward the P7 pocket where it forms two H bonds with the Asp 114 carboxylate, an H bond to Arg 97 N{{varepsilon}}, and additionally forms a water-mediated H bond to the backbone of the bound peptide (Phe 7N). The aromatic ring of Tyr 116 also packs against the long aliphatic side chain of Arg 97. The electrostatic surfaces of the area bounding the F pocket of B*4402 (E) and B*4405 (F) are depicted using the program GRASP(39). Electropositive (blue); electronegative (red).
- We next determined the structure of HLA-B*4405 complexed with EEFGRAFSF, an abundant shared ligand derived from HLA-DP, and compared this with the structure of B*4402/EEFGRAFSF (35) (Fig. 5).
- The polymorphism at 116 (Tyr to Asp) significantly impacts on the characteristics of the respective F pockets (Fig. 5, A–F).
- The H-bond networks involving residue 116 and the arrangement of aromatic residues in the F pockets of B*4402 and B*4405 are shown in Fig. 5, B and C, respectively.
- In B*4402, the presence of Asp 116 imparts a markedly electronegative potential on the F pocket (Fig. 5 E), whereas in B*4405 the F pocket is predominantly hydrophobic (Fig. 5 F).
- Nonetheless, the specificity, mode of P9-Phe binding, and architecture of the F pocket is largely maintained in the two structures (Fig. 5, B and C).
- The basis for the alternate selection of either Phe or Tyr in the F pocket of B*4402 was addressed by analyzing a new structure of B*4403 complexed to the natural peptide ligand EEPTVIKKY (Fig. 5 D).
- In the B*4403–P9 Tyr complex, the carboxylate of Asp 116 formed a H-bond with the P9 TyrO group (Fig. 5 D).
BMC Plant Biology
Extra large 
|
|
|
Boolean representation of signaling network The binary switches used to represent allele status at the NPR1 and NDR1 loci were rendered according to standard depictions in engineering texts. All other symbols are according to Genoud, Trevino Santa Cruz and Métraux [24]. Signal generators were rendered as rectangles with black boxes inside them set at one or zero. "Or" gates, indicating that either input is sufficient to give the specified output, were rendered as bullet shapes with concave left sides. "And" gates, indicating that both inputs are required to give the specified output, were rendered as bullet shapes with flat left sides. Signaling outputs that also serve as inputs to downstream events were rendered as open triangles. Branches in the pathway were indicated with filled circles to suggest the resemblance to contact points in electrical circuit diagrams.
- These details are presented in a model of the signaling network for induction of SAR, PR-1 gene expression and PR-5 gene expression by avirulent P. syringae bacteria (Figure 4).
- No attempt has been made to represent the kinetics of signaling events in Figure 4.
- However, the authors agree with Genoud et. al. (2001) that the scheme used in that work and in Figure 4 of this paper has advantages for representing the topology of signaling networks.
Toxicological sciences : an official journal of the Society of Toxicology.
Extra large 
|
|
|
FIG. 4. Ingenuity pathway analysis of 4-day 400 mg/kg/day ciprofibrate gene expression. The pathway least likely to have occurred by chance in the 4-day 400 mg/kg/day treatment condition is shown. Green indicates downregulation, and red indicates upregulation, with the more intense color indicating a greater change. The numbers under each gene indicate the fold-change, a negative number means downregulation, and a positive number indicates upregulation. In this map, all genes shown were statistically different from the control. An asterisk next to a gene abbreviation indicates that more than one probeset is reporting on a gene. The probeset showing the greatest disregulation is used. A line indicates that two genes products have shown binding, a line terminating in an arrow means one gene product acts on the other gene product, and a plus symbol indicates other networks contain the gene product. Full gene names are given in Supplementary Figure 3.
- Figure 4 shows the network least likely to have occurred by chance for the 4-day treatment condition is a network centered on MYC.