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Definitions

• NADH dehydrogenase subunit 2 (ND2) has been reported to associate with the tyrosine kinase Src (see Fig. 1A).
• Src is one of several Src family kinases (SFKs) in the NMDAR complex (i.e. Src, Fyn, Lyn and Yes) [5-7].
• Src is involved in the control of many functions, including cell adhesion, growth, movement and differentiation. Src is widely expressed in many cell types, and can have different locations within a cell. It appears that the subcellular location of Src can affect its function.
• Src can associate with cellular membranes, such as the plasma membrane, the perinuclear membrane and the endosomal membrane.
• Figure 10 Quantification of the co-localization of Src and NMDAR 2B in 14DIV hippocampal neurons with and without treatment with Tat-ND2 310-321 or Src 40-49-Tat.
• Figure 11 Quantification of the co-localization of Src and PSD95 in 14DIV hippocampal neurons with and without treatment with Tat-ND2 310-321 or Src 40-49-Tat.
• Figure 19 Sequence of ND2, with predicted membrane topology. The location of 310-321 is highlighted and predicted to be intracellular.
• Excitotoxicity is the pathological process by which neurons are damaged and killed by the overactivation of glutamate receptors, such as NMDA receptors.
• Aromatic rings of a nonnatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
• Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
• Carboxyl side groups e.g., aspartyl or glutamyl
• mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
• a stroke is a condition resulting from impaired blood flow in the CNS regardless of cause.
• Potential causes include embolism, hemorrhage, thrombosis and surgery.
• Some neuronal cells die immediately as a result of impaired blood flow. These cells release their component molecules including glutamate, which in turn activates NMDA receptors, which raise intracellular calcium levels, and intracellular enzyme levels leading to further neuronal cell death (the excitotoxicity cascade).
• the death of tissue from lack of oxygen is referred to as infarction.
• Infarction Volume i.e., the volume of dead neuronal cells resulting from stroke in the brain
• An ischemic stroke refers more specifically to a type of stroke that caused by blockage of blood flow to the brain.
• the underlying condition for this type of blockage is most commonly the development of fatty deposits lining the vessel walls. This condition is called atherosclerosis. These fatty deposits can cause two types of obstruction.
• Cerebral thrombosis refers to a thrombus (blood clot) that develops at the clogged part of the vessel
• Cerebral embolism refers generally to a blood clot that forms at another location in the circulatory system, usually the heart and large arteries of the upper chest and neck.
• suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
• disintegrating agents can be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Effective dose of the present agents can provide benefit without causing substantial toxicity.
• Toxicity of the agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 5 0 (the dose lethal to 5 0% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
• Agents e.g., peptides or peptidomimetics, exhibiting high therapeutic indices are preferred (see, e.g., Fingl et l, 197 , ⁇ : The Pharmacological Basis of Therapeutics, Ch.l, p.l).
• Agents can initially be screened for a desired binding activity, for example, an ability to bind Src, or Src peptide including residues 40-49 of Src.
• an agent can be screened for ability to compete with ND2 or a ND2 peptide as described above (e.g., a peptide consisting of residues 310-321 of ND2) for ability to bind to Src or a peptide including residues 40-49 or 40-58 thereof. Binding can be assessed by ELISA, Fluorescence polarization, or Western blot among other methods. In some formats, one component of such a binding assay is immobilized. Several formats are possible as described in the Examples.
• a positive control can be the Tat-NR2B9c peptide, YGRKKRRQRRRKLSSIESDV (SEQ ID NO:6), previously shown to be effective.
• infarction volumes are usually determined 24 hr post treatment but can be determined at a later time such as 3,7,14 or 60 days.
• Disability index can be monitored over time, e.g., at 2 hr post treatment, 24 hr post treatment, one week and one month post treatment. Agents showing a statistically significant reduction in infarction volume and/or disability index relative to control animals not treated with the agents are identified as having activity useful for practicing the methods of the invention.
• Tests for inflammatory pain and hypersensitivity include the formalin paw test (Tjolsen et al. (1992), Pain 51: 5-17), the complete Freund's Adjuvant paw test (CFA), the test for formalin-induced facial pain (Clavelou et al. (1989), Neurosci. Lett. 103: 349-353), and paw tests upon administration of substances such as carageenan, capsaicin or bradykinin. Arthritic conditions can be simulated by various models, including injection of agents such as carageenan, uric acid or mustard oil or adjuvant into various joints.
• Flurothyl a hexafluorinated ether
• Flurothyl is a chemical inhalant used to induce a reproducible convulsive seizure pattern in rodents.
• rats or mice are placed in an airtight chamber into which centrally administered flurothyl diffuses; after 10-20 min flurothyl initially causes myoclonic jerks followed by severe clonic-tonic convulsions.
• Anxiety can be induced by placing an animal, such as a rat, in an unfamiliar environment and observing a response (e.g., crossing a grid of lines or selecting open or closed tubes).
• rats can be tested in an open field arena to determine both state of arousal and ability to habituate to a novel environment and assessed from crossing gridlines. A reduction in crossings indicates reduced anxiety.
• Rats can also be tested in a maze to assess anxiety/emotionality in rats.
• a suitable maze has 4 arms (two open, two closed: 1 5 cm width and 60 cm length) extending from a central platform and elevated l. 5 m from the floor.
• ND2 310- 321 has a superior ability to provide pain relief in the CFA model (Figure 17), disrupt the association between Src and NMDA R2 subunits better (Figure 13C - IP NR2B in the presence of Tat-ND2 or Src 40-49-Tat, probing with anti-Src), and reduce NMDA currents (Figure 14B).
• Example #6 ND2 INHIBITORS ARE EFFECTIVE AT REDUCING BRAIN
• Peripheral nerve injury (PNI) via the chronic sciatic nerve constriction model was induced in rodents by surgically implanting a polyethylene cuff (2 mm in length) around the sciatic nerve of rats or mice under isoflurane anesthesia.
• PWT mechanical paw withdrawal threshold
• ND2 peptides including myr-ND2, are effective inhibitors of pain.

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