WO1997040380A1 - Dosage de la leptine - Google Patents

Dosage de la leptine Download PDF

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Publication number
WO1997040380A1
WO1997040380A1 PCT/US1997/006505 US9706505W WO9740380A1 WO 1997040380 A1 WO1997040380 A1 WO 1997040380A1 US 9706505 W US9706505 W US 9706505W WO 9740380 A1 WO9740380 A1 WO 9740380A1
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WIPO (PCT)
Prior art keywords
leptin
promoter region
promoter
reporter gene
response element
Prior art date
Application number
PCT/US1997/006505
Other languages
English (en)
Inventor
Charles I. Rosenblum
Leonardus Van Der Ploeg
Sajjad A. Qureshi
Doris F. Cully
John W. Hess
Michael R. Tota
Fang Chen
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9611785.8A external-priority patent/GB9611785D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to EP97924507A priority Critical patent/EP0914611A4/fr
Priority to JP09538203A priority patent/JP2000511406A/ja
Publication of WO1997040380A1 publication Critical patent/WO1997040380A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention relates to an assay for determining the presence of compounds which bind to a leptin receptor. This invention also relates to an assay for quantifying the amount of a leptin receptor binding agent present in a sample.
  • Leptin is a product of the Ob gene (Zhang et al., 1995 Nature 372:425-432). In its absence, mice develop extreme obesity resembling morbid obesity of humans. They also develop diabetes resembling human type D diabetes (Zhang et al., 1995 Nature 372:425- 432), and become infertile (Chehab et ai, 1996 Nature Genetics 12:318- 320). Repletion of leptin leptin reverses these pathologies.
  • This invention relates to a method for determining if a leptin receptor binding compound is present in a sample comprising: contacting the sample with a cell which comprises a) nucleic acids comprising a promoter region, said promoter region comprising a promoter and at least one leptin response element; said promoter region operatively linked to a reporter gene; and b) nucleic acids encoding a leptin receptor; and determining if transcription of the reporter gene occurs.
  • Figure 1 is a graph showing functional assay of leptin using GTI -7 cells.
  • Cells were either: mock transfected with respect to ob-r, transfected with lean rat Ob-r cDNA, or transfected human Ob-r cDNA and showed induction of luciferase activity in cell expressing Ob-r treated with leptin.
  • Figure 2 is a graph showing that cells respond in a linear, dose-responsive manner to the addition of leptin as determined by increase in luciferase activity.
  • GTI -7 cells were transiently transfected with human Ob-r and treated with various amounts of human leptin.
  • Figure 3 is the DNA sequence for lean rat Ob-r.
  • Figure 4 is the DNA sequence for human Ob-r which has been placed in the pCMV4 vector.
  • Gen Bank accession number for human ob-r is U43168.
  • Nucleotides 141 -37-7-0 are used in pCMV4- Ob-4.
  • Figure 5 is the DNA sequence for fatty rat Ob-r.
  • Leptin receptor binding compound means a compound which binds to a leptin receptor.
  • Leptin mimetic means a compound other than leptin which binds to a leptin receptor and triggers a cascade of intracellular reactions which ultimately results in transcription of DNA under the control of leptin response elements.
  • Leptin response elements means DNA sequences located within a promoter region which are responsive to a leptin receptor binding event, and in the presence of such an event, allow for transcription of the DNA under the control of the promoter.
  • Promoter region means DNA located upstream of a protein or peptide encoding sequence, and includes at least one response element.
  • Promoter includes full length promoters, minimal promoters and promoters which are less than full length, but include more nucleic acids than minimal promoters.
  • Ob-r means Ob-receptor.
  • One aspect of this invention is a cellular "trans" activation assay which can determine if a particular compound can bind to a leptin receptor.
  • the binding of a ligand to its membrane -bound receptor initiates an intercellular cascade of signals.
  • a specific transcriptional control element is activated, leading to the transcription of DNA.
  • the binding of native leptin with its native leptin receptor initiates a cascade which activates leptin response elements, leading to the transcription of DNA.
  • One such leptin response element which has been identified in accordance with this invention is an IRF-1 derived garnma-interferon activation sequence. IRF-1 derived activation sequences have been described in the art (Pine et al., 1994 EMBO J. 13:158-167) but their responsiveness to leptin-leptin receptor binding events has been heretofore unknown.
  • a promoter region is constructed which contains a promoter of choice and one or more leptin response elements.
  • the promoter region may contain be a hybrid promoter region, i.e. contain a promoter and response elements which do not naturally occur together, or they may be a naturally occurring promoter region.
  • the promoter is a well-characterized promoter, such as the he ⁇ es simplex virus thymidine kinase promoter, but any promoter which is known to function in the host cell chosen may be used. It is also preferred that the promoter be a minimal promoter, so that there are no transcriptional control sequences which may influence the activity of the leptin response element(s).
  • the leptin-response element is preferably placed proximal to the promoter region.
  • Intervening sequences may be present between the leptin response element and the promoter, provided they do not interfere with the functioning of the leptin response element.
  • a suitable leptin response element is an LRF-1 derived gamma interferon activation sequence 5'-CTGATTTCCC CGAAATGACG-3 * .
  • the promoter region comprises a plurality of IRF- 1 derived gamma interferon activation sequences; and more preferably it comprises at least three such sequences.
  • a plurality of leptin response elements are present, they are preferably joined in tandem. However, there may be interviewing sequences present provided they do not interfere with the functioning of the leptin response elements.
  • a reporter gene may be any gene which encodes a peptide which is easily detected, or otherwise allows for easy detection of transcription or translation. It generally encodes a protein which does naturally occur in the host cell or only is produced in small amounts by the host cell. Examples of well known reporter genes include: chloramphenicol acetyl transferase (CAT), green fluorescent protein (GFP), luciferase (either bacterial or firefly), and other enzyme-based detection systems such as ⁇ -galactosidase, alkaline phosphatase, and the like. In a particularly preferred embodiment, luciferase is a reporter gene. In alternative embodiments, the mRNA transcribed from the reporter gene DNA may be measured rather than the translation product.
  • the reporter gene construct which comprises a) the promoter region which contains a promoter and at least one leptin response element and b) the reporter gene operatively linked to the promoter region forms yet another aspect of this invention. It is preferably placed in an appropriate vector and in used to transfect a host cell.
  • This vector comprises yet another aspect of this invention.
  • the vector may be any known vector, including plasmids, cosmids and viral vectors which can function in a chosen host cell.
  • the host cell may be any cell or cell line which is conveniently cultured.
  • a preferred host cell will be eukaryotic, preferably mammalian, and in particularly preferred embodiments it is a mouse hypothalamic cell (such as GTI-7), a mammalian neuroblastoma cell line, mouse fibroblast cell lines such as NIH/3T3 (ATCC CRL 1658) or L (ATCC CCL 1), Chinese hamster ovary CHO-Kl cells (ATCC CCL 61) or human embryonic kidney- derived 293 cells (ATCC CRL 1573).
  • the host cell should either have an abundance of leptin receptors on its surface or it can be transformed to express an abundance of leptin receptors on its surface, the latter situation being the preferred one. Any leptin receptor may be used; human and rodent ones are preferred. Nucleic acids encoding human leptin receptors have been described (Tartaglia et al., 1995 Cell 83: 1263- 1271 which is hereby inco ⁇ orated by reference) and are shown in Figure 4. Mouse genes, can also be used, especially the position 26 to 2775, based on the sequence given in Gen Bank.
  • Nucleic acids encoding leptin receptors from wild-type YdXs, fatty rats (fa/fa genotype) (shown in Figure 3) and rats heterozygous for the ob receptor gene (shown in Figure 5) are described in co-pending U.S. application Serial Nos. and
  • Another aspect of this invention is a set of vectors suitable for transfecting a host cell so that it can be used in the assays of this invention.
  • the set of vectors comprises a first vector which contains a leptin receptor construct.
  • the leptin receptor construct includes nucleic acids encoding a desired leptin receptor or mutated form of the receptor. It may be under the control of its native promoter or any other desired heterologous promoter. Optionally it may also contain other expression-control elements, such as enhancers and sequences which assist in expressing the receptor on the membrane.
  • the set of vectors also comprises a second vector comprising the receptor gene construct described previously.
  • compounds which are suspected of being leptin receptor ligands can be assayed.
  • cells which express a leptin receptor are transfected with the reporter gene construct described previously and is transfected with a leptin receptor construct, if the cell does not naturally express an abundance of the leptin receptor.
  • the putative leptin receptor ligand is placed in contact with the transfected cells, and the presence of the reporter gene transcription or translation product is detected. This may be compared to the amount of transcription or translation measured in a control assay, where an identically transfected cell is placed in contact with leptin.
  • a further advantage of the assay of this invention is that it is dose-responsive; i.e. as more leptin ligand-leptin receptor binding occurs, transcription and/or translation of reporter gene increases, and therefore allows for a quantitative determination of leptin receptor binding activity.
  • a counterscreen may also be employed as a part of this assay.
  • a second cell of the same cell type is transfected with the reporter gene construct described previously, but is not transfected with the leptin receptor gene construct.
  • the putative leptin receptor ligand is placed in contact with the transfected cells and the presence of of the reporter gene transcription or translation is detected.
  • Those putative leptin receptor ligands which activate the reporter gene construct only in the presence of leptin receptor are determined to be specific leptin agonists.
  • leptin agonists and antagonists may be identified.
  • a leptin agonist is a compound which binds to the leptin receptor, such as a leptin mimetic, and produces a cellular response which is at least about equivalent to that of leptin, and which may be greater than that of leptin. Such compounds would be useful in situations where leptin insufficiency causes obesity, diabetes or infertility.
  • leptin antagonists may be identified.
  • a leptin antagonist is a compound which can bind to the leptin receptor, but produces a lesser response than that of native leptin. Such compounds would be useful in the treatment of anorexia and cachexia.
  • novel leptin response elements can be identified by inserting various putative leptin response elements proximal to a promoter which controls the transcription of a reporter gene.
  • a cell transfected with this construct and expressing natural or recombinant leptin receptors is contacted with leptin.
  • the resulting transcription of the reporter gene is compared with that which occurs when the leptin response element is an LRF-1 derived gamma interferon activation sequence.
  • Novel leptin response elements which are identified using this assay form yet another aspect of this invention.
  • the potency of a leptin preparation can be determined, and quantified.
  • the leptin-containing preparation is contacted with the cell containing the reporter gene construct and which is expressing leptin receptors (either recombinant or native).
  • the amount of transcription and/or translation of the reporter gene is measured and is compared to that obtained using preparations of known potency.
  • the biological activity of various leptin receptors can be studied and compared.
  • a first cell is transfected with a reporter gene construct and a first leptin receptor.
  • the first leptin receptor can be any leptin receptor or variant or mutant whose activity is to be determined.
  • the first cell is put into contact with leptin and the reporter gene transcription or translation is measured. This amount is compared to that obtained under the same assay conditions using a second leptin receptor whose activity is known or is otherwise to be used as a reference.
  • AH32 an expression vector transactivated by leptin, was constructed as follows. Two complimentary oligonucleotides containing the Stat Binding Element (SBE) from the IRF- 1 gene (Sims et ai, 1993 Mol Cell Biol. 13:690-702; and Pine et al., 1994 EMBO J. 13: 158-167; both of which are hereby inco ⁇ orated by reference) were synthesized, kinased and annealed using standard molecular biology techniques (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • SBE Stat Binding Element
  • Plasmid pTKLuc contains a promoter sequence from -35 to + 10 of the he ⁇ es simplex virus thymidine kinase gene (McKnight et al., 1982 Science, 217:316-324) upstream of the firefly luciferase gene found in pZLuc (Schadlow, et al., 1992 Mol. Biol Cell 3:941 -951).
  • the ligated DNA was used to transform E. coli using DH5 ⁇ competent cells (Gibco/BRL, Gaithersburg, MD). DNA obtained from the transformed colonies was analyzed by restriction digest analysis and the orientation and number of copies of SBE oligonucleotides found in each of the transformants was confirmed by sequencing through the insert using a sequencing kit (US Biochemical, Cleveland, OH). Clone AH32 was found to contain 3 copies of the SBE inserted in the same orientation and was used for further studies with a human fibroblast cell line (WI- 38 VA13 subline 2RA; ATCC No. CCL 75.1).
  • the standard PCR reaction mix in a final volume of 20 ⁇ l, consisted of 40 ng of template (rat cDNA), 200 ng of primers, 500 mM dNTPs, 1 X Buffer 3 from the Expand kit, and 0.2 ml of the Expand kit enzyme mix. Reactants were assembled in thin walled reaction tubes.
  • the amplification protocol was 1 cycle of 92°C for 30 sec, followed by 35 cycles at 92°C for 30 sec, 54°C for 1 min. and 68°C for 5 min. using a Perkin Elmer 480 Thermal Cycler.
  • the DNA sequence of the full length products was determined using ABI PRISM Dye terminator cycle sequencing with AmpliTaq DNA polymerase, FS (Perkin Elmer, Foster City), and a number of PCR induced errors were detected. To eliminate these PCR induced mutations, restriction fragments from clones containing the correct DNA sequence were assembled. Three different restriction fragments were used to construct a full length clone derived from fa/fa hypothalamic cDNA, with a C at nucleotide 880.
  • the expression construct was obtained by ligating a fragment from a hind III polylinker site to a Nde I site at nucleotide 502, a fragment from the Nde I site at 502 to a Sac I site at 1522, and a fragment from the Sac I site at 1522 to a Xba I polylinker site into the Hind lU and Xba I sites of pcDNA 3 (Invitrogen, San Diego, CA).
  • the wild type (lean) clone, with an A at nucleotide 880 was constructed by replacing the Nde I by Sac I fragment from 502 to 1522 with a fragment derived from lean hypothalamic cDNA.
  • pCHI 10 a vector expressing ⁇ -galactosidase under control of the SV40 early promoter (Hall et al., 1983, J Mol Appl Gen 2: 101- 109), was obtained from Pharmacia Biotech, Inc. (Piscataway, NJ).
  • GTI -7 cells a mouse hypothalamic cell line, a gift of P. L. Mellon (Liposits et al., 1991 Endocrinology 129: 1575-1583) were maintained in Dulbecco-modified Eagle Medium (DMEM) (Gibco/BRL, Gaithersburg, MD), 10 % bovine calf serum, 50 ⁇ g/ml streptomycin, and 50 U/ml penicillin (growth medium) at 37°C in an atmosphere of 95% air, 5% C ⁇ 2- Cells were seeded at approximately 4x lO ⁇ /cm ⁇ and passaged before confluence.
  • DMEM Dulbecco-modified Eagle Medium
  • Cells were washed twice with phosphate buffered saline. The DNA-liposome complex was applied to cells. Cells were incubated for 6 hours at which time 2 ml of growth medium was added to cells. 16 hours later, the transfection mixture was removed and 2 ml fresh growth medium was added.

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Abstract

Dosage de la leptine par transactivation. Les éléments de réponse à la leptine sont situés à proximité d'un promoteur, tandis que la région du promoteur est liée de manière fonctionnelle au gène reporter. Lorsque la leptine se lie au récepteur, le gène reporter est transcrit.
PCT/US1997/006505 1996-04-22 1997-04-18 Dosage de la leptine WO1997040380A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97924507A EP0914611A4 (fr) 1996-04-22 1997-04-18 Dosage de la leptine
JP09538203A JP2000511406A (ja) 1996-04-22 1997-04-18 レプチンアッセイ

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US1605196P 1996-04-22 1996-04-22
US60/016,051 1996-04-22
GBGB9611785.8A GB9611785D0 (en) 1996-06-06 1996-06-06 Leptin assay
GB9611785.8 1996-06-06
US3100296P 1996-11-15 1996-11-15
US60/031,002 1996-11-15

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WO1997040380A1 true WO1997040380A1 (fr) 1997-10-30

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JP (1) JP2000511406A (fr)
CA (1) CA2252443A1 (fr)
WO (1) WO1997040380A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020158A1 (fr) * 1996-11-01 1998-05-14 Smithkline Beecham Plc Procede pour la detection de composes modulant les effets de la proteine de l'obesite (ob)
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
WO2001027319A2 (fr) * 1999-10-10 2001-04-19 Yeda Research And Development Co. Ltd. Dosage de la leptine
US6310034B1 (en) 1993-05-21 2001-10-30 Ut-Battelle, Llc Agouti polypeptide compositions
JP2002521496A (ja) * 1998-07-28 2002-07-16 フラームス・インテルウニフェルシタイル・インスティチュート・フォール・ビオテヒノロヒー レプチン介在性遺伝子誘導
US7063958B1 (en) 1996-01-16 2006-06-20 The Rockefeller University Nucleic acids db, the receptor for leptin
US7084252B1 (en) 1996-01-16 2006-08-01 The Rockefeller University DB, the receptor for leptin
US7148004B1 (en) 1997-01-16 2006-12-12 The Rockefeller University Oligonucleotides of the OB-R isoforms and methods of diagnosing body weight
US7619079B2 (en) 1996-02-14 2009-11-17 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002639A1 (fr) * 1990-08-07 1992-02-20 The Salk Institute Biotechnology/Industrial Associates, Inc. Techniques et compositions de detection et d'evaluation de la transduction intracellulaire d'un signal extracellulaire
WO1996038586A1 (fr) * 1995-05-30 1996-12-05 Smithkline Beecham Plc Procede de detection de composes modulant les effets de la proteine ob
US5643748A (en) * 1994-09-14 1997-07-01 Progenitor, Inc. HU-B1.219, a novel human hematopoietin receptor

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002639A1 (fr) * 1990-08-07 1992-02-20 The Salk Institute Biotechnology/Industrial Associates, Inc. Techniques et compositions de detection et d'evaluation de la transduction intracellulaire d'un signal extracellulaire
US5643748A (en) * 1994-09-14 1997-07-01 Progenitor, Inc. HU-B1.219, a novel human hematopoietin receptor
WO1996038586A1 (fr) * 1995-05-30 1996-12-05 Smithkline Beecham Plc Procede de detection de composes modulant les effets de la proteine ob

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, September 1992, Vol. 10, HODGSON J., "Receptor Screening and the Search for New Pharmaceuticals", pages 973-977. *
BIOCHEM. BIOPHYS. RES. COMM., May 1996, Vol. 222, IIDA et al., "Phenotype-Linked Amino Acid Alteration in Leptin Receptor cDNA from Zucker Fatty (fa/fa) Rat", pages 19-26. *
CELL, 09 February 1996, Vol. 84, CHEN et al., "Evidence that the Diabetes Gene Encodes the Leptin Receptor: Identification of a Mutation in the Leptin Receptor Gene in Db/Db Mice", pages 491-495. *
CELL, 29 December 1995, Vol. 83, TARTAGLIA et al., "Identification and Expression Cloning of a Leptin Receptor, OB-R", pages 1263-1271. *
ENDOCRINOLOGY, November 1996, Vol. 137, No. 11, ROSENBLUM et al., "Functional STAT 1 and 3 Signaling by the Leptin Receptor (OB-R); Reduced Expression of the Rat Fatty Leptin Receptor in Transfected Cells", pages 5178-5181. *
FEBS LETTERS, August 1996, Vol. 392, WANG et al., "A Novel Leptin Receptor Isoform in Rat", pages 87-90. *
NATURE MEDICINE, May 1996, Vol. 2, No. 5, CIOFFI et al., "Novel B219/OB Receptor Isoforms: Possible Role of Leptin in Hematopoiesis and Reproduction", pages 585-589. *
NATURE, 15 February 1996, Vol. 379, LEE et al., "Abnormal Splicing of the Leptin Receptor in Diabetic Mice", pages 332-335. *
PROC. NATL. ACAD. SCI. U.S.A., August 1996, Vol. 93, BAUMANN et al., "The Full-Length Leptin Receptor Has Signalling Capabilities of Interleukin 6-Type Cytokine Receptors", pages 8374-8378. *
PROC. NATL. ACAD. SCI. U.S.A., June 1996, Vol. 93, GHILARDI et al., "Defective STAT Signaling by the Leptin Receptor in Diabetic Mice",pages 6231-6235. *
See also references of EP0914611A4 *
THE EMBO J., 1994, Vol. 13, No. 1, PINE et al., "Tyrosine Phosphorylated p91 Binds to a Single Element in the ISGF2/IF-1 Promoter to Mediate Induction by IFNalpha and IFNkappa and is Likely to Autoregulate the p91 Gene", pages 158-167. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6310034B1 (en) 1993-05-21 2001-10-30 Ut-Battelle, Llc Agouti polypeptide compositions
US6514747B2 (en) 1993-05-21 2003-02-04 Ut-Battelle, Llc Agouti polynucleotide compositions and methods of use
US7521258B2 (en) 1994-08-17 2009-04-21 The Rockefeller University Methods of detecting, measuring, and evaluating modulators of body weight in biological samples, and diagnostic, monitoring, and therapeutic uses thereof
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US7812137B2 (en) 1996-01-16 2010-10-12 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
US7612171B2 (en) 1996-01-16 2009-11-03 The Rockefeller University DB, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
US7063958B1 (en) 1996-01-16 2006-06-20 The Rockefeller University Nucleic acids db, the receptor for leptin
US7084252B1 (en) 1996-01-16 2006-08-01 The Rockefeller University DB, the receptor for leptin
US7619079B2 (en) 1996-02-14 2009-11-17 The Rockefeller University Db, the receptor for leptin, nucleic acids encoding the receptor, and uses thereof
WO1998020158A1 (fr) * 1996-11-01 1998-05-14 Smithkline Beecham Plc Procede pour la detection de composes modulant les effets de la proteine de l'obesite (ob)
US7148004B1 (en) 1997-01-16 2006-12-12 The Rockefeller University Oligonucleotides of the OB-R isoforms and methods of diagnosing body weight
JP2002521496A (ja) * 1998-07-28 2002-07-16 フラームス・インテルウニフェルシタイル・インスティチュート・フォール・ビオテヒノロヒー レプチン介在性遺伝子誘導
KR100863580B1 (ko) 1999-10-10 2008-10-15 예다 리서치 앤드 디벨럽먼트 캄파니 리미티드 렙틴 분석법
WO2001027319A2 (fr) * 1999-10-10 2001-04-19 Yeda Research And Development Co. Ltd. Dosage de la leptine
WO2001027319A3 (fr) * 1999-10-11 2001-06-21 Yeda Res & Dev Dosage de la leptine

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CA2252443A1 (fr) 1997-10-30
EP0914611A4 (fr) 2002-10-16
EP0914611A1 (fr) 1999-05-12

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