Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-25
2003-11-18
Ponnaluri, Padmashri (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007930, C435S287300, C435S288500, C435S004000, C435S007100, C436S538000
Reexamination Certificate
active
06649358
ABSTRACT:
BACKGROUND OF THE INVENTION
Model systems which mimic receptor binding and other cell-based assays are of increasing importance in molecular biology. Phenomena such as transporter activity, pre- and post-synaptic cell interactions, chemotactic response, enzyme-ligand binding and the like are of relevance to pharmacology, clinical chemistry and basic research.
For example, one clinically relevant system involves the interaction of cells at synapses. At least two general types of synapses exist in nature. In electrical synapses, gap junctions connect cells which are in communication. Gap junctions permit direct transmission of electrical impulses from a presynaptic cell to a postsynaptic cell.
In the more common chemical synapse, an axon terminal of a presynaptic cell contains vesicles filled with a neurotransmitter, such as epinephrine or acetylcholine, which is released by exocytosis when a nerve impulse reaches the axon terminal. The vesicles release their contents into the synaptic cleft and the transmitter diffuses across the synaptic cleft. After a brief lag time (e.g., about 0.5 ms) the transmitter binds to receptors on postsynaptic cells. This typically causes a change in ion permeability and electrical potential in the postsynaptic cell. Excitatory signals induce an action potential in the postsynaptic neuron. Inhibitory signals prevent production of an action potential in the postsynaptic neuron. Both inhibitory and excitatory signals can exist simultaneously in the same synaptic cleft, depending on the cell types, neurotransmitters, etc. Similarly, a postsynaptic cell can be in simultaneous contact with multiple presynaptic cells, each of which can transmit both excitatory and inhibitory signals to the postsynaptic cell.
The presence of transmitter in the synapse is regulated in a variety of ways, thereby controlling the signal received by the postsynaptic cell. For example, some cells and vesicles actively transport transmitter out of the synapse, thereby reducing the presence of the transmitter in the junction. Similarly, oxidases and other enzymes degrade some neurotransmitters in the synapse. Neurotransmitters can also diffuse away from the synapse. For a review of neurotransmitter and transporter systems, see,
Neurotransmitter Transporters: Structure, Function and Regulation
(1997) M. E. A. Reith, ed. Human Press, Towata N.J., and the references cited therein.
Transporters have a variety of important biological roles. For example, the Na
+
/Cl
−
dependent transporters (e.g., the monoamine transporters, as well as betaine, creatine, GABA, glycine, proline and taurine carriers) are the primary sites of action for a variety of drugs of both therapeutic and abuse potential. For example, among the monoamine transporters, inhibition of the dopamine transporter (DAT) is linked to euphoric and reinforcing properties of psychomotor stimulants such as cocaine and amphetamines. The major classes of therapeutic antidepressants act by inhibiting the norepinephrine and serotonin transporters (NET and SERT) and many of these compounds have proved clinically useful in the treatment of panic, stress, obsessive compulsive disorders, and other conditions.
Chemotactic responses have also long been known to play significant roles in various biologicial systems. Chemotaxis is the capacity of a motile cell to respond to chemical changes in its environment by directed movement. The migration of a motile cell exhibiting a chemotactic response can be either up or down a concentration gradient of a chemotactic factor. For example, phagocytic cells like macrophages are attracted by and move toward various substances generated in an immune response, whereas other motile cells including certain bacteria can move either toward an attractant (e.g., assorted sugars) or away from various repellents (e.g., phenol). For further discussion of chemotaxis and related components, including adhesion and chemotactic factors, see, Kuby,
Immunology,
3
rd
Ed. W. H. Freeman and Company, New York (1997) and Stryer,
Biochemistry,
4
th
Ed., W. H. Freeman and Company, New York (1995).
Aside from methods and devices for modeling transporter activity and chemotactic responses, general binding assays for studying, e.g., enzyme-ligand binding interactions, receptor-ligand binding interactions, and the like are also useful, e.g., in modeling biological systems.
In general, existing in vitro systems for studying transmitters, transporters, presynaptic and postsynaptic cells, and other aspects of cell signaling do not provide ideal high-throughput methods and devices for modeling and mimicking transmitter diffusion, transporter activity, transmitter activity, and the like. More generally, cell-cell signaling, which is central to biological activity, is not ideally modeled using existing technologies and additional high throughput methods of screening for modulators of signaling activities are desirable. Furthermore, progress in the study of chemotaxis and various binding activities has also been impeded by in vitro assays that are tedious to perform and whose results have been difficult to quantify. As such, automated and quantitative assays for all of these important biological processes are desirable.
The present invention provides these and other features by providing high-throughput microscale systems for modeling transporter activity, transmitter degradation activity, transmitter activity, cell signaling, and detection of modulators (inhibitors and enhancers) of transporter or transmitter degradation activity. The present invention also relates to high-throughput systems for modeling gradient induced activities, e.g., chemotactic responses, and for assessing general binding activities. These and many other features which will be apparent upon complete review of the following disclosure.
SUMMARY OF THE INVENTION
The invention provides methods, devices, kits, reagents and related materials for modeling various important biological processes. For example, the present invention is optionally used to determine the activity of transporter components such as neurotransmitter transporters (for example, the neurotransmitter acetylcholine is specifically internalized by cells via endocytosis of acetylcholine from the synaptic cleft during the recovery period following signal transmission). The invention is also optionally used to assess gradient induced activities (e.g., study chemotactic responses) and to evaluate the binding activity of, e.g., various biological components. The methods are typically conducted in a microscale format using a microfluidic system which includes or is coupled to sources of the relevant assay components.
In the transporter-related methods and assays of the invention, a first component which includes transporter activity is flowed through a first channel. A second component which produces a detectable signal upon exposure to a transportable molecule or set of transportable molecules is flowed into the first channel. The transportable molecule is flowed into the first channel and a signal produced by contacting the second component with the transportable molecule is then detected. Typically, the level of signal product is inversely related to transporter activity.
A variety of formats for the methods are appropriate. The first and second components are typically flowed sequentially (a typical configuration) or simultaneously in the first channel. The second component optionally is flowed into contact with the transportable molecule in the presence or absence of the first component (for example, if flowed in the absence of the first component, the resulting signal serves as a positive control for the signal produced by contacting the second component with the transportable molecule).
Known activity modulators are optionally incorporated into assay schemes as controls for modulation of a particular transporter. For example, paraxetine, citalopram, fluxetine, imipramine, amitriptyline, mazindol, cocaine, desipramine, nomifensine, GBR12909, D-amphetamine, L-amphetamine, nortripty
Hodge C. Nicholas
Parce J. Wallace
Wada H. Garrett
Caliper Technologies Corp.
McKenna Donald R.
Ponnaluri Padmashri
Quine Intellectual Property Law Group
Tran My Chau T
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