Table 1. Demonstration models.

Name

Description

Simple reaction

Conversion of a A to B

Simple enzymatic reaction

Substrate to product conversion by an enzyme

Bistable

A network that shows bistability due to positive feedback between MAPK, PLA2 and PKC.(1)

cAMP

Ligand activation of the cAMP cascade and PKA.(2)

Chemotaxis

Phosphorelay cascade that controls the rapid chemotactic response in E.coli to the attractant aspartate and the repellent Ni2+ ions.(3)

CamKII

Activation of calcium/calmodulin-dependent protein kinase II (CaMKII) by calmodulin following calcium entry into the cell.(4)

G protein activation

Generic G-protein coupled receptor signalling model that looks at the kinetics of the G protein activation and receptor phosphorylation(5, 6)

MAPK-EGFR

EGFR stimulated activation of MAPK and subsequent inactivation of EGFR input(1)

MAPK oscillations

Oscillations in the mitogen-activated protein kinase (MAPK) cascades due to negative feedback loop combined with intrinsic ultrasensitivity of the MAPK cascade(7)

nNOS

Catalysis and activation in neuronal nitric Oxide Synthase (nNOS) and in its interaction with CaMCa4(8)

RanGTPase

Kinetics of the RanGTPase system involved in receptor-mediated nuclear transport(9)

Ras

Ras GTPase activation by GTPase Activating Proteins (10, 11)

Repressilator

Artificial network of transcription regulators in E.coli which results in sustained oscillations (12)

 

MATERIALS

 

1. GENESIS/Kinetikit software, included in download. Approximately 1.5 MB download and 4 MB uncompressed.

 

2. Set of simulation models in GENESIS (.g) format, a sample of which come with the download.

 

3. Optional:

    Additional simulation models. May be downloaded from the databases DOQCS (http://doqcs.ncbs.res.in) and SIGPATH (http://icb.med.cornell.edu/crt/SigPath/index.xml)

 

 

EQUIPMENT

PC with Linux operating system (Kernel 2.4 or later, e.g., Red Hat Linux 7.2 and higher).

 

 

INSTRUCTIONS

 

Installing the software.

  1. Access the site (INSERT STKE WEBSITE ADDRESS) or its mirror at (http://www.ncbs.res.in/~bhalla/kkit/download.html)
  2. Download the stke demo file. Unpack it into a convenient destination directory, using the command

tar xzf kkitdemo.tgz

This will produce a directory named kinetic_demo. These steps are sufficient for running the demonstration simulations.

 

 

Running demos

  1. From the kinetic_demo directory, type

./demo

 Several model schematics will flash past as the system loads the models.

  1. Select one of the demonstration models from the graphical menu. It will automatically load the stimulus sequence and commence the simulation.
  2. To select a different demonstration, click the ‘Select demo…’ button which is located on the upper right of the reaction schematic.

 

Stochastic simulations.

  1. Select the Simple Enzyme or Simple Reaction demo.
  2. Choose the ‘Mixed Stochastic’ integration method from the ‘Options’ menu. The other stochastic methods do not work in demo mode, but they will work (albeit slowly) with individual models as described below.
  3. Optionally, click the ‘Overlay plots’ toggle in the ‘Options’ menu. This will let you compare successive runs.
  4. Click on the green ‘Start stimulus’ button to run the same stimulus sequence using stochastic calculations.
  5. The other demos may also be run in the Mixed Stochastic mode. It may be necessary to toggle the ‘Manual dt selection’ in the ‘Options’ menu to run more accurately, but slowly.

 

Beyond demos: Modifying and manually running a model.

  1. Select a demonstration model. Click on the ‘Schematic’ button to expose the edit window. You can now double-click on any of the icons to pop up a window that will let you set your model parameters. In complex models it may be necessary to zoom in or pan the window to see the icons. Zooming is done using the angle bracket < > keys, and panning with the arrow keys.
  2. To update the value in a dialog, it is necessary to have the mouse cursor on the dialog, and to hit return or click the mouse on the dialog button to complete the entry.
  3. Click on the green ‘Start’ button to run the model without further input. If you do this you should set the desired runtime of the simulation in the ‘Runtime’ log. Alternatively:
  4. Click on the green ‘Start stimulus ’ button to run the same stimulus sequence again.
  5. Click on the red ‘Reset’ button to restore the simulation to its initial state.
  6. Should the graph axes need rescaling, double click on the axes to pop up a dialog box.
  7. The ‘File ’ menu item lets you save any changes to your current model.
  8. There is an extensive on-line help file, accessed through the ‘Help’ menu. It describes the basics of using Kinetikit.

 

Running other models

  1. Download a model from one of the databases, or use a model file you have edited and saved yourself. Genesis/kinetikit model files are indicated by a .g suffix.
  2. From the Linux command line, type

genesis modelname.g

It will load up the model and you can edit it and run it in the regular way. Predefined stimulus inputs will not work in such cases.

  1. Kinetikit provides several features to run models in batch mode (without graphics). To run in text-only mode, type

genesis –nox modelname.g

This mode still allows fully interactive text control of the simulation using the scripting language. The next level of automation is to run it completely without supervision using a model file that specifies everything including model, inputs, and outputs:

genesis –nox –notty modelname.g

This mode is used for example, in running simulations in parallel on a computing cluster.

 

Merging models.

  1. Composite simulations may be built up by successively loading models. The loader assumes that identically named molecules are, in fact, identical, and avoids duplications while preserving connections. Using the models provided with the demo we can connect the cAMP signaling pathway to the EGFR-MAPK pathway. From the command line in the kinetic_demo directory, type in:

genesis models/EGFR_MAPK.g

This creates the EGFR_MAPK model, which itself is rather large. This model has a fixed value of PKA as a regulatory input. Now from the genesis prompt type:

include models/cAMP.g

This loads in the activation stages for PKA, and will result in a large composite model including all the represented pathways. Now the PKA activity is no longer fixed, but is in turn a function of G-protein activity. The composite MAPK activity is a function now both of EGF and G-protein coupled receptor inputs. The composite model can be edited and saved in the usual way.

 

Installing for heavy use.

  1. If you wish to set up GENESIS/Kinetikit for extensive use, move the genesis binary file from kinetic_demo/genesis to some location in your executable path, such as ~/bin or /usr/local/bin.
  2. Copy the .simrc file to your home directory:

cp kinetic_demo/.simrc&nsbp; ~

Edit it so that the paths following the SIMPATH and GENESIS_HELP fields are set to refer to your installation directory. Note that the preceding period in the name .simrc will make the file invisible to the usual Unix directory listing command ls.

These changes will enable kinetikit to be run from any directory by typing

genesis kkit

or

genesis modelname

  1. The complete GENESIS simulator including many neuronal demos and an older version of kinetikit is available as source code from http://genesis-sim.org. This can be compiled to run on several UNIX-related platforms.

 

 

 

 

 

REFERENCES

 

1.   U. S. Bhalla, R. Iyengar, Science 283, 381-387 (1999).

2.   U. S. Bhalla, in Methods in Enzymology J. D. Hildebrandt, R. Iyengar, Eds. (Academic Press, 2002), vol. 345, pp. 3-23.

3.   D. Bray, R. B. Bourret, M. I. Simon, Mol Biol Cell 4, 469-82 (May, 1993).

4.   W. R. Holmes, J. Comput. Neurosci. 8, 65-85 (2000).

5.   L. D. Shea, R. R. Neubig, J. J. Linderman, Life Sci 68, 647-58 (Dec 29, 2000).

6.   P. J. Woolf, J. J. Linderman, Biophys J 84, 3-13 (Jan, 2003).

7.   B. N. Kholodenko, Eur J Biochem 267, 1583-1588 (2000).

8.   J. Santolini, S. Adak, C. M. Curran, D. J. Stuehr, J Biol Chem 276, 1233-43 (Jan 12, 2001).

9.   D. Gorlich, M. J. Seewald, K. Ribbeck, Embo J 22, 1088-100 (Mar 3, 2003).

10.   R. A. Phillips, J. L. Hunter, J. F. Eccleston, M. R. Webb, Biochemistry 42, 3956-65 (Apr 8, 2003).

11.   S. E. Neal, J. F. Eccleston, A. Hall, M. R. Webb, J Biol Chem 263, 19718-22 (Dec 25, 1988).

12.   M. B. Elowitz, S. Leibler, Nature 403, 335-8 (Jan 20, 2000).