text:
|Publication number
|Publication type
|Application number
|Publication date
|Aug 31, 2010
|Priority date
|Mar 1, 2006
|Fee status
|Also published as
|Publication number
|Azad Bonni, Esther B. E. Becker
|Original Assignee
|President And Fellows Of Harvard College
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (5), Non-Patent Citations (31), Classifications (15), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application claims priority to U.S. Ser. No. 60/778,536 filed Mar. 1, 2006, which is incorporated herein by reference in its entirety.
This invention was funded in part by the U.S. Government under grant number NS41021 awarded by the National Institute of Neurological Disorders and Stroke (NINDS). The Government has certain rights in the invention.
Apoptosis of neurons is a fundamental process in the development of the nervous system and contributes to the pathogenesis of several neurologic disorders. Since neurons are postmitotic cells that last the entire lifespan of an organism, specific mechanisms have evolved to regulate apoptosis in neurons. Since the mechanisms underlying neuron-specific mechanisms of apoptosis remains poor, there is a dearth of treatment modalities for neurologic disorders that involve excessive neurodegenerative disorders.
The invention provides a method of reducing neural cell apoptosis by contacting a neural cell (e.g., granule neuron) with an agent that reduces the level or activity of the prolyl isomerase Pin1. Inhibitors such as substrate analogs and RNA interference vectors or products thereof preferentially reduce apoptosis in neural cells compared to non-neural cells. For example, the inhibitor reduces apoptosis at least 20%, 50%, 100%, 2 fold, 5-fold and up to ten fold in neural cells as compared to non-neural cells. By reducing neural cell apoptosis, this agent is useful for treating or preventing neurologic or neurodegenerative disorders including Alzheimer's disease, multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, stroke, cerebral ischemic disease, Huntington's disease, spinal muscular atrophy, stroke, brain trauma, spinal cord injury, diabetic neuropathy, and other neurodegenerative diseases. For example, the agent reduces apoptosis by reducing binding of Pin1 to BIMEL, e.g., a BIMEL domain contain a phosphorylated serine residue at amino acid position 65 of BIMEL. Exemplary agents are small molecule inhibitors and RNA interfering agents. A small molecule inhibitor is a compound that is less than 2000 daltons in mass. The molecular mass of the inhibitory compounds is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons. In some embodiments, the inhibitor is a peptide. For example, the peptide is at least 8, 10, 20, 30, 40 residues in length and inhibits binding of endogenous Pin1 to BIMEL. In other embodiments, the inhibitor is not a peptide or proteinaceous in nature.
The invention also provides methods for identifying a candidate compound for reducing or preventing apoptosis in a neural cell. These methods involve the steps of: (a) contacting a cell expressing a Pin1 gene with a candidate compound; and (b) measuring Pin1 gene expression or protein activity in the cell. A candidate compound that decreases the expression or the activity of Pin1 relative to such expression or activity in a cell that has not been contacted with the candidate compound is useful for reducing or preventing neural cell apoptosis. For example, the candidate compound reduces binding of Pin1 to BIMEL, specifically phosphorylated Ser65 BIMEL. Optionally, the Pin1 gene is a Pin1 fusion gene and the Pin1-expressing cell is a mammalian cell (e.g., a rodent or human cell). In other embodiments, step (b) involves the measurement of the level of Pin1 mRNA or protein.
Alternatively, the method involves the steps of: (a) contacting a Pin1 protein with a candidate compound; and (b) determining whether the candidate compound binds the Pin1 protein and/or reduces Pin1 activity. Candidate compounds that bind and reduce Pin1 activity are identified as compounds useful for reducing or preventing neural cell apoptosis. Preferably, the candidate compound reduces binding of Pin1 to BIMEL, specifically the phosphorylated Ser65 BIMEL.
In yet another screening approach, a method for identifying a candidate compound for reducing or preventing neural cell apoptosis involves the steps of: (a) contacting a Pin1 protein (e.g., human Pin1 protein) with a candidate compound; and (b) determining whether the candidate compound decreases binding of Pin1 to BIMEL. The candidate compound is first contacted with Pin1, BIMEL, or is simultaneously contacted with both proteins or fragments thereof, e.g., a fragment of BIMEL containing phosphorylated Ser65. Candidate compounds that decrease such binding reduce or prevent neural cell apoptosis.
In all foregoing aspects of the invention, candidate compounds identified as being useful for reducing or preventing neural cell apoptosis are useful to treat or prevent neural disorders.
By "reduce the expression or activity of Pin1" is meant to reduce the level or biological activity of Pin1 relative to the level or biological activity of Pin1 in an untreated control. The level or activity is preferably reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative to an untreated control. Since Pin1 binds and stabilizes BIMEL, a reduction in the biological activity of Pin1 is, for example, a reduction in the activity of BIMEL, in turn resulting in a reduction in apoptosis. For example, the binding interaction between Pin1 and BIMEL is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% relative to an untreated control, thereby reducing apoptosis and ultimately treating or reducing neural or neurodegenerative disorders. Thus, as used herein, the term "activity" with respect to a Pin1 polypeptide includes any activity which is inherent to the naturally occurring Pin1 protein, such as binding and stabilizing BIMEL, activation of neural apoptosis, or both, as detected by any standard method.
By "treating or preventing a neurologic disorder" is meant ameliorating any of the conditions or symptoms associated with the disorder before or after it has occurred including, for example, seizures, headaches, and memory loss. Alternatively, alleviating a symptom of a disorder may involve reducing visible areas of neuronal cell death relative to an untreated control. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique. A patient who is being treated for a neurologic disorder is one who a medical practitioner has diagnosed as having such a condition. Diagnosis may be by any suitable means. Diagnosis and monitoring may involve, for example, detecting the presence of destroyed or dying neurons in a biological sample (e.g., tissue biopsy, blood test, or urine test), detecting the presence of amyloid plaques, detecting the level of a surrogate marker of the neurologic disorder in a biological sample, or detecting symptoms associated with the neurologic disorder. A patient in whom the development of a neurologic disorder is being prevented may or may not have received such a diagnosis. One in the art will understand that these patients may have been subjected to the same standard tests as described above or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., family history or genetic predisposition).
As used herein, by "Pin1" is meant a polypeptide that forms a complex with the BIMEL and is involved in various signaling pathways including mitochondrial apoptosis. The Pin1 proteins of the invention are substantially identical to the naturally occurring Pin1 (e.g., accession numbers AAC50492 (human PIN1 (SEQ ID NO: 10) and BAA87038 (murine PIN1 (SEQ ID NO: 11), the sequences of which are hereby incorporated by reference). Neurologic disorders are treated or prevented when Pin1 activity or expression is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% below control levels as measured by any standard method (e.g.,. Northern blot analysis).
By a "Pin1 gene" is meant a nucleic acid that encodes a Pin1 protein.
By "Pin1 fusion gene" is meant a Pin1 promoter and/or all or part of a Pin1 coding region operably linked to a second, heterologous nucleic acid sequence. In preferred embodiments, the second, heterologous nucleic acid sequence is a reporter gene, that is, a gene whose expression may be assayed; reporter genes include, without limitation, those encoding glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), alkaline phosphatase, and beta-galactosidase.
By "substantially identical," when referring to a protein or polypeptide, is meant a protein or polypeptide exhibiting at least 75%, but preferably 85%, more preferably 90%, most preferably 95%, or even 99% identity to a reference amino acid sequence. For proteins or polypeptides, the length of comparison sequences will generally be at least 20 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids, and most preferably 50 amino acids or the full length protein or polypeptide. Nucleic acids that encode such "substantially identical" proteins or polypeptides constitute an example of "substantially identical" nucleic acids; it is recognized that the nucleic acids include any sequence, due to the degeneracy of the genetic code, that encodes those proteins or polypeptides. In addition, a "substantially identical" nucleic acid sequence also includes a polynucleotide that hybridizes to a reference nucleic acid molecule under high stringency conditions.
By "high stringency conditions" is meant any set of conditions that are characterized by high temperature and low ionic strength and allow hybridization comparable with those resulting from the use of a DNA probe of at least 40 nucleotides in length, in a buffer containing 0.5 M NaHPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at a temperature of 65� C., or a buffer containing 48% formamide, 4.8�SSC, 0.2 M Tris-Cl, pH 7.6, 1� Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42� C. Other conditions for high stringency hybridization, such as for PCR, Northern, Southern, or in situ hybridization, DNA sequencing, etc., are well known by those skilled in the art of molecular biology. See, e.g., F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998, hereby incorporated by reference.
By "substantially pure" is meant a nucleic acid, polypeptide, or other molecule that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. For example, a substantially pure polypeptide may be obtained by extraction from a natural source, by expression of a recombinant nucleic acid in a cell that does not normally express that protein, or by chemical synthesis.
The term "isolated DNA" is meant DNA that is free of the genes which, in the naturally occurring genome of the organism from which the given DNA is derived, flank the DNA. Thus, the term "isolated DNA" encompasses, for example, cDNA, cloned genomic DNA, and synthetic DNA.
By "an effective amount" is meant an amount of a compound, alone or in a combination, required to reduce or prevent the neurologic disorder in a mammal. The effective amount of active compound(s) varies depending upon the route of administration, age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen.
By a "candidate compound" is meant an agent to be evaluated as a Pin1 inhibitor. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof.
The term "pharmaceutical composition" is meant any composition, which contains at least one therapeutically or biologically active agent and is suitable for administration to the patient. Any of these formulations can be prepared by well-known and accepted methods of the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th edition, (ed. A. R. Gennaro), Mack Publishing Co., Easton, Pa., 2000.
The invention provides significant advantages over standard therapies for treatment, prevention, and reduction, or alternatively, the alleviation of one or more symptoms associated with neurologic disorders, because it preferentially targets neural cells compared to non-neural cells. In addition, the screening methods allow for the identification of therapeutics that modify the injury process rather than merely mitigating the symptoms.
Cited publications including sequences defined by GENBANK™ accession numbers are incorporated herein by reference.
Other features, objects, and advantages of the invention will be apparent from the description of the drawings.
The invention is based on the discovery that the prolyl isomerase Pin1 plays a key role in neural apoptosis. In particular, we report that the prolyl isomerase Pin1 interacts with the Ser65-phosphorylated BH3-only protein, BIMEL, in neurons, in turn causing neural cell death.
Proteins of the BH3-only family couple apoptotic signals from distinct cellular compartments and signaling pathways to the mitochondrial cell death machinery. The BH3-only protein BIMEL resides at the mitochondria in neurons and mediates neuronal cell death following growth factor deprivation as well as activation of the p75 cell death receptor. These apoptotic stimuli activate c-Jun N-terminal kinase (JNK). JNK in turn phosphorylates BIMEL at the distinct site of Ser65. Whereas phosphorylation of the BIMEL at Ser65 suppresses cell death in non-neural cells, this event triggers apoptosis in neurons. In neurons, Pin1 is enriched at the mitochondrial membrane, where it forms a physical complex with the neuron-specific JNK scaffold protein JIP3. Activation of JNK signaling induces the dissociation of Pin1 from JIP3 and concomitantly promotes Pin1 binding to phosphorylated BIMEL. The interaction of Pin1 with phosphorylated BIMEL stabilizes BIMEL and thereby activates neuronal apoptosis. Accordingly, our finding of a neural-specific mechanism of cell death whereby Pin1 couples phosphorylation of BH3-only proteins to activation of the mitochondrial apoptotic machinery provides a rationale for administering inhibitors of Pin1 to reduce or inhibit neural apoptosis, thereby treating or preventing neural disorders. An exemplary inhibitor reduces binding between Pin1 and BIMEL.
The experiments described herein were performed using the following Materials and Methods.
Pin1 plasmids were employed. Point mutations in Pin1 were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), and verified by sequencing. FLAG-tagged JIP3 fragments 1-442, 420-815, 800-1337, and 144-241 were generated by PCR and subcloned into the 3�FLAG CMV-7.1 vector (Sigma). The U6/pin1 RNAi plasmid was designed to specifically target the 21-nucleotide region GAGACCTGGGTGCCTTCAGCA (SEQ ID NO: 1) in Pin1 mouse, rat, and human mRNA. The targeted region in Pin1 showed no significant homology with any other gene by BLAST. The pLentiLox3.7 (pLL3.7) vector was used to generate the Pin1 RNAi lentivirus.
Cell Culture, Transfections, and Infections
Transfections in 293T cells were performed by a calcium phosphate transfection method. Primary cerebellar granule neurons were prepared from 6-day old Long-Evans rats and transfected using a calcium phosphate method as described by Konishi et al. Mol Cell 9: 1005-1016, 2002. Lentivirus was generated in 293T cells by co-transfecting pLL3.7 and viral packaging vectors as described by Rubinson et al., Nat. Genet. 33: 401-406, 2003. Neurons were infected with lentivirus on P6 (8 h after plating) and harvested 4 days later for analysis.
Biochemical Assays
GST-pulldown assays were performed as described by Ryo et al., Mol. Cell 12: 1413-1426, 2003. Immunoprecipitations and subcellular fractionations were performed as described by Konishi et al., Science 303: 1026-1030, 2004. The postnuclear supernatant was further centrifuged at 10,000 g for 30 min to pellet the mitochondrially-enriched heavy membrane (HM) fraction. Antibodies to BIM (Stressgen); phospho65 BIM antibody (Biosource); HA, HSP60, JIP3, GST (Santa Cruz Biotechnology); FLAG (Sigma); P-c-Jun, MKK7, Pin1 (Cell Signaling); SP1, JNK (Upstate); GFP (Molecular Probes) are available commercially.
Survival Assays in Cerebellar Granule Neurons
Survival assays in granule neurons were done as described by Becker et al., J. Neurosci. 24: 8762-8770, 2004. Cell death was assessed in transfected neurons based on the integrity of neurites and the morphology of the nucleus as determined using the DNA dye bisbenzimide Hoechst 33258 (Sigma). Cell counts were done in a blinded manner (n=100 cells per condition) and analyzed for statistical significance by ANOVA followed by Fisher's protected least significance difference post-hoc test. At least 3 independent experiments were used for statistical analysis.
Therapeutic Agents
An inhibitor of Pin1 is any agent having the ability to reduce the expression or the activity of Pin1 in a cell. The inhibitor preferentially inhibits neural cell death. The control cell is a cell that has not been treated with the Pin1 activator. Pin1 expression or activity is determined by any standard method in the art, including those described herein. Pin1 inhibitors include polypeptides, polynucleotides, small molecule antagonists, or siRNA. For example, a Pin1 inhibitor reduces Pin1 activity by reducing binding between Pin1 and BIMEL.
Alternatively, the Pin1 inhibitor is a dominant negative protein or a nucleic acid encoding a dominant negative protein that interferes with the biological activity of Pin1. A dominant negative protein is any amino acid molecule having a sequence that has at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence identity to at least 10, 20, 35, 50, 100, or more than 150 amino acids of the wild type protein to which the dominant negative protein corresponds. For example, a dominant-negative Pin1 has mutation such that it no longer activates downstream pathways. Specifically, a dominant-negative Pin1 binds BIMEL less efficiently than the naturally-occurring Pin1 polypeptide and therefore fails to activate apoptosis.
The dominant negative protein may be administered as an expression vector. The expression vector may be a non-viral vector or a viral vector (e.g., recombinant retrovirus, recombinant lentivirus, recombinant adeno-associated virus, or a recombinant adenoviral vector). Alternatively, the dominant negative protein may be directly administered as a recombinant protein systemically or to the affected area using, for example, microinjection techniques.
The Pin1 inhibitor is an antisense molecule, an RNA interference (siRNA) molecule, a small molecule antagonist that targets Pin1 expression or activity, or a vector that directs production of such inhibitory compositions. By the term "siRNA" is meant a double stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into a cell are used, including those in which DNA is a template from which an siRNA RNA is transcribed. The siRNA includes a sense Pin1 nucleic acid sequence, an anti-sense Pin1 nucleic acid sequence or both. Optionally, the siRNA is constructed such that a single transcript has both the sense and complementary antisense sequences from the target gene, e.g., a hairpin. Binding of the siRNA to a Pn1 transcript in the target cell results in a reduction in Pin1 production by the cell. The length of the oligonucleotide is at least 10 nucleotides and may be as long as the naturally-occurring Pin1 transcript. Preferably, the oligonucleotide is 19-25 nucleotides in length. Most preferably, the oligonucleotide is less than 75, 50, 25 nucleotides in length.
Small molecules includes, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heterorganic and organomettallic compounds) having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 2,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Useful small molecules may reduce Pin1 expression or activity by reducing the interaction between Pin1 and Ser65 phosphorylated BIMEL. Inhibitors of prolyl isomerases, e.g., Pin1, include small molecules such as 5-hydroxy-1,4-naphthoquinone (juglone) (Chao et al, 2001, Nucleic Acids Res. 29 (3): 767-773) as well as peptide based compositions such as phosphoSer-cis (or trans) Pro isostere or other conformationally locked pSer-Pros substrate analogues of Pin1. Other Pin1 inhibitors include PiA (2,7-dimethylbenzo[Imn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetrone and PiB(diethyl-1,3,6,8-tetrahydro-1,3,6,8-tetraoxobenzo[Imn][3,8]phenanthroline-2,7-diacetate) (Uchida et al, 2003, Chemistry & Biology 10: 15-24). Daum et al. have reported that aryl indanyl ketones and derivates (compounds (R)-9 and rac-9 pictured below) also inhibit Pin1 (Daum et al. 2006, Angew. Chem. Int. Ed. 45: 7454-7458; hereby incorporated by reference).
Competitive substrate inhibitors also include peptides based on the Pin1 substrate peptide WFYSPR (SEQ ID NO: 2), which requires a phosphorylated serine residue N-terminal to the proline residue for activity. For example, an inhibitory peptide contains the amino acid sequence YGRKKRRQRRRWFYpSPR (SEQ ID NO: 3) (with a phosphorylated Ser at position 15). Other inhibitory compounds are described in PCT/US99/12544 (hereby incorporated by reference), e.g., a compound with the formaul A-X-R in which A is a radica that mimics the steric and electronic properties of a phosphoserine or phosphothreonine, X is a spacer or absent, and R is a ring structure. Other Pin1 inhibitors include peptides with the sequence Ac-Lys(Nε-biotinoyl)-Ala-Ala-Bth-
A biologically active dose of a Pin1 inhibitor is a dose that will reduce neural apoptosis. Desirably, the Pin1 inhibitor has the ability to reduce the expression or activity of Pin1 in neuronal cells (e.g., granule neurons) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% below untreated control levels. The levels or activity of Pin1 in cells is measured by any method known in the art, including, for example, Western blot analysis, immunohistochemistry, ELISA, and Northern Blot analysis. Alternatively, the biological activity of Pin1 is measured by assessing the expression or activity of any of the molecules involved in Pin1 signaling. The biological activity of Pin1 is determined according to its ability to reduce neural cell apoptosis. Preferably, the agent that reduces the expression or activity of Pin1 can reduce neural cell apoptosis or neurodegeneration by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% below untreated control levels. The agent of the present invention is therefore any agent having any one or more of these activities.
Optionally, the subject is administered one or more additional therapeutic regiments. The additional therapeutic regimens may be administered prior to, concomitantly, or subsequent to administration of the Pin1 inhibitor. For example, the Pin1 inhibitor and the additional agent are administered in separate formulations within at least 1, 2, 4, 6, 10, 12, 18, or more than 24 hours apart. Optionally, the additional agent is formulated together with the Pin1 inhibitor. When the additional agent is present in a different composition, different routes of administration may be used. The agent is administered at doses known to be effective for such agent for treating, reducing, or preventing the progression of the neural disorder.
Concentrations of the Pin1 inhibitor and the additional agent depends upon different factors, including means of administration, target site, physiological state of the mammal, and other medication administered. Thus treatment dosages may be titrated to optimize safety and efficacy and is within the skill of an artisan. Determination of the proper dosage and administration regime for a particular situation is within the skill of the art.
Pin1 inhibitors are administered in an amount sufficient to reduce neural apoptosis or neurodegeneration. Such reduction includes the alleviation of one or more of symptoms associated with the neural disorder being treated or prevented. Administration of the Pin1 inhibitor reduces the neurodegeneration associated with the neural disorder or alleviates one or more symptoms associated with the disorder by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to an untreated subject.
Treatment is efficacious if the treatment leads to clinical benefit such as, a reduction of the symptoms of a neurologic disorder in the subject. When treatment is applied prophylactically, "efficacious" means that the treatment retards or prevents the neurodegenerative process. Efficacy may be determined using any known method for diagnosing or treating the neural disorder.
Therapeutic Administration
The invention includes administering to a subject a composition that includes a compound that reduces Pin1 expression or activity (referred to herein as an "Pin1 inhibitor" or "therapeutic compound"). As described herein, this inhibitor may reduce binding between Pin1 and BIMEL.
An effective amount of a therapeutic compound is preferably from about 0.1 mg/kg to about 150 mg/kg. Effective doses vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and coadministration with other therapeutic treatments including use of other agents or therapeutic agents for treating, preventing or alleviating a symptom of a neurodegenerative disorder. A therapeutic regimen is carried out by identifying a mammal, e.g., a human patient suffering from (or at risk of developing) a neural disorder, using standard methods.
The pharmaceutical compound is administered to such an individual using methods known in the art. Preferably, the compound is administered orally, rectally, nasally, topically or parenterally, e.g., subcutaneously, intraperitoneally, intrathecally, intramuscularly, and intravenously. The compound is administered prophylactically, or after the detection of the neurologic injury. Compounds are also delivered locally to make direct contact with a site of injury or disease. The compound is optionally formulated as a component of a cocktail of therapeutic drugs to treat the neural disorder. Examples of formulations suitable for parenteral administration include aqueous solutions of the active agent in an isotonic saline solution, a 5% glucose solution, or another standard pharmaceutically acceptable excipient. Standard solubilizing agents such as PVP or cyclodextrins are also utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
The therapeutic compounds described herein are formulated into compositions for other routes of administration utilizing conventional methods. For example, the Pin1 inhibitor is formulated in a capsule or a tablet for oral administration. Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing mixtures of a therapeutic compound with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The compound is administered in the form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent. Other formulations include an ointment, suppository, paste, spray, patch, cream, gel, resorbable sponge, or foam. Such formulations are produced using methods well known in the art.
Where the therapeutic compound is a nucleic acid encoding a protein, the therapeutic nucleic acid is administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See, e.g., Joliot, et al., 1991. Proc Natl Acad Sci USA 88:1864-1868), and the like. A nucleic acid therapeutic is introduced intracellularly and incorporated within host cell DNA or remain episomal.
For local administration of DNA, standard gene therapy vectors used. Such vectors include viral vectors, including those derived from replication-defective hepatitis viruses (e.g., HBV and HCV), retroviruses (see, e.g., WO 89/07136; Rosenberg et al., 1990, N. Eng. J. Med. 323(9):570-578), adenovirus (see, e.g., Morsey et al., 1993, J. Cell. Biochem., Supp. 17E,), adeno-associated virus (Kotin et al., 1990, Proc. Natl. Acad. Sci. USA 87:2211-2215,), replication defective herpes simplex viruses (HSV; Lu et al., 1992, Abstract, page 66, Abstracts of the Meeting on Gene Therapy, September 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and any modified versions of these vectors. The invention may utilize any other delivery system which accomplishes in vivo transfer of nucleic acids into eucaryotic cells. For example, the nucleic acids may be packaged into liposomes, e.g., cationic liposomes (Lipofectin), receptor-mediated delivery systems, non-viral nucleic acid-based vectors, erythrocyte ghosts, or microspheres (e.g., microparticles; see, e.g., U.S. Pat. No. 4,789,734; U.S. Pat. No. 4,925,673; U.S. Pat. No. 3,625,214; Gregoriadis, 1979, Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press,). Naked DNA may also be administered.
DNA for gene therapy can be administered to patients parenterally, e.g., intravenously, subcutaneously, intramuscularly, and intraperitoneally. DNA or an inducing agent is administered in a pharmaceutically acceptable carrier, i.e., a biologically compatible vehicle which is suitable for administration to an animal e.g., physiological saline. A therapeutically effective amount is an amount which is capable of producing a medically desirable result, e.g., a decrease of a Pin1 gene product in a treated animal. Such an amount can be determined by one of ordinary skill in the art. As is well known in the medical arts, dosage for any given patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages may vary, but a preferred dosage for intravenous administration of DNA is approximately 106 to 1022 copies of the DNA molecule. Typically, plasmids are administered to a mammal in an amount of about 1 nanogram to about 5000 micrograms of DNA. Desirably, compositions contain about 5 nanograms to 1000 micrograms of DNA, 10 nanograms to 800 micrograms of DNA, 0.1 micrograms to 500 micrograms of DNA, 1 microgram to 350 micrograms of DNA, 25 micrograms to 250 micrograms of DNA, or 100 micrograms to 200 micrograms of DNA. Alternatively, administration of recombinant adenoviral vectors encoding the Pin1 inhibitor into a mammal may be administered at a concentration of at least 105, 106, 107, 108, 109, 1010, or 1011 plaque forming unit (pfu).
Pin1 gene products are administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used, e.g. packaged in liposomes. Such methods are well known to those of ordinary skill in the art. It is expected that an intravenous dosage of approximately 1 to 100 moles of the polypeptide of the invention would be administered per kg of body weight per day. The compositions of the invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.
Pin1 inhibitors are effective upon direct contact of the compound with the affected tissue or may alternatively be administered systemically (e.g., intravenously, rectally or orally). The Pin1 inhibitor may be administered intravenously or intrathecally (i.e., by direct infusion into the cerebrospinal fluid). For local administration, a compound-impregnated wafer or resorbable sponge is placed in direct contact with CNS tissue. The compound or mixture of compounds is slowly released in vivo by diffusion of the drug from the wafer and erosion of the polymer matrix. Alternatively, the compound is infused into the brain or cerebrospinal fluid using standard methods. For example, a burr hole ring with a catheter for use as an injection port is positioned to engage the skull at a burr hole drilled into the skull. A fluid reservoir connected to the catheter is accessed by a needle or stylet inserted through a septum positioned over the top of the burr hole ring. A catheter assembly (described, for example, in U.S. Pat. No. 5,954,687) provides a fluid flow path suitable for the transfer of fluids to or from selected location at, near or within the brain to allow administration of the drug over a period of time.
One in the art will understand that the patients treated according to the invention may have been subjected to the tests to diagnose a subject as having a neurologic disorder or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors (e.g., genetic predisposition). Reduction of neurodegenerative symptoms or damage may also include, but are not limited to, alleviation of symptoms (e.g., headaches, nausea, skin rash), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, and amelioration or palliation of the disease state. Treatment may occur at home with close supervision by the health care provider, or may occur in a health care facility.
Screening Assays
The present invention provides screening methods to identify compounds that can inhibit the expression or activity of Pin1. Useful compounds include any agent that inhibits the biological activity or reduces the cellular level of Pin1. For example, useful compounds are identified by detecting an attenuation of the expression or activity of any of the molecules involved in Pin1 signaling. For example, a useful compound reduces binding between Pin1 and Ser65 phosphorylated BIMEL. The screening assays may also identify agents that reduce neural cell apoptosis.
A number of methods are available for carrying out such screening assays. According to one approach, candidate compounds are added at varying concentrations to the culture medium of cells expressing Pin1. Gene expression of Pin1 is then measured, for example, by standard Northern blot analysis, using any appropriate fragment prepared from the nucleic acid molecule of Pin1 as a hybridization probe or by real time PCR with appropriate primers. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. If desired, the effect of candidate compounds may, in the alternative, be measured at the level of Pin1 polypeptide using the same general approach and standard immunological techniques, such as Western blotting or immunoprecipitation with an antibody specific to Pin1 for example. For example, immunoassays may be used to detect or monitor the level of Pin1. Polyclonal or monoclonal antibodies which are capable of binding to Pin1 may be used in any standard immunoassay format (e.g., ELISA or RIA assay) to measure the levels of Pin1. Pin1 can also be measured using mass spectroscopy, high performance liquid chromatography, spectrophotometric or fluorometric techniques, or combinations thereof.
As a specific example, mammalian cells (e.g., rodent cells) that express a nucleic acid encoding Pin1 are cultured in the presence of a candidate compound (e.g., a peptide, polypeptide, synthetic organic molecule, naturally occurring organic molecule, nucleic acid molecule, or component thereof). Cells may either endogenously express Pin1 or may alternatively be genetically engineered by any standard technique known in the art (e.g., transfection and viral infection) to overexpress Pin1. The expression level of Pin1 is measured in these cells by means of Western blot analysis and subsequently compared to the level of expression of the same protein in control cells that have not been contacted by the candidate compound. A compound which promotes a decrease in the level of Pin1 activity as a result of reducing its synthesis or biological activity is considered useful in the invention.
Alternatively, the screening methods of the invention may be used to identify candidate compounds that decrease the biological activity of Pin1 by reducing neural cell apoptosis by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to an untreated control. As another alternative, candidate compounds are identified for their ability to reduce binding between Pin1 and BIMEL. A candidate compound may be tested for its ability to reduce such binding in neural cells that naturally express Pin1 and BIMEL or after transfection with cDNA for Pin1 and BIMEL, or in cell-free solutions containing Pin1 and BIMEL, as described further below. The effect of a candidate compound on the binding or activation of BIMEL can be tested by radioactive and non-radiaoctive binding assays, competition assays, and receptor signaling assays.
Given its ability to decrease the biological activity of Pin1, such a molecule may be used, for example, as a therapeutic agent to treat, reduce, or prevent a neural disorder, or alternatively, to alleviate one or more symptoms associated with such a disorder. As a specific example, a candidate compound may be contacted with two proteins, the first protein being a polypeptide substantially identical to Pin1 and the second protein being BIMEL (i.e., a protein that binds the Pin1 polypeptide under conditions that allow binding). According to this particular screening method, the interaction between these two proteins is measured following the addition of a candidate compound. A decrease in the binding of Pin1 to BIMEL following the addition of the candidate compound (relative to such binding in the absence of the compound) identifies the candidate compound as having the ability to inhibit the interaction between the two proteins, and thereby having the ability to reduce Pin1 activity. The screening assay of the invention may be carried out, for example, in a cell-free system or using a yeast two-hybrid system. If desired, one of the proteins or the candidate compound may be immobilized on a support as described above or may have a detectable group.
Alternatively, or in addition, candidate compounds may be screened for those which specifically bind to and thereby inhibit Pin1. The efficacy of such a candidate compound is dependent upon its ability to interact with Pin1. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays. For example, a candidate compound may be tested in vitro for interaction and binding with Pin1 and its ability to modulate neural cell apoptosis may be assayed by any standard assays (e.g., those described herein).
For example, a candidate compound that binds to Pin1 may be identified using a chromatography-based technique. For example, a recombinant Pin1 may be purified by standard techniques from cells engineered to express Pin1 and may be immobilized on a column. Alternatively, the naturally-occurring Pin1 may be immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for Pin1 is identified on the basis of its ability to bind to Pin1 and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected. Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography).
Screening for new inhibitors and optimization of lead compounds may be assessed, for example, by assessing their ability to modulate Pin1 activity using standard techniques. Compounds which are identified as binding to Pin1 with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention.
Potential therapeutic agents include organic molecules, peptides, peptide mimetics, polypeptides, and antibodies that bind to a nucleic acid sequence encodes Pin1 or a Pin1 peptide and thereby inhibit or extinguish their activity. Inhibitory agents also include small molecules that bind to and occupy domains of Pin1 or BIMEL that interact with each other. Other potential agents include antisense molecules.
This invention is based in part on the experiments described in the following examples. These examples are provided to illustrate the invention and should not be construed as limiting.
Pin1 Interacts with Phosphorylated BIMEL in Vitro and in Vivo.
The phosphorylation of BIMEL at Ser65 triggers apoptosis specifically in neurons but not in non-neural cells. A critical regulatory post-phosphorylation event is the prolyl isomerization of proteins at sites of proline-dependent phosphorylation. A major function of Pin1 is to drive cells through mitosis in proliferating cells. Pin1 is also highly expressed in the nervous system. To assess whether Pin1 binds to Ser65-phosphorylated BIMEL in vitro, we used a GST pulldown assay. We expressed BIMEL in 293T cells alone or together with a constitutively active form of the kinase MEKK1 (MEKK1Δ), an upstream activator of JNK. Expression of MEKK1Δ results in the robust phosphorylation of BIMEL at Ser65, which is reflected by a mobility shift of BIMEL (
We next determined whether endogenous Pin1 and BIMEL interact in neurons. Endogenous BIMEL co-immunoprecipitated with Pin1 in lysates of granule neurons that were starved for two hours to activate JNK signaling but not in lysates of neurons kept under survival conditions (conditioned medium, CM) (
Pin1 is Localized at the Mitochondria in Neurons and Associates with the Neuron-Specific JNK Signaling Scaffold Protein JIP3
We next determined the subcellular site of the Pin1-BIMEL interaction in neurons. BIMEL resides at the mitochondria in neurons (
We next determined the mechanism by which Pin1 is tethered to the mitochondria in neurons and thus facilitates the interaction of Pin1 with Ser65-phosphorylated BIMEL. We ruled out that activation of JNK signaling might recruit Pin1 to the mitochondria independently of cell type since robust activation of the JNK signaling pathway by treatment of 293T cells with sorbitol did not lead to appreciable changes in the subcellular localization of Pin1 (
In other experiments, we found that endogenous JIP3 in granule neurons robustly interacted with recombinant GST-Pin1 or GST-Pin1 WW in vitro (
To determine whether the binding of Pin1 to JIP3 is regulated by an apoptotic stimulus, we measured the interaction of endogenous Pin1 and JIP3 in granule neurons that were left in full medium (conditioned medium, CM) or that were starved for two hours. Pin1 co-immunoprecipitated with JIP3 in full medium (
Pin1 Contributes to Survival Factor Withdrawal-Induced and BIMEL-Induced Apoptosis in Neurons
The interaction of Pin1 with Ser65-phosphorylated BIMEL in neurons led us to investigate whether Pin1 might mediate the ability of Ser65-phosphorylated BIMEL to induce apoptosis specifically in neurons. We first determined the role of Pin1 in apoptosis of granule neurons. Expression of exogenous Pin1 in granule neurons significantly induced cell death (
To assess the role of endogenous Pin1 in neuronal apoptosis, we employed a vector-based RNA interference (RNAi) method. The expression of Pin1 hairpin RNAs (hpRNAs) effectively reduced endogenous Pin1 expression in 293T cells and primary neurons (
To ensure specificity of the Pin1 knockdown-induced phenotype, we performed a rescue experiment. We generated an expression construct encoding a Pin1 rescue that is insensitive to the Pin1 RNAi (Pin1-Res). Pin1 knockdown reduced the expression of Pin1, but failed to effectively reduce the levels of Pin1-Res (
Experiments were carried out to determine whether Pin1 is required for BIMEL to induce cell death in neurons. The expression of BIMEL on its own potently induced apoptosis in neurons, knockdown of Pin1 significantly inhibited BIMEL-induced apoptosis (
Pin1 Isomerase Activity is Required for Neuronal Apoptosis and Stabilizes BIMEL Protein in Neurons
Having identified that Pin1 is an important mediator of apoptosis downstream of JNK-BIMEL signaling in neurons, we next investigated the mechanism underlying Pin1-induced neuronal cell death. Based on the observation that Pin1-Res reverses the Pin1 RNAi-induced survival phenotype, structure-function analyses of Pin1 in the background of Pin1 knockdown were carried out. Pin1 contains an amino-terminal WW domain that binds to specific phosphoSer/Thr-Pro motifs and a carboxyl-terminal enzymatic peptidyl prolyl isomerase (PPI) domain that catalyzes the cis-trans isomerization of the phosphoSer/Thr-Pro bonds. To determine whether binding of phosphoproteins through its WW domain is sufficient for the apoptotic function of Pin1, we expressed a Pin1 WW domain that contains an additional mutation (S16A), thereby allowing Pin1 to bind tightly to its substrates. As the Pin1 hpRNAs target the C-terminal PPI domain of Pin1, the Pin1 WW S16A mutant is insensitive to the Pin1 RNAi. In contrast to Pin1-Res, expression of Pin1 WW S16A failed to rescue the Pin1 RNAi-induced survival phenotype in granule neurons, indicating that the WW domain is not sufficient for the apoptotic function of Pin1 in neurons (
To assess the role of the PPI domain in Pin1-mediated cell death, we generated two catalytically impaired Pin1-Res constructs by mutating the critical catalytical sites C113A and H59A. The C113A and H59A mutations significantly reduce Pin1's PPI activity without affecting Pin1-substrate binding. We expressed the Pin1-Res mutants C113A and H59A in the background of Pin1 RNAi. In contrast to Pin1-Res, both Pin1-Res mutants failed to reverse the Pin1 RNAi-induced survival phenotype in neurons (
The requirement of the PPI domain for Pin1-mediated neuronal cell death suggests that a conformational change in the Pin1 substrate BIMEL may be required for its function in neuronal apoptosis. This raises the question of how the binding and consequent isomerization of Ser65-phosphorylated BIMEL by Pin1 might induce BIMEL-dependent apoptosis in neurons. Phosphorylation of BIMEL at Ser65 by ERK1/2 in nonneural cells is reported to promote degradation of BIMEL by the proteasome. To assess the effect of Pin1 on BIMEL protein levels, we induced knockdown of Pin1 in granule neurons using a Pin1 RNAi lentivirus. Four days after lentiviral infection, neurons were starved for two hours to activate JNK signaling. Pin1 protein levels were significantly reduced upon induction of Pin1 RNAi (65�3% reduction compared to control vector-infected neurons; n=3; ANOVA; p<0.0001) (
The data define a mechanism by which JNK-induced phosphorylation of the BH3-only protein BIMEL at Ser65 triggers apoptosis specifically in neurons. Our results show that the prolyl isomerase Pin1 interacts with Ser65-phosphorylated BIMEL in neurons. A significant proportion of Pin1 in neurons but not in non-neural cells is localized at the mitochondrial membrane, where BIMEL resides. Pin1 associates with the neuron-specific JNK signaling scaffold protein JIP3. Upon exposure of neurons to apoptotic stimuli that induce JNK signaling, Pin1 dissociates from JIP3 and concomitantly interacts with JNK-phosphorylated BIMEL. The interaction of Pin1 with Ser65-phosphorylated BIMEL stabilizes BIMEL and thereby promotes neuronal apoptosis. These findings define a neuron-specific cell death mechanism, whereby Pin1 mediates activation of the mitochondrial cell death machinery.
These results illustrate that the phosphorylation of BIMEL at Ser65 elicits strikingly distinct responses depending on cell type, leading to apoptosis in neurons and cell survival in proliferating non-neural cells. The phosphorylation of BIMEL at Ser65 is mediated by ERK1/2 in non-neural cells and thought to lead to the ubiquitin-dependent proteasomal degradation of BIMEL. Our findings however indicate that the binding of Pin1 to Ser65-phosphorylated BIMEL and its consequent isomerization in neurons may act as a molecular switch that protects BIMEL from ubiquitination and degradation (
The identification of Pin1 's interaction with JIP3 has important ramifications for both Pin1 and JIP3 function in neurons. Studies of JIP3 in Drosophila, C. elegans, and mice point to functions of JIP3 in kinesin-dependent anterograde axonal transport. JIP3 also interacts with the dynactin/dynein motor complex and thus engages in retrograde transport of JNK signaling in response to nerve injury. Our findings provides support for Pin1 as a link between JIP3-propagated axonal injury signals and the activation of the cell death machinery. JIP3 and Pin1 have an intimate colocalization pattern in subcellular fractions in addition to the mitochondria in neurons. The JIP3 signaling complex participates in the regulation of other Pin1 functions in the nervous system, by counteracting the degenerative effects of hyperphosphorylated tau on the neuronal cytoskeleton. In addition to its role downstream of Ser65-phosphorylated BIMEL, Pin1 has a more general role in neuronal cell death, e.g., in dividing cells, Pin1 promotes cell cycle progression by acting on substrates of the mitotic kinase Cdc2. Cdc2 induces apoptosis in neurons and Pin1 may contribute to Cdc2-induced cell death in neurons.
Our results provide a basis for manipulating the expression or activity of Pin1 at the mitochondrial membrane and its interactions with JIP3 in the treatment of neurologic diseases.
|Cited Patent
|Filing date
|Publication date
|May 18, 1970
|Dec 7, 1971
|Alza Corp
|Drug-delivery device
|Aug 6, 1985
|Dec 6, 1988
|La Jolla Cancer Research Foundation
|Vitronectin specific cell receptor derived from mammalian mesenchymal tissue
|Aug 14, 1987
|May 15, 1990
|Clinical Technologies Associates, Inc.
|Delivery systems for pharmacological agents encapsulated with proteinoids
|Apr 28, 1995
|Sep 21, 1999
|Medtronic, Inc.
|Burr hole ring with catheter for use as an injection port
|Jun 4, 1999
|Dec 16, 1999
|The Salk Institute For Biological Studies
|Peptidyl-prolyl cis-trans isomerase inhibitors and uses therefor
|Accession No. AAC50492, May 1996.
|Accession No. BAA87038, Nov. 1999.
|Becker et al., "Characterization of the c-Jun-Terminal Kinase-BimEL Signaling Pathway in Neuronal Apoptosis", J. Neurosci., 24(40):8762-8770 (2004).
|Becker et al., "Pin1 Mediates Neural-Specific Activation of the Mitochondrial Apoptotic Machinery", Neuron, 49:655-662 (2006).
|Chao et al., "Juglone, an Inhibitor of the Peptidyl-Prolyl Isomerase Pin1, also directly blocks Transcription", Nuc. Acids Res., 29(3):767-773 (2001).
|Daum et al., "Aryl Indanyl Ketones: Efficient Inhibitors of the Human Peptidyl Prolyl cisl trans Isomerase Pin1", Angew. Chem. Int. Ed., 45:7454-7458 (2006).
|Etzkorn, F., "Synthesized Molecules Studied as Weapon to Stop Cell Division in Cancer Cells", EurekAlert, http://www.eurkalert.org/pub-releases/2004-08/vt-sms081604.php, 3 pages (2004).
|Etzkorn, F., "Synthesized Molecules Studied as Weapon to Stop Cell Division in Cancer Cells", EurekAlert, http://www.eurkalert.org/pub—releases/2004-08/vt-sms081604.php, 3 pages (2004).
|Gregoriadis, "Liposomes", Drug Carriers Biol. Med., (Academic Press), p. 287-341 (1979).
|Holzer et al., Inverse Association of Pin1 and tau accumulation in Alzheimer's disease hippocampus, 2002, Acta Neuropathol, 104, pp. 471-481.
|Joliot et al., "Antennapedia Homobox Peptide Regulates Neural Morphogenesis", Proc. Natl. Acad. Sci. USA, 88:1864-1868 (1991).
|Konishi et al., "Cdc2 Phosphorylation of BAD Links the Cell Cycle to the Cell Death Machinery", Mol. Cell, 9:1005-1016 (2002).
|Konishi et al., "Cdh 1-APC Controls Axonal Growth and Patterning in the Mammalian Brain", Science, 303:1026-1030 (2004).
|Kotin et al., "Site-Specific Integration by Adeno-Associated Virus", Proc. Natl. Acad. Sci. USA, 87:2211-2215 (1990).
|Lu et al., "Gene Transfer into Hepatocytes Using a Defective Herpes Simplex Viral Vector", Abstracts of the Meeting on Gene Therapy, Sep. 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Abstract, p. 66 (1992).
|Mahato et al., Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA, Jan. 2005, Expert Opinion on Drug Delivery, vol. 2, No. 1, pp. 3-28.
|Morsey et al., "Efficient Adenoviral Gene Transduction in Human and Mouse Hepatocytes In Vitro and in Mouse Liver In Vivo",J. Cell. Biochem., Abstract SZ 109, Supp. 17E:226 (1993).
|Pastorino et al. "The Prolyl Isomerase Pin1 Regulates Amyloid Precursor Protein Processign and Amyloid-beta Production" Nature, 440(23):528-534 (2006).
|Pastorino et al. "The Prolyl Isomerase Pin1 Regulates Amyloid Precursor Protein Processign and Amyloid-β Production" Nature, 440(23):528-534 (2006).
|Rosenberg et al., "Gene Transfer into Humans-Immunotherapy of Patients with Advanced Melanoma, Using Tumor-Infiltrating Lymphocytes Modified by Retroviral Gene Transduction", N. Eng. J. Med., 323(9):570-578 (1990).
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|Ryo et al., "Regulation of NF-κB Signaling by Pin1-Dependent Prolyl Isomerization and Ubiquitin-Mediated Proteolysis of p65/RelA", Mol. Cell., 12:1413-1426 (2003).
|Ryo et al., Stable Suppression of Tumorigenicity by Pin1-Targeted RNA Interference in Prostate Cancer, 2005, Clin Cancer Res, 11(20), 2005.
|Scherer et al., Approaches for the sequence-specific knockdown of mRNA, 2003, Nat. Biotechnol., 21(12), pp. 1457-1465.
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|Zhang et al., Targeted Gene Silencing by Small Interfering RNA-Based Knock-Down Technology, 2004, Current Pharmaceutical Biotechnology, vol. 5, p. 1-7.
|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|Jun 22, 2007
Owner name: PRESIDENT AND FELLOWS OF HARVARD COLLEGE, MASSACHU
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BONNI, AZAD M.;BECKER, ESTHER B.E.;REEL/FRAME:019470/0345
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|Jan 29, 2009
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
Free format text: CONFIRMATORY LICENSE;ASSIGNOR:HARVARD UNIVERSITY;REEL/FRAME:022171/0838
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Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF
Free format text: CONFIRMATORY LICENSE;ASSIGNOR:HARVARD UNIVERSITY;REEL/FRAME:024694/0362
Effective date: 20070126
|Feb 28, 2014
|Fee payment
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text:
i love that this recipe doesn't call for free oils of any kind- i couldn't find a "bean" recipe without it until now!
Rating4/5 (from 3 ratings)4
YieldServes 4
1 cup Sunflower seeds
½ cup Almonds
¼ cup Cashews
1 clove garlic, or more to taste
Braggs, to taste
¼ teaspoon mustard powder, celery, cumin
1 cup sun dried tomatoes, soaked 2-4 hours OPTIONAL
1 teaspoon Brewers Yeast, OPTIONAL
Recipe Directions
Soak your nuts and seeds (and sundried tomatoes if going that route) drain all the liquid then add all the rest of the ingredients into the food processor, I like it a little chunky, so I dont blend all the way.
Try it with the Spanish Rice, Sour Cream, Torilla and Taco Filling recipes from this site for a FIESTA!
Ambikalee's ThoughtsBy ambikalee
This is a combination recipe that is great for refried beans. The original is from Robyn Boyd and is in her book RawSome (mostly raw with some cooked…has kid approved recipes) Her version used 2 cups of almonds only and the rest of the OPTIONAL ingredients and she used Wholeearth Latin AMerica Spice blend instead of the herbs listed. This version is from my friend Beth who is a Raw Chef and likes to keep the power of the spice in your hands!
Print This Recipe (PDF)
Click the button below to download the printable PDF.
Top voted
I made this last night and it was Delicious!!! We could not stop eating it. I added to a burrito we had for dinner. Thanks for sharing.
I am out of nama shoya (sub for braggs) and did not use the yeast. This was really good. We had it in corn tortillas with avacados and salsa.
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subset: commoncrawl


text:
items tagged with Behind the Music
Written By: Mike Schulz
Section: Movies
Category: Reviews
2005-11-23 00:00:00
This summer, I was fortunate enough to catch a special screening of writer/director Don Roos' Happy Endings at the University of Iowa, but decided to hold off on a review until the film made it to our area.
Read More About A Summer Sleeper Trumps A Trio Of New Releases: "Happy Endings," "Walk The Line," "Harry Potter & The Goblet Of Fire," And "Kiss Kiss Bang Bang"...
Written By: Mike Schulz
Section: Movies
Category: Reviews
2004-10-13 00:00:00
Metallica: Some Kind of Monster has the sort of title guaranteed to repel viewers who might love it the most. This warts-and-all documentary, chronicling the two-plus years devoted to creating Metallica's St. Anger CD, is like the best episode of Behind the Music ever made, offering an intimate look at the relationship between guitarist James Hetfield and drummer Lars Ulrich, and detailing the nightmare involved in getting the group recording again after a five-year hiatus. The movie will be Mecca for metal fans, yet its appeal isn't totally insular. Audiences who may be loath to sit through a doc on any heavy-metal group might not realize what directors Joe Berlinger and Bruce Sinofsky have fashioned here; Some Kind of Monster is one of the finest recordings of the collaborative artistic process ever committed to film, a hard-edged and endlessly fascinating look at the excruciating work that goes into the making of an album. And for those for whom documentaries are even less appealing than heavy metal, it must be said that the film is one of the funniest and most shockingly touching screen works of the year, This Is Spinal Tap with actual human beings at its core. It's a thrilling experience.
Read More About "Metallica: Some Kind Of Monster" Transcends Metal And The Documentary: Also, "Shark Tale," "Friday Night Lights," And Ladder 49"...
Written By: Mike Schulz
Section: Movies
Category: Reviews
2003-11-26 00:00:00
Although I'm sure some of you are wondering if Dr. Suess' The Cat in the Hat is as thoroughly obnoxious as its trailers indicate, I'll begin assessing the major holiday releases next week. Until then, here's a look at the pre-Thanksgiving leftovers ... .
Read More About Fall Leftovers Full Of Turkeys: "Looney Tunes: Back In Action," "Dirty Pretty Things," "Tupac Resurrection," And "Brother Bear"...
There are 3 items tagged with Behind the Music. You can view all our tags in the Tag Cloud


text:
Jessica Chastain
Is Andrew Dominik's 'Blonde' Ever Going to Happen?
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- @templeofreviews
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- @FakeRobHunter
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- By Caitlin Hughes
- December 21, 2012
A fair amount of critics are touting Kathryn Bigelow's Zero Dark Thirty as her masterpiece. While Bigelow has definitely directed films in her decades of filmmaking that are comparable to the overall quality of Zero Dark Thirty, it is great that between this and her Oscar-winning The Hurt Locker, she...
Are All Those Critical Lauds Not Doing It For You? Fine, Here's a New 'Zero Dark Thirty' Trailer
- By Kate Erbland
- @katerbland
- December 13, 2012
If you've somehow managed to avoid the massive praise that's already been heaped on Kathryn Bigelow's Zero Dark Thirty – so far, they include four Golden Globe nominations, three National Board of Review wins, inclusion in AFI's list of the best films of the year, countless critics associations nominations, and...
'Zero Dark Thirty' Trailer: That's It, Jessica Chastain Is a Movie Star Now
- By Kate Erbland
- @katerbland
- October 11, 2012
Sure, this latest trailer for Kathryn Bigelow's Zero Dark Thirty is a lot of things: cool, collected, awesome, awesome, exciting, cool all over again, bold, vibrant, but it's also dominated by one overwhelming force that encompasses all those adjectives. Jessica Chastain. Did we somehow miss that this film, Bigelow and...
'Mama' Trailer: Let's Just Stop Adopting Feral Children, Okay?
- By Kate Erbland
- @katerbland
- September 13, 2012
Blanket statement: let's stop associating with feral children. It really hasn't worked out too well, you know? Cinematically speaking, we have Nell from Nell (heartbreaking), Howie Mandel's character from Walk Like a Man (embarrassing), Jason Vorhees from Friday the 13th (yup, he lived in the woods), The Penguin from Batman Returns...
'Lawless' Director John Hillcoat Stays Violent, But Goes Digital for 'Lawless'
- By Jack Giroux
- @JackGi
- August 28, 2012
Director John Hillcoat isn't entirely known for crowd-pleasing studio fare. After putting The Road and The Proposition under his belt, Hillcoat showed he's the type of filmmaker never to shy away from bleakness. One would think that's what made Lawless such a difficult project to get off the ground, but surprisingly, Hillcoat has made a real summer...
Review: Brutal Bootlegging Tale 'Lawless' Doesn't Go Down Quite So Smoothly
The twelve-year run of prohibition in the United States was a period that punctuated social imparity, religious activism, and was a launchpad for some of the biggest names in organized crime. Basically, it's a mixed bag of deeply interesting subject matter that is spot-on perfect for the big screen. Director...
For Exploring Race Relations Without Getting Preachy, 'The Landlord' Trumps 'The Help'
- By Nathan Adams
- @templeofreviews
- July 31, 2012
In 2011, director Tate Taylor adapted Kathryn Stockett's novel "The Help," a story about the relationship between the wealthy whites and the poor blacks who raised their children of 60s-era Mississippi, into a feature film. When all was said and done, Taylor's film made nearly ten times its production budget,...


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Thank you!
Appetizers/snacks for jewelry party
Thank you!
foodblog: http://kyannsmealplans.blogspot.com/
Comments (6)
check out my post "girls nite out"...there are a ton of ideas on it! :)
Married to the Love of my life 6/3/06 DS 8/26/08 YDS 3/15/11
<3 Hearts for Carter--Miss you Carter <3
actually it's girls nite food....to be more exact! :)
Married to the Love of my life 6/3/06 DS 8/26/08 YDS 3/15/11
<3 Hearts for Carter--Miss you Carter <3
i think these look like fun! family.go.com/food/pkg-family-appro...
i walked right out! Took my eyes out of my head, put them in my purse and drove home blind!
Tinkerbomb 4/22/04
Kikilicious 3/24/09
Something I always do is get lunch meat - I prefer the kind from Sam's Club or from the deli, super thin sliced. Roll it cut it in half and organize around a big plate. I usually do 2 diff meats. I also get 2 diff blocks cheese and cube/slice them myself and have a side plate of crackers.
Fruit Dip- 1 8 oz package cream cheese, 1 C sour cream, 1 pkg vanilla pudding (sm box), 1 C milk, 2 tsp vanilla, 2 tsp lemon juice, 3/4 C brown sugar.
- beat cream cheese adn brown sugar. then add each ingred one at a time, beating after each addition.
-Chill and serve w/ apple slices, peach, pineapple...
Both of these always get devoured the fastest in a crowd. :)
4x GOLD (. y .) <-- as if they're that perky still
How about a big antipasto platter with italian meats, cheeses, olives, marinated artichoke hearts, marinated mushrooms, roasted red peppers, hot pickled vegetable salad with sliced baguettes.
don't forget something chocolate
I'm a Gemini so I have a sweet side and a mean side. I'm also half Yankee, half Rebel and half Italian. Which side you will you get today?


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The Feast of the Ascension at Westminster Abbey / O'Donnell, Quinney, The Choir of Westminster Abbey
1 CD / Hyperion / 2008-05-13Works: Charles Villiers Stanford: Coelis Ascendit Hodie, motet for chorus, Op. 38/2; Bernard Rose: Preces and Responses; Responses and Dismissal; Joseph Barnby: The earth is the Lord's; Benjamin Britten: Fe...
Performers: Brett, Francis; Dobell, Mark; Empitt, Julian; Grote, Ashley; MacDonald, Robert [Bass Voice]; Quinney, Robert; Stocker, Julian; Trevelyan-Jones, Warren; Wallington, Lawrence
Conductors: O'Donnell, James [Organ]
Ensemble: Westminster Abbey Choir
"The first thing that hits you about the singing is the celebratory tone. The boys might be singing Stanford's Caelos ascendit hodie, but they could...continue...
read more Login or Create an Account to write a review
The English Anthem Vol 2 / Scott, St. Paul's Cathedral Choir
1 CD / Hyperion / 1992-03-01Works: Jonathan Battishill: O Lord, Look Down from Heaven, anthem for chorus & organ; John Stainer: How Beautiful Upon the Mountain, anthem for chorus & organ; Charles Villiers Stanford: Motets (3) for chor...
Performers: Lucas, Andrew
Conductors: Music], John Scott [Choral
'Spellbinding performances of some of the great classics of the repertoire. Buy this one; you'll enjoy every moment' (Organists' Review)...continue...
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If you've ever been part of a birth board, then you should read this and know its true:
If you haven't, read it and know what to expect.
Mama's are vicious, we all love our LO's to death and always think we know best, and we'll argue til our fingers bleed on here to get our point across, but eventually we all come together.
Good luck mama's!
~*~*~CaT, wife to Aaron 8/13/11,
mama to Orin 12/6/11~*~*~
***Expecting BABY BOY #2 8/30/13***
Mommy to Delaini, 3 and Bailie, 2 and expecting our Rainbow baby in August 2013 after two MMC in 2012!
Ahhhh people hormones are NOT the problem. Different personalities are!!!
Mama to my boys:
A (9/5/2010) and L (8/27/2013)
All bb's are the same. I didn't post much in my first bc I was afraid of the backlash. I quickly learned, grew a spine and took the opinions for what they're worth. If you don't like what a bunch of strangers think about your name list, why your ms suddenly disappeared, what a mucus plug looks like, etc...then don't post.
J & D - happily married since 11/1/08
V - born 12/8/11 via CS, ERF to 3y8m, FF in Frontier 90
J - born 8/5/13 via RCS, ERF in Graco 4Ever
I HATE it when someone has an opinion that differs from mine or makes different choices!
Both born peacefully at home:
ODS 7.2010 YDS 8.2013 (Hypnobabies)
I am the diaper sniffer! CD Swap Feedback
Go to July 2010 board, them ladies were ruthless... Hah! this is nothing compared to that... whew...
my baby girl (aybrie mae.) 7.23.10
team blue (is due) 8.26.13
Jill and Nic had Beautiful Simone 9/16/10 on mommy's b-day Rainbow Betty born 7/31/13 Nolan Ryan HAS ARRIVED!August 2015! I wish to remain anony- MOOSE
LOL... I didnt mean for this to start drama... I am a SPEECH AND DEBATE coach who welcomes differing opnions and fosters unique personalities. I just saw some mean remarks that werent EVEN expressing opinions on ANY matter. They were just mean. Doesnt really matter. The mean responses to this post certainly proved my point :). Thats ok... Im over 35 and on baby #3, perhaps I should know internet haggling a bit better by now lol...
Learn more
Look how far your child has come.
Review your child's growth milestones.
Read about 31 months : Week 4.
Your growing child
Your 32-month-old: It's rhyme time
Your 32-month-old: Let's pretend
Your 32-month-old: Nap-time strategies
Your 32-month-old: Intro to manners
Your 33-month-old: Taming fears
Learn more
What's next for your growing child?
Get a glimpse of what's to come.
Read about 33 months : Week 2.


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Java Architecture for XML Binding (JAXB)
Java Architecture for XML Binding (JAXB)
documents: Java Architecture for XML Binding (JAXB). It allows java developer
for mapping between the Java classes and the XML representations. JAXB allow us
JAXB Create XML File And Get Data From XML
the Java code and JAXB codes
has made easy to work with XML files. We can use...JAXB Create XML File And Get Data From XML
In this section we will read about how to create XML file and how to convert
XML file's data to Java Object
The Beginners Guide to JAXB
Java Architecture for XML Binding (JAXB) project provides a standard API... of JAXB and its importance in
binding XML to Java classes.
(Java Architecture for
The Beginners Guide to JAXB part 3
will create a java object tree and then send it to a file as xml document...;));
System.out.println("java tree converted into xml &...
java tree converted into xml & filed
Overriding jaxb binding at client side?
Overriding jaxb binding at client side? How can override jaxb binding at client side? I have a webservice client which creates xmlgregoriancalendar.... Overriding jaxb binding at client side
The Beginners Guide to JAXB
binding compiler classes
jaxb-impl.jar (formerly named jaxb-ri.jar)
The JAXB RI binding runtime framework classes
Supporting classes...
The JAXB RI is shipped with experimental DTD support, which lets you compile XML
Unmarshalling Jaxb
Unmarshalling Jaxb How to unmarshal very large xml to java objects usin jaxb/sax parser
Java Architecture for XML Binding
Java Architecture for XML Binding
Today, XML has emerged as the standard for exchanging data across disparate systems, and Java technology
XML data binding
XML data binding hello,
What is XML data binding?
XML data binding refers to the process of representing the information in an XML document as an object in computer memory
What XML JavaObject-XML transformation tools to use? JDOM, Dom4J, XOM, XStream, JAXB, JiBX, PojoXml
, JAXB, JiBX, PojoXml I just need some simple and stable xml tools to transform java objects to XML and back from XML to Java Objects. Provided the JAVA Objects are predefined and the xml schema/DTD is predefined. What is the best
Binding Error in Spring - Spring
Binding Error in Spring Error:
Neither BindingResult nor plain target object for bean name 'loginBean' available as request attribute
I am...;
Password :
My Maping in xml
An Overview of the XML-APIs
independent to vendor's implementation .
JAXB: Java Architecture for XML...;
Here we have listed all the major
Java APIs
for XML...: Java API for XML Processing
This API provides a common interface
Hibernate Architecture
Hibernate Architecture - Understand the architecture of the Hibernate ORM
Here we will discuss about the architecture of very extensible used Hibernate
ORM framework. Hibernate is based on the Java technologies
Data binding with data model in Flex 3
Data binding with data model:-
In this example we have used data model for binding data for objects.
<?xml version="1.0"?>
<mx:Application xmlns:mx="http://www.adobe.com/2006/mxml
Flex 3 data binding process and example
binding at run time in the Action
<?xml version...Data binding in Flex 3:-
Data binding is a process to pass data from one... of the application. There are
many type of data binding in Flex 3. The source
Binding in Flex
Binding in Flex Hi.......
Explain how binding works in mxml components?
Please give the answer with example.
Thanks Ans:
Binding in MXML
Please see the following code:
<s:Button id="btn" label
Binding in flex
Binding in flex Hi.........
Please tell me the Difference between...:
1. When we define a data binding in MXML it will use at compile time and we define a data binding in action script it will use at run time.
2. you use
Data Binding with XML Data Model in Flex4
Data Binding with XML DataModel in Flex4:
The <fx:XML> tag contains...;
title="Data Binding with XML
DataModel"... compiles the <fx:XML> tag into literal XML data. You can use
this data
Context binding
Context binding What is meant by Context binding?
A binding context is an object that holds data that you can reference from your... a hierarchy of binding contexts. The root level of the hierarchy refers to the viewModel
JSF Login Example By Using Component Instance Binding
JSF Login Example By Using Component
Instance Binding... value or component instance. Both types of
binding requires use... instance binding the property
holds the local value of the component
add XMl to JTable
add XMl to JTable Hi..
i saw the program of adding add XMl to JTable using DOM parser,but i need to do that in JAXB ,is it possible to do? help me
JavaScript Frameworks
XMLas XMLas is a tool for binding xml... is a Javascript API which encapsulates XML functionality in
browser-independent... is to have a robust and
solid logging API which is very simmilar to the Java logging
EAI - Service Oriented Architecture,EAI Architecture,Enterprise Application Integration,Enterprise Application Integration EAI
(Message Oriented Middleware), DMA (Distributed Messaging
Architecture), EDA (Event Driven Architecture), SOA (Service Oriented
Architecture), eAI (eBusiness... of integration frameworks like J2EE and DotNet. EIA
Architecture consists of integration
value and binding attribute
value and binding attribute What is the difference between value and binding attribute of jsf tag
value and binding attribute
value and binding attribute What is the difference between value and binding attribute in jsf
Log4J Architecture - Introduction to the Log4J architecture
Log4J Architecture - Introduction to the Log4J architecture... the architecture of Log4J is important.
Three main component of Log4J
The architecture... by using xml file. Using xml for Log4J is quite
popular and it is recommended
JSP Page Architecture and its life cycle
In this article, we are discussing the fundamentals of JSP architecture and it's life cycle. In short, Java Server Pages(JSP) is a high level extension of servlet, which is used to deliver HTML and XML documents. Using JSP one can easily


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User Comments
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Is this "Coire nan Leac" or "Coire na Lice"? Sorry to be a pedant but I'm into these things ;-)
Geoffrey Michaels - 16/Mar/08
Geoffrey Michaels - 16/Mar/08
Nominative! (Typing error corrected.) I seem to remember 'leac' occurs at least once elsewhere on this ridge line. A number of possible naming reasons are apparent. Not only are there numerous erratics in this corrie but these are added to with many marker stones on the way from the gorge up to the base of the faint ridge that disappears out of shot in the top right. Also, along the same route, and below in Coire Odhar, there are many large areas of smooth exposed rock.
Jim Fraser - 17/Mar/08
Jim Fraser - 17/Mar/08
Nice photo - we camped here one Easter a few years back on a backpack coming up from Glen Quoich to the south. It was about the only dry(ish) place for a tent. Passed below some interesting looking crags - see http://www.geograph.org.uk/browse.php?p=726788 Anything done on them - it's a long walk in?
Lankyman - 31/Dec/09
Lankyman - 31/Dec/09
Probably not. Most of the interest around there is in the corries on the north side in winter.
Jim Fraser - 31/Dec/09
Jim Fraser - 31/Dec/09


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Head Teacher's foreword
"As the Head Teacher of Boldon School, I am delighted to introduce the school to you and hope that you find this website helpful. Our brand new state of the art school opened for teaching on Monday 6th November 2006 when we moved from our very old buildings.
The students and staff who work here are very fortunate to be able to use excellent facilities including:
Each year group has their own 'breakout' area where they can relax. The 'Boldon Bistro', operated by Catering Academy, provides nutritious food at lunchtime.
At Boldon School, every student will experience a varied curriculum which fulfils statutory requirements but at the same time is tailored to meet individual need. Students begin KS4 in Year 9 and this maximizes achievement opportunities for every young person.
Thank you for visiting our website, I hope that you find all the information you require. You are very welcome to visit our school, please telephone or email and make an appointment."
Elizabeth M Hayes
Head Teacher
Our aim is to encourage all students to take advantage of the opportunities offered by the school to attain their full potential in all areas of school life – the academic, the social, the physical and the personal – so that they are ready both to play a productive part in a democratic society and to move forward to the next stage of education, training or career with confidence, as young people of responsibility and judgment.


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Complete Collection of Girls Seating
Child Wingback Chair - Toile Black by Angel Song
Our lovely Black Toile Wingback Child's Chair is a great chair for your little girl to relax in after a long day. Classic Black Toile covers the seat back, chair back, arms, and sides. Black and Ivory Check is used for the seat and skirt. The front arm panels and lower front panel are covered with Ivory Diamond Pintuck. This chair is highlighted with black and ivory cording and fringe. This is a truly unique children's chair unparalleled by any other manufacturer. Your little girl will love relaxing in this chair after a long day of school. Matching ottoman (sold separately) features a removable lid for storing toys, dolls, books, or any of the sort. Adult Empire, Adult Princess, Adult Park Avenue also available in matching chair style and fabric collection. This chair is meticulously handmade by experienced craftsmen in the USA to ensure the finest quality possible.
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The Looking Glass, Leicester - pub details
Address: 68-70 Braunstone Gate, Leicester, Leicestershire, LE3 5LG [map] [gmap]
Tel: 0871 951 1000 (ref 28640) - calls cost 10p per minute plus network extras
Leicester (1 mile), South Wigston (3.6 miles), Narborough (5.1 miles)
Pub facilities/features:
- Live music
- Late licence
Are you the Licensee? Click here.
> Post a comment > Mail a friend
other pubs nearby:
O Bar, Leicester (0.0 miles), Talk Bar and Grill, Leicester (0.0 miles), Black Horse, Leicester (0.0 miles), Pump and Tap, Leicester (0.1 miles), Western, Leicester (0.2 miles) - see more nearby pubs
user reviews of the Looking Glass, Leicester
please note - reviews on this site are purely the opinion of site visitors, so don't take them too seriously.
5 most recent reviews of 6 shown - see all reviews
|this is one of the better bars along the trendy braunstone gate. serves a huge range of spirits and continental beers. Also has live music on most evenings. run by the same people who have the Rutland & Derby.
Hot_Stuff - 10 Oct 2008 15:39
|Was rather underimpressed. Crowded, uncomfortable, found a perch eventually downstairs but it was claustrophobic. Kept getting jostled which really pisses me off. Enormous range of spirits which could have kept me amused all night, but not cheap. Soon moved on to more congenial quarters in the city.
pressforaction - 22 Jan 2008 18:02
|Crap cocktails, that's for sure. Good range of spirits and imported lagers, ale selection poor. Can have good live acts on. An odd place, that is not entirely conducive to relaxing drinking.
insekt - 27 Nov 2006 18:08
|I found my visit very pleasent, live music free on a friday night is a real a plus and with a huge range of drinks to choose from I can`t think of anything better!
anonymous - 25 Oct 2006 17:17
|Very elitist and not very comfortable. An old clientel, nothing special on the cocktail menu and far too expensive. What are you paying for? A "try-too-hard" interior and dirty facilities? Grossly unimpressed.
anonymous - 16 Aug 2006 00:53
see more reviews


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Editor rating : 8
Price: 56,900
Editor rating : 8
Price: 24,999
Editor rating : 8
Price: 14,999
Editor rating : 7.5
Price: 89,900
Editor rating : 7.5
Price: 39,990
Washington: Rocks analysed by NASA's Mars Curiosity rover have provided solid evidence that the red planet once had flowing rivers or streams, researchers say.
Despite satellite images that show vast networks of channels, past Mars rover missions have shown limited evidence for flowing water on Mars.
Now, rocks analysed by the rover team, including Linda Kah, associate professor of Earth and planetary sciences at The University of Tennessee, Knoxville suggest that the environment was drastically different than today's cold and dry conditions, with the potential to support life.
Since its landing in August last year, the Curiosity has been looking for clues to whether the Martian surface has ever had environments capable of sustaining, or potentially evolving, life.
Critical evidence may include hydrated minerals or water-bearing minerals, organic compounds, or other chemical ingredients related to life.
Scientists of the Mars Science Laboratory mission used images collected from the rover's MastCam, which includes two high-resolution cameras mounted onto its mast.
As the rover moved from its landing site to its current location in "Yellowknife Bay," the cameras captured images of large rock formations composed of many rounded pebbles cemented into beds several centimetres thick.
While such deposits are very common on Earth, the presence of these types of rocks on Mars has great significance for the
red planet.
"These (rock formations) point to a past on Mars that was warmer, and wet enough to allow water to flow for many kilometres across the surface of Mars," said Kah, who helped work the cameras.
The clasts, or pebbles within the rock formation, appear to have been rounded by erosion while carried through water, such as in a stream or river. The size and orientation of the pebbles suggest they may have been carried by one or more shallow, fast-moving streams.
Using published abrasion rates and taking into consideration reduced gravity, the scientists estimate the pebbles were moved at least a few kilometres.
Analysing the grain size distribution and similar rock formations, the scientists believe the river was less than a meter deep and the water's average velocity was 0.2 to 0.75 meters per second.
"These rocks provide a record of past conditions at the site that contrasts with the modern Martian environment, whose atmospheric conditions make liquid water unstable," said Kah.
"Finding ancient river deposits indicates sustained liquid water flows across the landscape, and raises prospects of once habitable conditions," Kah said.
The study was published in the journal Science.
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