Disclosed are therapeutic treatment methods, compositions and devices for maintaining neural pathways in a mammal, including enhancing survival of neurons at risk of dying, inducing cellular repair of damaged neurons and neural pathways, and stimulating neurons to maintain their differentiated phenotype. In one embodiment, the invention provides means for stimulating CAM expression in neurons. The invention also provides means for evaluating the status of nerve tissue, including means for detecting and monitoring neuropathies in a mammal. The methods, devices and compositions include a morphogen or morphogen-stimulating agent provided to the mammal in a therapeutically effective concentration.

The present invention relates to methods for enhancing the survival of neuronal cells in vivo and to methods, compositions and devices for maintaining neural pathways in vivo. More particularly, the invention provides methods for enhancing survival of neuronal cells at risk of dying, including methods for redifferentiating transformed cells of neural origin and methods for maintaining phenotypic expression of differentiated neuronal cells. The invention also provides means for repairing damaged neural pathways, including methods for stimulating axonal growth over extended distances, and methods for alleviating immunologically-related nerve tissue damage. In a particular embodiment of the invention, this invention provides a method for stimulating cell adhesion molecule expression in cells, and particularly nerve cell adhesion molecule expression in neurons. Finally, the invention provides means for evaluating nerve tissue stasis and identifying neural dysfunction in a mammal.
The mammalian nervous system comprises a peripheral nervous system (PNS) and a central nervous system (CNS, comprising the brain and spinal cord), and is composed of two principal classes of cells: neurons and glial cells. The glial cells fill the spaces between neurons, nourishing them and modulating their function. Certain glial cells, such as Schwann cells in the PNS and oligodendrocytes in the CNS, also provide a protective myelin sheath that surrounds and protects neuronal axons, which are the processes that extend from the neuron cell body and through which the electric impulses of the neuron are transported. In the peripheral nervous system, the long axons of multiple neurons are bundled together to form a nerve or nerve fiber. These, in turn, may be combined into fascicles, wherein the nerve fibers form bundles embedded, together with the intraneural vascular supply, in a loose collagenous matrix bounded by a protective multilamellar sheath. In the central nervous system, the neuron cell bodies are visually distinguishable from their myelin-ensheathed processes, and are referenced in the art as grey and white matter, respectively.
During development, differentiating neurons from the central and peripheral nervous systems send out axons that must grow and make contact with specific target cells. In some cases, growing axons must cover enormous distances; some grow into the periphery, whereas others stay confined within the central nervous system. In mammals, this stage of neurogenesis is complete during the embryonic phase of life and neuronal cells do not multiply once they have fully differentiated.
Accordingly, the neural pathways of a mammal are particularly at risk if neurons are subjected to mechanical or chemical trauma or to neuropathic degeneration sufficient to put the neurons that define the pathway at risk of dying. A host of neuropathies, some of which affect only a subpopulation or a system of neurons in the peripheral or central nervous systems have been identified to date. The neuropathies, which may affect the neurons themselves or the associated glial cells, may result from cellular metabolic dysfunction, infection, exposure to toxic agents, autoimmunity dysfunction, malnutrition or ischemia. In some cases the cellular dysfunction is thought to induce cell death directly. In other cases, the neuropathy may induce sufficient tissue necrosis to stimulate the body""s immune/inflammatory system and the mechanisms of the body""s immune response to the initial neural injury then destroys the neurons and the pathway defined by these neurons.
Currently no satisfactory method exists to repair the damage caused by these neuropathies, which include multiple sclerosis, amyotrophic lateral sclerosis (ALS), Huntington""s chorea, Alzheimer""s disease, Parkinson""s disease (parkinsonism), and metabolically derived disorders, such as hepatic encephalopathy. Current attempts to counteract the effects of severe traumatic or neural degenerative lesions of the brain and/or spinal cord have to date primarily involved implantation of embryonic neurons in an effort to replace functionally, or otherwise compensate for, lost or deficient neurons. Currently, however, human fetal cell transplantation research is severely restricted. Administration of neurotrophic factors such as nerve growth factor and insulin-like growth factor also have been suggested to stimulate neuronal growth within the CNS. (See, for example, Lundborg, (1987) Acta Orthop. Scand. 58:145-169 and U.S. Pat. No. 5,093,317.) Administration of neurotrophic factors to the CNS requires bypassing the blood-brain barrier. The barrier may be overcome by direct infusion, or by modifying the molecule to enhance its transport across the barrier, as by chemical modification or conjugation, or by molecule truncation. Schwann cells also have been grafted to a site of a CNS lesion in an attempt to stimulate and maintain growth of damaged neuronal processes (Paino et al. (1991) Exp. Neurology 114(2):254-257).
Where the damaged neural pathway results from CNS axonal damage, autologous peripheral nerve grafts have been used to bridge lesions in the central nervous system and to allow axons to make it back to their normal target area. In contrast to CNS neurons, neurons of the peripheral nervous system can extend new peripheral processes in response to axonal damage. This regenerative property of peripheral nervous system axons is thought to be sufficient to allow grafting of these segments to CNS axons. Successful grafting appears to be limited, however, by a number of factors, including the length of the CNS axonal lesion to be bypassed, and the distance of the graft sites from the CNS neuronal cell bodies, with successful grafts occurring near the cell body.
Within the peripheral nervous system, this cellular regenerative property of neurons has limited ability to repair function to a damaged neural pathway. Specifically, the new axons extend randomly, and are often misdirected, making contact with inappropriate targets that can cause abnormal function. For example, if a motor nerve is damaged, regrowing axons may contact the wrong muscles, resulting in paralysis. In addition, where severed nerve processes result in a gap of longer than a few millimeters, e.g., greater than 10 millimeters (mm), appropriate nerve regeneration does not occur, either because the processes fail to grow the necessary distance, or because of misdirected axonal growth. Efforts to repair peripheral nerve damage by surgical means have met with mixed results, particularly where damage extends over a significant distance. In some cases, the suturing steps used to obtain proper alignment of severed nerve ends stimulates the formulation of scar tissue which is thought to inhibit axon regeneration. Even where scar tissue formation has been reduced, as with the use of nerve guidance channels or other tubular prostheses, successful regeneration generally still is limited to nerve damage of less than 10 millimeters in distance. In addition, the reparative ability of peripheral neurons is significantly inhibited where an injury or neuropathy affects the cell body itself or results in extensive degeneration of a distal axon.
Mammalian neural pathways also are at risk due to damage caused by neoplastic lesions. Neoplasias of both the neurons and glial cells have been identified. Transformed cells of neural origin generally lose their ability to behave as normal differentiated cells and can destroy neural pathways by loss of function. In addition, the proliferating tumors may induce lesions by distorting normal nerve tissue structure, inhibiting pathways by compressing nerves, inhibiting cerbrospinal fluid or blood supply flow, and/or by stimulating the body""s immune response. Metastatic tumors, which are a significant cause of neoplastic lesions in the brain and spinal cord, also similarly may damage neural pathways and induce neuronal cell death.
One type of morphoregulatory molecule associated with neuronal cell growth, differentiation and development is the cell adhesion molecule (xe2x80x9cCAMxe2x80x9d), most notably the nerve cell adhesion molecule (N-CAM). CAMs belong to the immunoglobulin super-family and mediate cell-cell interactions in developing and adult tissues through homophilic binding, i.e., CAM-CAM binding on apposing cells. A number of different CAMs currently have been identified. Of these, the most thoroughly studied to date are N-CAM and L-CAM (liver cell adhesion molecules), both of which have been identified on all cells at early stages of development, as well as in different adult tissues. In neural tissue development, N-CAM expression is believed to be important in tissue organization, neuronal migration, nerve-muscle tissue adhesion, retinal formation, synaptogenesis, and neural degeneration. Reduced N-CAM expression also is thought to be associated with nerve dysfunction. For example, expression of at least one form of N-CAM, N-CAM-180, is reduced in a mouse dysmyelinating mutant (Bhat (1988) Brain Res. 452:373-377). Reduced levels of N-CAM also have been associated with normal pressure hydrocephalus (Werdelin (1989) Acta Neurol. Scand. 79:177-181), and with type II schizophrenia (Lyons et al., (1988) Biol. Psychiatry 23:769-775.) In addition, antibodies to N-CAM have been shown to disrupt functional recovery in injured nerves (Remsen (1990) Exp. Neurobiol. 110:268-273).
It is an object of this invention to provide methods for enhancing survival of neurons at risk of dying in a mammal. Another object is to provide methods for maintaining neural pathways in vivo at risk of injury, or following damage to nerve tissue due to mechanical or chemical trauma, a neuropathy, or a neoplastic lesion. Another object is to provide compositions and devices for repairing gaps in a neural pathway of the peripheral nervous system. Yet another object is to provide a means for redifferentiating transformed cells defining neural pathways, particularly transformed cells of neural origin. Another object is to provide a means for stimulating CAM expression, particularly N-CAM expression in a cell. Yet another object is to provide methods for monitoring the status of nerve tissue by monitoring fluctuations in protein levels present in nerve tissue, serum and/or cerebrospinal fluid. These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.
The present invention provides methods and compositions for maintaining neural pathways in a mammal in vivo, including methods for enhancing the survival of neural cells.
In one aspect, the invention features compositions and therapeutic treatment methods that comprise the step of administering to a mammal a therapeutically effective amount of a morphogenic protein (xe2x80x9cmorphogenxe2x80x9d), as defined herein, upon injury to a neural pathway, or in anticipation of such injury, for a time and at a concentration sufficient to maintain the neural pathway, including repairing damaged pathways, or inhibiting additional damage thereto.
In another aspect, the invention features compositions and therapeutic treatment methods for maintaining neural pathways in a mammal in vivo which include administering to the mammal, upon injury to a neural pathway or in anticipation of such injury, a compound that stimulates in vivo a therapeutically effective concentration of an endogenous morphogen within the body of the mammal sufficient to maintain the neural pathway, including repairing damaged pathways or inhibiting additional damage thereto. These compounds are referred to herein as morphogen-stimulating agents, and are understood to include substances which, when administered to a mammal, act on tissue(s) or organ(s) that normally are responsible for, or capable of, producing a morphogen and/or secreting a morphogen, and which cause the endogenous level of the morphogen to be altered. The agent may act, for example, by stimulating expression and/or secretion of an endogenous morphogen.
In particular, the invention provides methods for enhancing the survival of neurons at risk of dying, including protecting neurons from the tissue destructive effects associated with the body""s immune/inflammatory response to a nerve injury. The invention also provides methods for stimulating neurons to maintain their differentiated phenotype, including inducing the redifferentiation of transformed cells of neuronal origin to a morphology characteristic of untransformed neurons. In one embodiment, the invention provides means for stimulating production of cell adhesion molecules in cells, particularly nerve cell adhesion molecules (N-CAM) in neurons. The invention also provides methods, compositions and devices for stimulating cellular repair of damaged neurons and neural pathways, including regenerating damaged axons of the peripheral and central nervous systems. In addition, the invention also provides means for evaluating the status of nerve tissue, and for detecting and monitoring neuropathies in a mammal by monitoring fluctuations in the morphogen levels or endogenous morphogen antibody levels present in a mammal""s serum or cerebrospinal fluid.
As used herein, a xe2x80x9cneural pathwayxe2x80x9d describes a nerve circuit for the passage of electric signals from a source to a target cell site. The pathway includes the neurons through which the electric impulse is transported, including groups of interconnecting neurons, the nerve fibers formed by bundled neuronal axons, and the glial cells surrounding and associated with the neurons.
In one aspect of the invention, the morphogens described herein are useful in repairing damaged neural pathways of the peripheral nervous system. In particular, the morphogens are useful for repairing damaged pathways, including transected or otherwise damaged nerve fibers (nerves) requiring regeneration of neuronal processes, particularly axons, over extended distances to bridge a gap in the nerve itself, or between the nerve and a post-synaptic cell. Specifically, the morphogens described herein are capable of stimulating complete axonal nerve regeneration, including vascularization and reformation of the protective myelin sheath. The morphogen preferably is provided to the site of injury dispersed in a biocompatible, bioresorbable carrier material capable of maintaining the morphogen at the site and, where necessary, means for directing axonal growth from the proximal to the distal ends of a severed neuron or nerve. For example, means for directing axonal growth may be required where nerve regeneration is to be induced over an extended distance, such as greater than 10 mm. Many carriers capable of providing these functions are envisioned. For example, useful carriers include substantially insoluble materials or viscous solutions prepared as disclosed herein comprising laminin, hyaluronic acid or collagen, or other suitable synthetic, biocompatible polymeric materials such as polylactic, polyglycolic or polybutyric acids and/or copolymers thereof. The currently preferred carrier comprises an extracellular matrix composition, such as one described herein derived, for example, from mouse sarcoma cells. Also envisioned as especially useful are brain tissue-derived extracellular matrices.
In a particularly preferred embodiment, the morphogen is provided to the site as part of a device wherein the morphogen is disposed in a nerve guidance channel which spans the distance of the damaged pathway. The channel acts both as a protective covering and a physical means for guiding growth of a neuronal process such as an axon. Useful channels comprise a biocompatible membrane or casing, which may be tubular in structure, having a dimension sufficient to span the gap or break in the nerve to be repaired, and having openings adapted to receive severed nerve ends. The casing or membrane may be made of any biocompatible, nonirritating material, such as silicone or a biocompatible polymer such as polyethylene or polyethylene vinyl acetate. The casing also may be composed of biocompatible, bioresorbable polymers, including, for example, collagen, hyaluronic acid, polylactic, polybutyric and polyglycolic acids. In a currently preferred embodiment, the outer surface of the channel is substantially impermeable.
The morphogen may be disposed in the channel in association with a biocompatible carrier material, or it may be adsorbed to or otherwise associated with the inner surface of the casing, such as is described in U.S. Pat. No. 5,011,486, provided that the morphogen is accessible to the severed nerve ends. Additionally, although the nerve guidance channels described herein generally are tubular in shape, it should be evident to those skilled in the art that various alternative shapes may be employed. The lumen of the guidance channels may, for example, be oval or even square in cross section. Moreover the guidance channels may be constructed of two or more parts which may be clamped together to secure the nerve stumps. Nerve endings may be secured to the nerve guidance channels by means of sutures, biocompatible adhesives such as fibrin glue, or other means known in the medical art.
The morphogens described herein also are envisioned to be useful in autologous peripheral nerve segment implants to bypass damaged neural pathways in the central nervous system, such as in the repair of damaged or detached retinas, or other damage to the optic nerve. Here the morphogen is provided to the site of attachment to stimulate axonal growth at the graft site, particularly where the damaged axonal segment to be bypassed occurs far from the neuronal cell body.
The morphogens described herein also are useful for enhancing survival of neuronal cells at risk of dying, thereby preventing, limiting or otherwise inhibiting damage to neural pathways. Non-mitotic neurons are at risk of dying as a result of a neuropathy or other cellular dysfunction of a neuron or glial cell inducing cell death, or following a chemical or mechanical lesion to the cell or its surrounding tissue. The chemical lesions may result from known toxic agents, including lead, ethanol, ammonia, formaldehyde and many other organic solvents, as well as the toxins in cigarette smoke and opiates. Excitatory amino acids, such as glutamate also may play a role in the pathogenesis of neuronal cell death (see Freese et al. (1990) Brain Res. 521:254-264). Neuronal cell death also is thought to be a significant contributing factor in a number of neurodegenerative diseases, including Alzheimer""s disease, Huntington""s chorea, and Parkinson""s disease, amyotrophic lateral sclerosis and multiple sclerosis. The etiology of these neuropathies may be metabolic, as results in hepatic encephalopathy, infectious, toxic, autoimmune, nutritional or ischemic. In addition, ethanol and a number of other toxins also have been identified as significant contributing factors in neurodegenerative diseases. The morphogens described herein may be provided to cells at risk of dying to enhance their survival and thereby protect the integrity of the neural pathway. The morphogens may be provided directly to the site, or they may be provided systemically. Alternatively, as described above, an agent capable of stimulating endogenous morphogen expression and/or secretion, preferably in cells associated with the nerve tissue of interest, may be administered to the mammal.
In another aspect of the invention, the method disclosed is useful for redifferentiating transformed cells, particularly transformed cells of neuronal or glial origin, such that the morphogen-treated cells are induced to display a morphology characteristic of untransformed cells. Where the transformed cells are cells of neuronal origin, morphogen treatment preferably induces cell rounding and cell aggregation (clumping), cell-cell adhesion, neurite outgrowth formation and elongation, and N-CAM production. The methods described herein are anticipated to substantially inhibit or reduce neural cell tumor formation and/or proliferation in nerve tissue. It is anticipated that the methods of this invention will be useful in substantially reducing the effects of various carcinomas of nerve tissue origin such as retinoblastomas, neuroblastomas, and gliomas or glioblastomas. In addition, the method also is anticipated to aid in inhibiting neoplastic lesions caused by metastatic tissue. Metastatic tumors are one of the most common neoplasms of the CNS, as they can reach the intracranial compartment through the bloodstream. Metastatic tumors may damage neural pathways for example, by distorting normal nerve tissue structure, compressing nerves, blocking flow of cerebrospinal fluid or the blood supply nourishing brain tissue, and/or by stimulating the body""s immune response.
In another aspect of the invention, the morphogens described herein are useful for providing neuroprotective effects to alleviate neural pathway damage associated with the body""s immune/inflammatory response to an initial injury to nerve tissue. Such a response may follow trauma to nerve tissue, caused, for example, by an autoimmune dysfunction, neoplastic lesion, infection, chemical or mechanical trauma, disease, by interruption of blood flow to the neurons or glial cells, for example following ischemia or hypoxia, or by other trauma to the nerve or surrounding material. For example, the primary damage resulting from hypoxia or ischemia-reperfusion following occlusion of a neural blood supply, as in an embolic stroke, is believed to be immunologically associated. In addition, at least part of the damage associated with a number of primary brain tumors also appears to be immunologically related. Application of the morphogen directly to the cells to be treated, or providing the morphogen to the mammal systemically, for example, intravenously or indirectly by oral administration, may be used to alleviate and/or inhibit the immunologically related response to a neural injury. Alternatively, administration of an agent capable of stimulating morphogen expression and/or secretion in vivo, preferably at the site of injury, also may be used. Where the injury is to be induced, as during surgery or other aggressive clinical treatment, the morphogen or agent may be provided prior to induction of the injury to provide a neuroprotective effect to the nerve tissue at risk.
In still another aspect, the invention described herein provides methods for supporting the growth and maintenance of differentiated neurons, including inducing neurons to continue expressing their phenotype. It is anticipated that this activity will be particularly useful in the treatment of nerve tissue disorders where loss of function is caused by reduced or lost cellular metabolic function and cells become senesent or quiescent, such as is thought to occur in aging cells and to be manifested in Alzheimer""s disease. Application of the morphogen directly to cells to be treated, or providing it systemically by parenteral or oral administration stimulates these cells to continue expressing their phenotype, significantly inhibiting and/or reversing the effects of the cellular metabolic dysfunction, thereby maintaining the neural pathway at risk. Alternatively, administration of an agent capable of stimulating endogenous morphogen expression and/or secretion in vivo may be used.
In still another aspect, the invention provides methods for stimulating CAM expression levels in a cell, particularly N-CAM expression in neurons. CAMs are molecules defined as carrying out cell-cell interactions necessary for tissue formation. CAMs are believed to play a fundamental regulatory role in tissue development, including tissue boundary formation, embryonic induction and migration, and tissue stabilization and regeneration. Altered CAM levels have been implicated in a number of tissue disorders, including congenital defects, neoplasias, and degenerative diseases.
In particular, N-CAM expression is associated with normal neuronal cell development and differentiation, including retinal formation, synaptogenesis, and nerve-muscle tissue adhesion. Inhibition of one or more of the N-CAM isoforms is known to prevent proper tissue development. Altered N-CAM expression levels also are associated with neoplasias, including neuroblastomas (see infra), as well as with a number of neuropathies, including normal pressure hydrocephalous and type II schizophrenia. Application of the morphogen directly to the cells to be treated, or providing the morphogen to the mammal systemically, for example, parenterally, or indirectly by oral administration, may be used to induce cellular expression of one or more CAMs, particularly N-CAMs. Alternatively, administration of an agent capable of stimulating morphogen expression and/or secretion in vivo, preferably at the site of injury, also may be used to induce CAM production.
CAMs also have been postulated as part of a morphoregulatory pathway whose activity is induced by a to date unidentified molecule (See, for example, Edelman, G. M. (1986) Ann. Rev. Cell Biol. 2:81-116). Without being limited to any given theory, the morphogens described herein may act as the inducer of this pathway.
Finally, modulations of endogenous morphogen levels may be monitored as part of a method of detecting nerve tissue dysfunction. Specifically, modulations in endogenous morphogen levels are anticipated to reflect changes in nerve tissue status. Morphogen expression may be monitored directly in biopsied cell samples, in cerebrospinal fluid, or serum. Alternatively, morphogen levels may be assessed by detecting changes in the levels of endogenous antibodies to the morphogen. For example, one may obtain serum samples from a mammal, and then detect the concentration of morphogen or antibody present in the fluid by standard protein detection means known to those skilled in the art. As an example, binding protein capable of interacting specifically with the morphogen of interest such as an anti-morphogen antibody may be used to detect a morphogen in a standard immunoassay. The morphogen levels detected then may be compared to a previously determined standard or reference level, with changes in the detected levels being indicative of the status of the tissue.
In one preferred embodiment of the invention, the morphogen or morphogen-stimulating agent is administered systemically to the individual, e.g., orally or parenterally. In another embodiment of the invention, the morphogen may be provided directly to the nerve tissue, e.g., by injection to the cerebral spinal fluid or to a nerve tissue locus.
In any treatment method of the invention, xe2x80x9cadministration of morphogenxe2x80x9d refers to the administration of the morphogen, either alone or in combination with other molecules. For example, the mature form of the morphogen may be provided in association with its precursor xe2x80x9cproxe2x80x9d domain, which is known to enhance the solubility of the protein. Other useful molecules known to enhance protein solubility include casein and other milk components, as well as various serum proteins. Additional useful molecules which may be associated with the morphogen or morphogen-stimulating agent include tissue targeting molecules capable of directing the morphogen or morphogen-stimulating agent to nerve tissue. Tissue targeting molecules envisioned to be useful in the treatment protocols of this invention include antibodies, antibody fragments or other binding proteins which interact specifically with surface molecules on nerve tissue cells.
Still another useful tissue targeting molecule is part or all of the morphogen precursor xe2x80x9cproxe2x80x9d domain, particularly that of OP-1 or GDF-1. These proteins are found naturally associated with nerve tissue but also may be synthesized in other tissues and targeted to nerve tissue after secretion from the synthesizing tissue. For example, while the protein has been shown to be active in bone tissue, the primary source of OP-1 synthesis appears to be the tissue of the urogenic system (e.g., renal and bladder tissue), with secondary expression levels occurring in the brain, heart and lungs (see below.) Moreover, the protein has been identified in serum, saliva and various milk forms. In addition, the secreted form of the protein comprises the mature dimer in association with the pro domain of the intact morphogen sequence. Accordingly, the associated morphogen pro domains may act to target specific morphogens to different tissues in vivo.
Associated tissue targeting or solubility-enhancing molecules also may be covalently linked to the morphogen using standard chemical means.
Finally, the morphogens or morphogen-stimulating agents provided herein also may be administered in combination with other molecules known to be beneficial in maintaining neural pathways, including, for example, nerve growth factors and anti-inflammatory agents.
Where the morphogen is intended for use as a therapeutic for disorders of the CNS, an additional problem must be addressed: overcoming the so-called xe2x80x9cblood-brain barrierxe2x80x9d, the brain capillary wall structure that effectively screens out all but selected categories of molecules present in the blood, preventing their passage into the brain. The blood-brain barrier may be bypassed effectively by direct infusion of the morphogen or morphogen- stimulating agent into the brain. Alternatively, the morphogen or morphogen-stimulating agent may be modified to enhance its transport across the blood-brain barrier. For example, truncated forms of the morphogen or a morphogen-stimulating agent may be most successful. Alternatively, the morphogen or morphogen-stimulating agent may be modified to render it more lipophilic, or it may be conjugated to another molecule which is naturally transported across the barrier, using standard means known to those skilled in the art, as, for example, described in Pardridge, Endocrine Reviews 7:314-330 (1986) and U.S. Pat. No. 4,801,575.
Accordingly, as used herein, a functional xe2x80x9canalogxe2x80x9d of a morphogen refers to a protein having morphogenic biological activity but possessing additional structural differences compared to a morphogen as defined herein, e.g., having additional chemical moieties not normally a part of a morphogen. Such moieties (introduced, for example, by acylation, alkylation, cationization, or glycosylation reactions, or other means for conjugating the moiety to the morphogen) may improve the molecule""s solubility, absorption, biological half-life, or transport, e.g., across the blood-brain barrier.
Among the morphogens useful in this invention are proteins originally identified as osteogenic proteins, such as the OP-1, OP-2 and CBMP2 proteins, as well as amino acid sequence-related proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, see Lee (1991) PNAS 88:4250-4254), all of which are presented in Table II and Seq. ID Nos.5-14), and the recently identified 60A protein (from Drosophila, Seq. ID No. 24, see Wharton et al. (1991) PNAS 88:9214-9218.) The members of this family, which include members of the TGF-xcex2 super-family of proteins, share substantial amino acid sequence homology in their C-terminal regions. The proteins are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a xe2x80x9cproxe2x80x9d domain that is cleaved to yield the mature sequence. The signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14:4683-4691.) Table I, below, describes the various morphogens identified to date, including their nomenclature as used herein, their Seq. ID references, and publication sources for the amino acid sequences for the full length proteins not included in the Seq. Listing. The disclosure of these publications is incorporated herein by reference.
xe2x80x9cOP-1xe2x80x9d
Refers generically to the group of morphogenically active proteins expressed from part or all of a DNA sequence encoding OP-1 protein, including allelic and species variants thereof, e.g., human OP-1 (xe2x80x9chOP-1xe2x80x9d, Seq. ID No. 5, mature protein amino acid sequence), or mouse OP-1 (xe2x80x9cmOP-1xe2x80x9d, Seq. ID No. 6, mature protein amino acid sequence.) The conserved seven cysteine skeleton is defined by residues 38 to 139 of Seq. ID Nos. 5 and 6. The cDNA sequences and the amino acids encoding the full length proteins are provided in Seq. Id Nos. 16 and 17 (hOP1) and Seq. ID Nos. 18 and 19 (mOP1.) The mature proteins are defined by residues 293-431 (hOP1) and 292-430 (mOP1). The xe2x80x9cproxe2x80x9d regions of the proteins, cleaved to yield the mature, morphogenically active proteins are defined essentially by residues 30-292 (hOP1) and residues 30-291 (mOP1).
xe2x80x9cOP-2xe2x80x9d
refers generically to the group of active proteins expressed from part or all of a DNA sequence encoding OP-2 protein, including allelic and species variants thereof, e.g., human OP-2 (xe2x80x9chOP-2xe2x80x9d, Seq. ID No. 7, mature protein amino acid sequence) or mouse OP-2 (xe2x80x9cmOP-2xe2x80x9d, Seq. ID No. 8, mature protein amino acid sequence). The conserved seven cysteine skeleton is defined by residues 38 to 139 of Seq. ID Nos. 7 and 8. The cDNA sequences and the amino acids encoding the full length proteins are provided in Seq. ID Nos. 20 and 21 (hOP2) and Seq. ID Nos. 22 and 23 (mOP2.) The mature proteins are defined essentially by residues 264-402 (hOP2) and 261-399 (mOP2). The xe2x80x9cproxe2x80x9d regions of the proteins, cleaved to yield the mature, morphogenically active proteins likely are defined essentially by residues 18-263 (hOP2) and residues 18-260 (mOP2). (Another cleavage site also occurs 21 residues upstream for both OP-2 proteins.)
xe2x80x9cCBMP2xe2x80x9d
refers generically to the morphogenically active proteins expressed from a DNA sequence encoding the CBMP2 proteins, including allelic and species variants thereof, e.g., human CBMP2A (xe2x80x9cCBMP2A(fx)xe2x80x9d, Seq ID No. 9) or human CBMP2B DNA (xe2x80x9cCBMP2B(fx)xe2x80x9d, Seq. ID No. 10). The amino acid sequence for the full length proteins, referred to in the literature as BMP2A and BMP2B, or BMP2 and BMP4, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro domain for BMP2 (BMP2A) likely includes residues 25-248 or 25-282; the mature protein, residues 249-396 or 283-396. The pro domain for BMP4 (BMP2B) likely includes residues 25-256 or 25-292; the mature protein, residues 257-408 or 293-408.
xe2x80x9cDPP(fx)xe2x80x9d
refers to protein sequences encoded by the Drosophila DPP gene and defining the conserved seven cysteine skeleton (Seq. ID No. 11). The amino acid sequence for the full length protein appears in Padgett, et al (1987) Nature 325: 81-84. The pro domain likely extends from the signal peptide cleavage site to residue 456; the mature protein likely is defined by residues 457-588.
xe2x80x9cVgl(fx)xe2x80x9d
refers to protein sequences encoded by the Xenopus Vgl gene and defining the conserved seven cysteine skeleton (Seq. ID No. 12). The amino acid sequence for the full length protein appears in Weeks (1987) Cell 51: 861-867. The prodomain likely extends from the signal peptide cleavage site to residue 246; the mature protein likely is defined by residues 247-360.
xe2x80x9cVgr-1 (fx)xe2x80x9d
refers to protein sequences encoded by the murine Vgr-1 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 13). The amino acid sequence for the full length protein appears in Lyons, et al, (1989) PNAS 86: 4554-4558. The prodomain likely extends from the signal peptide cleavage site to residue 299; the mature protein likely is defined by residues 300-438.
xe2x80x9cGDF-1 (fx)xe2x80x9d
refers to protein sequences encoded by the human GDF-1 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 14). The cDNA and encoded amino sequence for the full length protein is provided in Seq. ID. No. 32. The prodomain likely extends from the signal peptide clavage site to residue 214; the mature protein likely is defined by residues 215-372.
xe2x80x9c60Axe2x80x9d
refers generically to the morphogenically active proteins expressed from part or all of a DNA sequence (from the Drosophila 60A gene) encoding the 60A proteins (see Seq. ID No. 24 wherein the cDNA and encoded amino acid sequence for the full length protein is provided). xe2x80x9c60A(fx)xe2x80x9d refers to the protein sequences defining the conserved seven cysteine skeleton (residues 354 to 455 of Seq. ID No. 24.) The prodomain likely extends from the signal peptide cleavage site to residue 324; the mature protein likely is defined by residues 325-455.
xe2x80x9cBMP3(fx)xe2x80x9d
refers to protein sequences encoded by the human BMP3 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 26). The amino acid sequence for the full length protein appears in Wozney et al. (1988) Science 242: 1528-1534. The pro domain likely extends from the signal peptide cleavage site to residue 290; the mature protein likely is defined by residues 291-472.
xe2x80x9cBMP5(fx)xe2x80x9d
refers to protein sequences encoded by the human BMP5 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 27). The amino acid sequence for the full length protein appears in Celeste, et al. (1991) PNAS 87: 9843-9847. The pro domain likely extends from the signal peptide cleavage site to residue 316; the mature protein likely is defined by residues 317-454.
xe2x80x9cBMP6(fx)xe2x80x9d
refers to protein sequences encoded by the human BMP6 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 28). The amino acid sequence for the full length protein appear sin Celeste, et al. (1990) PNAS 87: 9843-5847. The pro domain likely includes extends from the signal peptide cleavage site to residue 374; the mature sequence likely includes residues 375-513.
The OP-2 proteins have an additional cysteine residue in this region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in addition to the conserved cysteine skeleton in common with the other proteins in this family. The GDF-1 protein has a four amino acid insert within the conserved skeleton (residues 44-47 of Seq. ID No. 14) but this insert likely does not interfere with the relationship of the cysteines in the folded structure. In addition, the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton.
The morphogens are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with other morphogens of this invention. Thus, as defined herein, a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 43-139 of Seq. ID No. 5, including functionally equivalent arrangements of these cysteines (e.g., amino acid insertions or deletions which alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure), such that, when the polypeptide chains are folded, the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra- or inter-chain disulfide bonds such that the protein is capable of acting as a morphogen as defined herein. Specifically, the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells, including the xe2x80x9credifferentiationxe2x80x9d of transformed cells. In addition, it is also anticipated that these morphogens are capable of inducing redifferentiation of committed cells under appropriate environmental conditions.
In one preferred aspect, the morphogens of this invention comprise one of two species of generic amino acid sequences: Generic Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID No. 2); where each Xaa indicates one of the 20 naturally-occurring L-isomer, a-amino acids or a derivative thereof. Generic Sequence 1 comprises the conserved six cysteine skeleton and Generic Sequence 2 comprises the conserved six cysteine skeleton plus the additional cysteine identified in OP-2 (see residue 36, Seq. ID No. 2). In another preferred aspect, these sequences further comprise the following additional sequence at their N-terminus:
Preferred amino acid sequences within the foregoing generic sequences include: Generic Sequence 3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. 4), Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31), listed below. These Generic Sequences accommodate the homologies shared among the various preferred members of this morphogen family identified in Table II, as well as the amino acid sequence variation among them. Specifically, Generic Sequences 3 and 4 are composite amino acid sequences of the following proteins presented in Table II and identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14.) The generic sequences include both the amino acid identity shared by the sequences in Table II, as well as alternative residues for the variable positions within the sequence. Note that these generic sequences allow for an additional cysteine at position 41 or 46 in Generic Sequences 3 or 4, respectively, providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids which influence the tertiary structure of the proteins.
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: xe2x80x9cRes.xe2x80x9d means xe2x80x9cresiduexe2x80x9d and Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or Val); Xaa at res.11 (Gln, Leu, Asp, His or Asn); Xaa at res.12=(Asp, Arg or Asn); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp, Ala or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at res.50=(His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala or Val); Xaa at res.53=(Asn, Lys, Ala or Glu); Xaa at res.54=(Pro or Ser); Xaa at res.55=(Glu, Asp, Asn, or Gly); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys or Leu); Xaa at res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr or Ala); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser or Asp); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr or Val); Xaa at res.71=(Ser or Ala); Xaa at res.72=(Val or Met); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr or Leu); Xaa at res.76=(Asp or Asn); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn or Tyr); Xaa at res.79=(Ser, Asn, Asp or Glu); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile or Val); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln or His); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln or Glu); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr or Ala); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly or Glu); and Xaa at res.97=(His or Arg);
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: xe2x80x9cRes.xe2x80x9d means xe2x80x9cresiduexe2x80x9d and Xaa at res.2=(Lys or Arg); Xaa at res.3=(Lys or Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu, Ser, His, Gly, Arg or Pro); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at res.13=(Leu or Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa at res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at res.54=(Val or Met); Xaa at res.55=(His or Asn); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala or Val); Xaa at res.58=(Asn, Lys, Ala or Glu); Xaa at res.59=(Pro or Ser); Xaa at res.60=(Glu, Asp, or Gly); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln or Glu); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr or Ala); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly or Glu); and Xaa at res.102=(His or Arg).
Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among all the morphogen protein family members identified in Table II. Specifically, Generic Sequences 5 and 6 are composite amino acid sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14), human BMP3 (Seq. ID No. 26), human BMP5 (Seq. ID No. 27), human BMP6 (Seq. ID No. 28) and 60(A) (from Drosophila, Seq. ID Nos. 24-25). The generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeltons (Generic Sequences 5 and 6, respectively), as well as alternative residues for the variable positions within the sequence. As for Generic Sequences 3 and 4, Generic Sequences 5 and 6 allow for an additional cysteine at position 41 (Generic Sequence 5) or position 46 (Generic Sequence 6), providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and containing certain critical amino acids which influence the tertiary structure of the proteins.
wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: xe2x80x9cRes.xe2x80x9d means xe2x80x9cresiduexe2x80x9d and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp, Glu or Lys); Xaa at res.8=(Leu, Val or Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn or Glu); Xaa at res.14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or Ser); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln or Asn); Xaa at res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu or Ile); Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val or Leu); Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val or Lys); Xaa at res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro or Ala); Xaa at res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa at res.72=(Val, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His or Val); Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa at res.97=(His or Arg).
wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: xe2x80x9cRes.xe2x80x9d means xe2x80x9cresiduexe2x80x9d and Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at res.7=(Tyr or Lys); Xaa at res.8=(Val or Ile); Xaa at res.9=(Ser, Asp or Glu); Xaa at res.11=(Arg, Gln, Ser, Lys or Ala); Xaa at res.12=(Asp, Glu, or Lys); Xaa at res.13=(Leu, Val or Ile); Xaa at res.16=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.17=(Asp, Arg, Asn or Glu); Xaa at res.19=(Ile or Val); Xaa at res.20=(Ile or Val); Xaa at res.21=(Ala or Ser); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.24=(Gly or Ser); Xaa at res.25=(Tyr or Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Gln, Leu, or Gly); Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.33=Glu, Lys, Asp, Gln or Ala); Xaa at res.35=(Ala, Ser, Pro, Gln or Asn); Xaa at res.36=(Phe, Leu or Tyr); Xaa at res.38=(Leu, Val or Met); Xaa at res.39=(Asn, Asp, Ala, Thr or Pro); Xaa at res.40 (Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.41=(Tyr, Cys, His, Ser or Ile); kaa at res.42=(Met, Phe, Gly or Leu); Xaa at res.43=(Asn, Ser or Lys); Xaa at res.44=(Ala, Ser, Gly or Pro); Xaa at res.45=(Thr, Leu or Ser); Xaa at res.49=(Ile, Val or Thr); Xaa at res.50=(Val, Leu or Ile); Xaa at res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser.); Xaa at res.53=(Leu or Ile); Xaa at res.54=(Val or Met); Xaa at res.55=(His, Asn or Arg); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57=(Ile, Met, Asn, Ala, Val or Leu); Xaa at res.58=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.59=(Pro, Ser or Val); Xaa at res.60=(Glu, Asp, Gly, Val or Lys); Xaa at res.61=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.62=.(Val,. Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at res.64=(Lys, Leu or Glu); Xaa at res.65=(Pro or Ala.); Xaa at res.68=(Ala or Val); Xaa at res.70=(Thr, Ala or Glu); Xaa at res.71=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser, Asp or Gly); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr, Val or Leu); Xaa at res.76=(Ser, Ala or Pro); Xaa at res.77=(Val, Met or Ile); Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr, Leu or His); Xaa at res.81=(Asp, Asn or Leu); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=.(Ser, Gln, Asn, Tyr or Asp); Xaa at res.84=(Ser, Asn, Asp, Glu or Lys); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile, Val or Asn); Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln, His or Val); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln, Glu or Pro); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr, Ala or Ile); Xaa at res.97=(Arg, Lys, Val, Asp or Glu); Xaa at res.98=(Ala, Gly, Glu or Ser); Xaa at res.100=(Gly or Ala); and Xaa at res.102=(His or Arg).
Particularly useful sequences for use as morphogens in this invention include the-C-terminal domains, e.g., the C-terminal 96-102 amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as well as proteins comprising the C-terminal domains of 60A, BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at least the conserved six or seven cysteine skeleton. In addition, biosynthetic constructs designed from the generic sequences, such as COP-1, 3-5, 7, 16, disclosed in U.S. Pat. No. 5,011,691, also are useful. Other sequences include the inhibins/activin proteins (see, for example, U.S. Pat. Nos. 4,968,590 and 5,011,691). Accordingly, other useful sequences are those sharing at least 70% amino acid sequence homology or xe2x80x9csimilarityxe2x80x9d, and preferably 80% homology or similarity with any of the sequences above. These are anticipated to include allelic and species variants and mutants, and biosynthetic muteins, as well as novel members of this morphogenic family of proteins. Particularly envisioned in the family of related proteins are those proteins exhibiting morphogenic activity and wherein the amino acid changes from the preferred sequences. include conservative changes, e.g., those as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol. 5, Suppl. 3, pp. 345-362, (M. O. Dayoff, ed., Nat""l BioMed. Research Fdn., Washington, D.C. 1979). As used herein, potentially useful sequences are aligned with a known morphogen sequence using the method of Needleman et al. ((1970) J.Mol.Biol. 48:443-453) and identities calculated by the Align program (DNAstar, Inc.). xe2x80x9cHomologyxe2x80x9d or xe2x80x9csimilarityxe2x80x9d as used herein includes allowed conservative changes as defined by Dayoff et al.
The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 (e.g., residues 43-139 of Seq. ID No. 5). These most preferred sequences include both allelic and species variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in another preferred aspect of the invention, useful morphogens include active proteins comprising species of polypeptide chains having the generic amino acid sequence herein referred to as xe2x80x9cOPXxe2x80x9d, which accommodates the homologies between the various identified species of OP1 and OP2 (Seq. ID No. 29).
The morphogens useful in the methods, composition and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and species variants of these proteins, naturally-occurring or biosynthetic mutants thereof, as well as various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein. The proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.
The morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions. Currently preferred host cells include E. coli or mammalian cells, such as CHO, COS or BSC cells. A detailed description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in copending U.S. patent application Ser. Nos. 752,764, filed Aug. 30, 1991, and 667,274, filed Mar. 11, 1991, the disclosure of which are incorporated herein by reference.
Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins capable of maintaining neural pathways in a mammal, including enhancing the survival of neurons at risk of dying and stimulating nerve regeneration and repair in a variety of mammals, including humans.
The foregoing and other objects, features and advantages of the present invention will be made more apparent from the following detailed description of the invention.

Morphogen-enhanced survival and repair of neural cells


The injection system has a pump for supplying high-pressure fuel to a storage volume for supplying a number of injectors. The pump has at least one reciprocating pumping element performing a compression stroke synchronously with each fuel injection. The injection system has at least one control device for controlling the amount of fuel supplied by the pump to the storage volume, and which is controlled by a chopper control unit to modulate the fuel volume introduced into the compression chamber during the intake stroke (Ps-Pi). The delivery start instant of the pumping element during the compression stroke being unequivocally defined by the fuel volume introduced.

1. Field of the Invention
The present invention relates to an internal combustion engine storage-volume fuel injection system.
2. Description of the Related Art
Modern internal combustion engine fuel injection systems normally comprise a pump for supplying high-pressure fuel to a common rail having a given fuel storage volume and for supplying a number of engine cylinder injectors. The pump comprises at least one reciprocating pumping element performing each time an intake stroke and a compression or delivery stroke.
As is known, for it to be atomized properly, the fuel must be brought to extremely high pressure, e.g. in the region of 1600 bars in maximum engine load conditions. Current regulations governing pollution by engine exhaust gas require that the fuel feed pressure to the injectors be reproducible as accurately as possible with respect to the electronic central control unit map. Pressure fluctuations, in the common rail, with respect to the set pressure can be limited if the volume of the common rail is over order of magnitude of the fuel quantity drawn by each injector per combustion cycle. Such a common rail, however, is invariably bulky and therefore difficult to accommodate on the engine.
To control pressure in the common rail as mapped in the central control unit, it has been proposed to fit, along the pump delivery conduit to the common rail, a bypass solenoid valve controlled by an electronic unit as a function of various engine operating parameters. It has also been proposed to operate the pumping element by means of cam operating synchronously with each injector.
In known systems of this sort, each pumping element has an instantaneous flow, the maximum value of which is less than the maximum value of each injector, so that, during each injection, only part of the injected fuel, about 20%, is normally supplied by the pump, the rest being supplied by the common rail. Systems of this sort therefore have the drawback of necessarily requiring a common rail of suitable size. Moreover, the pump operates permanently at the maximum flow rate, while the bypass solenoid valves simply provides for draining the surplus pumped fuel, in excess of that drawn by the injectors, into the tank, thus dissipating heat.

Internal combustion engine storage-volume fuel injection system


A pharmaceutical preparation for topical treatment of skin disorders, particularly psoriasis, comprising the ingredients of a latex extracted from the leaves of the Wrightia tinctoria R Br. plant, urea, and polyethylene glycol. The pharmaceutical preparation is also a hydrophilic ointment that is capable of delivering the active drugs without being greasy or irritating to the skin.

Psoriasis is among the dermatoses having a poorly understood etiology. The ailment is a chronic skin disease usually occurring in early to middle age and affecting approximately 2% of the US population. Psoriasis is characterized by epidermal hyperplasia and a greatly accelerated rate of epidermal turnover. The lesions are characteristically dry, well demarcated, red, slightly raised and scaly. Gentle scraping of the lesion removes scales and produces many pinpoint bleeding sites, the so-called "Auspitz sign". The lesions are discrete or confluent erythromatous plaques and papules covered with white or silvery scales found on the extensor surfaces such as the elbows, knees, back and scalp. No part of the body is exempt. The lesions may be localized or generalized.
The therapies of choice are generally limited to the topical application of fluorinated corticosteriods, keratolytics containing salicylates, coal tar, anthralin preparations and systemic therapy with corticosteroids and methotrexate. Methotrexate and drugs like it have severe side effects such as bone marrow depression and thus their therapeutic benefits must be weighed against potentially dangerous side effects. In addition, the formulations to be used are limited to the most common preparations such as, solutions, creams, and salves. Consequently, the manner of application for the dermatherapy is practically predetermined.
Typically the only pharmaceutical forms in which the therapeutic agents are available are lipophilic preparations for local therapy, due to the high instability of the active ingredients against air, oxygen, water, and alkalis. The strongly fattening lipophilic preparations have relatively low storage stability. Such preparations are necessarily inconvenient to use because the hydrophobic preparations are difficult to wash off.
A hydrophilic pharmaceutical preparation capable of delivering a therapeutic agent for the treatment of psoriasis is needed. The preparation would also need to have a high storage stability, and be innocuous, and non irritating to the skin. A therapeutic agent that is nontoxic and effective in treating psoriasis is also needed.

Herbal medication for the treatment of psoriasis


A spirogyra controlling and deodorant composition contains the cortex of Japanese cedar and/or Japanese cypress or an extract thereof. This serves to suppress or prevent the occurrence of spirogyra and maladous odor in water or on moist surface in fish rearing aquarium, pond, marsh, water culture or the like.

1. Field of the Invention
The present invention relates to a spirogyra controlling and deodorant composition that serves well for the purpose of suppressing or preventing the occurrence of spirogyra and maladous odor in water or on moist surface in fish rearing aquarium, water culture and by mulching pond and marsh, etc.
2. Prior art
In fish rearing, there is no effective means of controlling spirogyra and preventing maladous odor.
In water culture, there is a prior art method of controlling spirogyra, as illustrated in FIG. 3.
In this method, artificial urethane resin medium (2) is placed above a solution chamber (1); solution (3) is circulated by pumping it from a solution tank (4) to the solution chamber (1) by means of a circulatory pump (8); solution (3) is then fed through the solution chamber (1) to a grown plant (6) retained by the net-like retainer (5) of artificial medium (2), wherein the solution surface around the base of the grown plant (6) is covered with wool pills (7) for shading, to prevent the photosynthesis of spirogyra and thus suppress its occurrence.
The prior art method described above poses the following problems, 1) through 3).
1) The occurrence of spirogyra is suppressed only around the shaded base portion. Spirogyra that has occurred at the unshaded portion midway in the circulation path of the solution (3) is transferred, due to the circulation of solution (3), and clings to the net-like retainer (5) of artificial medium (2), adding maladous odor to the grown plant (6).
2) To remove spirogyra clinging to the net-like retainer (5) of artificial medium (2), it is necessary to remove the artificial medium (2) from the solution chamber (1). Also, clogging of the circulation path of the solution (3) by spirogyra necessitates disassembly of the circulation path unit. These operations hamper easy cleaning.
3) Since wool pills (7) are degreased and begin to absorb water in about 24 hours, and since spirogyra occurs on the surface of the pills (7) that have absorbed water, the pills must be renewed frequently, an undesirable requirement in that maintenance is troublesome and cost is high.
The object of the present invention is to provide a spirogyra controlling and deodorant composition capable of perfectly controlling spirogyra and maladous odor and retaining its controlling performance for a long time.

Spirogyra controlling and deodorant composition


A connecting system includes a connecting apparatus for connecting selectively a power source of a power supply circuit to a battery or to an electronic apparatus. When a turn-over switch is switched to the electronic apparatus, the information signals is transmitted between the connecting means and the electronic apparatus.

The present invention relates to a connecting system which is adaptable in supplying a power source to, for example, a video tape recorder having a camera provided integrally therewith and in connecting the video tape recorder to various video apparatuses.
In a conventional video tape recorder having a camera provided integrally therewith, input/output terminals for video signal and audio signal are provided such that a recorded content can be monitored on an available television receiver.
However, when a body of such a video tape recorder having a camera provided integrally therewith is connected to a television receiver, the connecting procedure is troublesome, causing the connection to tend to be erroneous.

Connecting system for supplying electrical power to a device in place of a battery along with the transmission of data signals from the device to monitoring apparatus, or supplying electrical power to a battery for the charging thereof


A frequency converter circuit for a double-fed asynchronous generator with a variable power output, which can be connected to a voltage network contains a rotor rectifier, which can be connected to the rotor of the asynchronous generator, a network frequency converter, which can be connected to the voltage network, and an intermediate circuit. The intermediate circuit contains a semiconductor switch arranged on the rotor rectifier, an intermediate circuit capacitor arranged on the network frequency converter, and a diode arranged between the semiconductor switch and the intermediate circuit capacitor. According to a method for operating such a frequency converter circuit, the semiconductor switch is kept closed during the sub-synchronous operation of the asynchronous generator, and during at least some periods of synchronous or super-synchronous operation of the asynchronous generator the semiconductor switch is opened.

1. Field of the Invention
The invention relates to a frequency converter circuits for use with wind power generating equipment and, more particularly, to such a circuit for a double-fed asynchronous generator with a variable power output, which can be connected to a voltage network, and a method for operating such a frequency converter circuit.
2. Description of the Related Art
Asynchronous generators with variable power output are widely used today in the field of energy generation. In particular, they are employed in connection with wind power generators, whose power output naturally greatly varies with the prevailing wind. A variable operating speed is of great advantage in connection with such asynchronous generators. A wind power generator with variable speed does permit better utilization of weak winds and exerts less mechanical stress on the tower of the wind power generator in strong winds.
A frequency converter circuit is required between an asynchronous generator and the voltage network for operating the former, into which the asynchronous generator is intended to feed energy. Frequency converter circuits for double-fed asynchronous generators in windmills are widely known and, as a rule, have a three-phase current frequency converter at the rotor side, intermediate circuit capacitors and a frequency converter on the network side. Such a classic frequency converter circuit for a double-fed asynchronous generator provides the two above mentioned advantages, but is comparatively expensive.
A simple solution regarding the double-fed asynchronous generator is known from U.S. Pat. No. 7,015,595, which offers a speed regulation in the sub-synchronous range. In this case "sub-/super-synchronous" refers to the so-called synchronous number of revolutions, or nominal rated speed of the asynchronous generator and means a number of rotor revolutions smaller/greater than the synchronous number of revolutions. The energy yield at low wind speeds is increased because of this. However, in case of a sudden increase in the wind speed, the windmill tower is not relieved.
A solution for windmills with an asynchronous generator with a short-circuited rotor and power electronic drive for smaller outputs and with an improved energy yield at low wind speeds is known from U.S. Pat. No. 7,012,409. Here, too, the windmill tower is not relieved in case of strong increases in wind speed.
An alternative option for tower relief is known from the prior art. Here, a resistor, which is switched on in case of higher wind speeds, is been inserted into the rotor circuit. Then the rotor current flows through the resistor. By means of this, the rotational speed of the windmill can be increased, additional wind energy is stored as additional kinetic energy of the windmill and the tower is mechanically relieved. Since the tower is mechanically relieved, the tower structure can be provided in a simpler and more cost-effective way. The "optislip" circuit with a resistor is completely retained in the rotor, or rotates along with it. Therefore no contact rings are provided. The rotor energy additionally heats the resistor, and therefore the rotor, and thus the generator. In actual use, the super-synchronous operation is briefly limited here to a few percent.

Frequency converter for a double-fed asynchronous generator with variable power output and method for its operation


A biological filtration system is provided for filtering a water supply. The filtration system includes a micron filter having an input and an output. Water is supplied from the water supply to the input of the micron filter. The filtration system also includes a canister filled with a biomedia material. The canister has an inlet end coupled to the output of the micron filter and an outlet end. Air is injected into the inlet end of the canister. Water from the outlet end of the canister is returned to the water supply.

The present invention relates to a biological filtration system for removing ammonia and nitrite from a water supply. More particularly, the present invention relates to a biological filtration system which improves the efficiency of the nitrification process of water passing through the filtration system.
Although the preferred embodiment of the present invention is disclosed with regard to filtering a water supply in a closed system such as an aquarium, it is understood that the present invention can be used for filtering other water supplies. In particular, by scaling of the sizes of the components of the biological filtration system of the present invention, the filtration system may be used to filter a municipal water supply or a water supply drawn from a well or reservoir.
Ammonia (NH.sub.3) or (NH.sub.3).sup.+4 depending upon the pH of water is a chemical compound that is very toxic. In a closed water supply system with a finite amount of water, such as in an aquarium, the ammonia level in the water can build up to toxic levels very rapidly. Ammonia builds up in the water supply from two sources. A first source of ammonia is animals which consume plant protein. This plant protein is broken down and excreted as urea, uric acid, or ammonia. Both urea and uric acid are quickly converted by bacteria into ammonia. A second source of ammonia is the break down of proteins and other nitrogen containing organic compounds by heterotrophic bacteria in a process called mineralization. Ammonia is a nonorganic compound which results from mineralization.
High levels of ammonia deplete oxygen through process of diffusion. Biological filtration is used to combat the problem of ammonia build up in the closed environment of an aquarium. Biological filtration is a process by which bacteria oxidize ammonia into a less toxic form. Oxidation of ammonia occurs in a two step process. In the first step, ammonia is oxidized by nitrifying bacteria in the genus Nitrosomonas or other related genera into nitrite (NO.sub.2). Nitrite is less harmful than ammonia but can still be dangerous in quantities greater than one part per million. Nitrite is further oxidized by nitrifying bacteria in the genus Nitrobacter or other related genera to form nitrate (NO.sub.3). Nitrate is diluted from closed system through periodic water changes.
Various types of biological filters are known. Wet/dry filtration systems work by running water over a vast surface area, usually a tray containing biomedia with water dripping over it. Nitrifying bacteria grow on the surface area of the biomedia. These filtration systems therefore provide a large surface area for nitrifying bacteria to grow and greater water surface coverage for the exchange of gases.
One known biological filter is a trickle filter. In a conventional trickle filter, water passes through a trickle section of the filter and takes on dissolved gases by diffusion. Water draining from the aquarium is coarsely prefiltered to keep out large chunks of detritus such as dead fish, snails, etc. Typically, the water passes over biomedia. Biomedia having a large surface area is commonly available. A standard formula for conventional trickle filters is that biomedia having a volume equal to about 10% of the total volume in the tank must be used in the trickle filter. For example, a trickle filter servicing a 55 gallon tank would need 5.5 gallon of biomedia. Commercial designs for trickle filters include a translucent or semi-translucent compartment for the biomedia. Therefore, owners can watch the water pass through the trickle filter. Most commercial designs for trickle filters use containers that tend to be short and fat in ratio. The water passing through the trickle filter tends to take the path of least resistance and "channels" through the biomedia in the trickle filter. This causes the water to miss entire areas of the biomedia, thereby reducing the overall efficiency of the filter.
An object of the present invention is to improve the efficiency of the nitrification process to reduce the amount of ammonia and nitrite present in a water supply.
Anther object of the present invention is to provide separate locations for the growth of heterotrophic bacteria colonies and for the growth of nitrifying bacteria colonies to provide an optimum environment for nitrifying bacteria to oxidize ammonia into nitrite and then into nitrate.
Yet another object of the present invention is to maintain saturated oxygen levels within a water supply while minimizing the amount of ammonia and nitrate in the water supply.
In the biological filtration system of the present invention, water is pumped from the water supply into a micron filter. The micron filter filters particles having a size larger than one micron (1/1000 of an inch) from the water. After passing through the micron filter, water proceeds to an array of filter canisters. Water passes through spray nozzles which spray the water into an inlet end of the canisters. The spray nozzles break up the water and mix it with air which is supplied to the inlet end of the canisters. Water hits biomedia stored in the array of canisters. Any commercially available biomedia or any other inert substance which has a large surface area on which bacteria can grow may be used in the canisters. Water passes over the biomedia and is further mixed with air. At the bottom of the canisters, the water is collected and passed through a tube back into the water supply.
The spray nozzles disperse the water over a larger area of the biomedia to reduce channeling as the water passes over the biomedia in the canisters. In addition, the air supply to the top of the canisters from the air pump increases the atmospheric pressure around the biomedia so that dissolved gases are forced into the water to the point of super-saturation. Because water passing through the biomedia is prefiltered to remove particles greater than one micron from the water, the filtration system of the present invention promotes substantially separate environments for the growth of heterotrophic bacteria colonies and nitrifying bacteria colonies. Heterotrophic bacteria are attracted to the food supply provided by the organic material trapped in the micron filter. Heterotrophic bacteria feeds on the organic detritus trapped in the micron filter. Nitrifying bacteria grow on the biomedia in the array of canisters. There is a negligible food supply for heterotrophic bacteria in the array of canisters. Therefore, the biological filtration system of the present invention provides an optimum environment for nitrifying bacteria colonies to grow by eliminating competition with heterotrophic bacteria that feed on organic detritus.
The present invention requires biomedia equal to only 1% to 3% of the total tank volume. Therefore, for a 55 gallon tank, the present invention requires only 1/2 to 11/2 gallons of biomedia. The canisters for holding the biomedia in the present invention are preferably completely opaque. Heterotrophic bacteria growth is partially light dependent. Therefore, the darkness inside the canisters further discourages competition between heterotrophic bacteria and nitrifying bacteria. The canisters are relatively tall and thin in ratio compared to commercially available trickle filters. This further reduces the likelihood that channeling will occur as water passes over the biomedia located in the canisters.
According to one aspect of the present invention, a biological filtration system is provided for filtering a water supply. The filtration system includes a micron filter having an input and an output, and means for supplying water from the water supply to the input of the micron filter. The filtration system also includes a canister filled with a biomedia material. The canister has an inlet end coupled to the output of the micron filter and an outlet end. The filtration system further includes means for injecting air into the inlet end of the canister, and means for returning water from the outlet end of the canister to the water supply.
In one embodiment of the invention, the means for supplying water from the water supply to the input of the micron filter comprises a canister filter. The canister filter filters the water and pumps the water from the water supply through the canister filter to the input of the micron filter.
According to another aspect of the present invention, a biological filtration system is provided for filtering a water supply. The filtration system includes an elongated canister having an opaque outer wall defining an interior region which contains biomedia material therein. The canister has an inlet end and an outlet end. The filtration system also includes means for supplying water from the water supply to the inlet end of the canister. The supplying means includes a spray nozzle located adjacent the inlet end of the canister for dispersing the water entering the inlet end of the canister so that water flows substantially evenly over the biomedia inside the canister. The filtration system further includes means for supplying air into the inlet end of the canister so that air mixes with the dispersed water from the spray nozzle, and means for returning water from the outlet end of the canister to the water supply.
In an illustrated embodiment, the canister has a generally cylindrical shape. The filtration system may include an array of canisters to increase the capacity of the filtration system.
According to yet another aspect of the invention, a biological filtration system is provided for filtering a water supply. The biological filtration system reduces the quantity of ammonia and nitrite in the water supply. The filtration system includes means for separating heterotrophic bacteria colonies from nitrifying bacteria colonies, and means for supplying water from the water supply to the separating means. The filtration system also includes a canister filled with a biomedia material for promoting growth of nitrifying bacteria. The canister has an inlet end coupled to the outlet of the separating means so that water from the separating means flows through the canister and an outlet end. The filtration system further includes means for injecting air into the inlet end of the canister to provide oxygen for promoting oxidation of ammonia and nitrite by the nitrifying bacteria growing on the biomedia in the canister. The filtration system still further includes means for returning water from the outlet end of the canister to the water supply.
In the illustrated embodiment, the separating means includes a micron filter having an input and an output. The micron filter traps detritus and heterotrophic bacteria entrained in the water inside the micron filter to promote growth of heterotrophic bacteria colonies inside the micron filter instead of in the canisters. The micron filter includes a removable micron filter cartridge so that the detritus and heterotrophic bacteria in the micron filter cartridge can be removed from the water supply, thereby reducing the content of heterotrophic bacteria in the water supply. Therefore, in the illustrated embodiment, the removable micron filter cartridge provides means for removing detritus from the water supply to reduce the quantity of ammonia in the water supply.
Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of a preferred embodiment exemplifying the best mode of carrying out the invention as presently perceived.


A method and apparatus to determine the pressure of a supercritical refrigerant within a heat exchanger of a transcritical vapor compression system. A plurality of measurements, e.g., temperature, are obtained at spaced locations on the heat exchanger and the location of the minimum temperature gradient, i.e., maximum specific heat value of the refrigerant, is determined (“the inflection point”). Obtaining the refrigerant temperature at the inflection point allows the refrigerant pressure to be determined. Alternatively, the temperature of the refrigerant at a second point can be determined together with the change in specific enthalpy between the inflection point and the second point to thereby determine the pressure of the refrigerant. The system can be regulated by controlling the location of the inflection point or by controlling the temperature difference of the refrigerant at the inflection point and a second point, e.g., the outlet of the heat exchanger.

The present invention relates to vapor compression systems and, more specifically, to determining the supercritical pressure within a heat exchanger in a transcritical vapor compression system.
In a typical vapor compression system, the refrigerant remains at subcritical pressures throughout the system. However, for some refrigerants, such as carbon dioxide, it is typical to operate the system as a transcritical vapor compression system wherein the refrigerant is at a supercritical pressure on the high pressure side of the system and at a subcritical pressure on the low pressure side of the system.
In such a transcritical system the refrigerant is compressed to a supercritical pressure in the compressor and then cooled in a heat exchanger, commonly called a gas cooler. After the refrigerant is cooled in the gas cooler, it is passed through an expansion device to lower its pressure from a supercritical pressure to a subcritical pressure. The low pressure refrigerant then enters an evaporator wherein the refrigerant absorbs thermal energy as it changes phase from a liquid to a vapor.
When a refrigerant is compressed to a supercritical pressure, i.e., a pressure above its critical pressure, the liquid and vapor phases of the refrigerant are indistinguishable and the refrigerant is commonly referred to as a gas. When the refrigerant is at a supercritical pressure, the phase of the refrigerant does not change by heating or cooling the refrigerant.
In a conventional vapor compression system wherein the refrigerant is not compressed to a supercritical pressure, when the pressure of the refrigerant in the condenser is monitored, i.e., the high pressure heat exchanger, it is typically directly measured by a pressure sensor that penetrates the structure forming the condenser. In a transcritical system, the pressure in the gas cooler will generally be substantially higher than that found in a conventional condenser and it is undesirable to penetrate the structure forming the gas cooler because such a penetration increases the possibility of a subsequent leak. Other methods of determining the pressure of a refrigerant which is at a subcritical pressure using the temperature or other physical parameter of the refrigerant are also known, however, such methods will generally not be applicable to a refrigerant at a supercritical pressure.
The Gibbs Phase Rule can be used to determine the degrees of freedom in a system and thereby indicate the number of parameters required to determine the thermodynamic state of the fluid system and states:p+f=c+2wherein, p=the number of phases; f=number of degrees of freedom in the system, i.e., the number of required parameters; and c=number of components in the thermodynamic system. Thus, a single phase system will have one more degree of freedom than a similar two phase system. For example, the temperature of a refrigerant can be used to determine the pressure of the refrigerant when the refrigerant is at a subcritical pressure and in a two phase state. For a refrigerant at a supercritical pressure and limited to a single phase, however, two physical parameters, such as temperature, pressure, specific volume or density, are required to determine any other thermodynamic property of the refrigerant.

Method and apparatus for determining supercritical pressure in a heat exchanger


A home video server system includes a source control library and a streaming library. A stream controller is connected between the source control library and the streaming library. Moreover, a plurality of network display terminals can be connected to the streaming library via a network connection. The source control library can choose between a plurality of media sources for streaming video content. When the sources for the streaming video content change, the connection between the network display terminal and the streaming library remains the same.

1. Field of the Invention
This invention pertains generally to home video server systems, and more particularly to managing video streams provided by home video server systems.
2. Description of Related Art
Televisions are ubiquitous and, in recent years, home video servers that operate in conjunction with televisions have been developed. A typical home video server can provide a variety of media content from various sources to be displayed at one or more televisions.
It is known that the media content can be delivered to a user's television via predefined paths or streams. The paths or streams used to deliver the media content can be built using various protocols (e.g., software modules or "stacks") that are based upon the type of content and the physical transport resources. Switching between the various video sources comprising the media content must be as fast and efficient as possible. However, switching between the video sources can be difficult due to the formats for the video sources, such as analog video, MPEG2 transport stream, MPEG2 program stream, MPEG-4, JVT, and others. Moreover, these video sources can require different resources from the home media server such as a digital encoder for analog video. Formats already in digital format may not require an encoder, but may require a format conversion depending on the requirements of the home media server and its connection to client display devices, such as televisions.
Current practice requires the entire video content stream (protocols, software modules, and/or the "stacks") to be torn down and rebuilt whenever source selection changes. This method was originally used for Internet streaming such as QuickTime and Real Networks. Later, this method was adopted and followed during the development of home media server products such as the NEC AX10, Pioneer DL-000, TiVo and Replay TV by SonicBlue.
Unfortunately, setting up the transport portion for such a connection can require a substantial set of information exchanges between the end user device and the server and as such, it can require an inordinate amount of time to establish the connection.
Accordingly, there is a need for a system and method for preserving the transport portion of the client streaming connection as well as other system resources across stream source selections. The present invention satisfies that need, as well as others, and overcomes deficiencies of prior approaches.

Apparatus and method for accommodating fast change of digital streaming sources and formats