CN1192248A - Method for the detection of compounds that modulate the effects of the obese protein - Google Patents
Method for the detection of compounds that modulate the effects of the obese protein Download PDFInfo
- Publication number
- CN1192248A CN1192248A CN96195983A CN96195983A CN1192248A CN 1192248 A CN1192248 A CN 1192248A CN 96195983 A CN96195983 A CN 96195983A CN 96195983 A CN96195983 A CN 96195983A CN 1192248 A CN1192248 A CN 1192248A
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- China
- Prior art keywords
- obesity
- compound
- albumen
- response element
- reporter gene
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- 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
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- 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
-
- 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
-
- 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
- C07K14/575—Hormones
- C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
Abstract
A method for the detection of a compound that mimics, potentiates or inhibits the physiological effect of the ob-protein, which method comprises: (a) for a compound which mimics the physiological effect of the ob-protein; assessing the effect of the compound upon an ob-protein activated signal transducer and activator of transcription (STAT) NDA response element coupled to a reporter gene; or (b) for a compound which potentiates or inhibits the physiological effect of the ob-protein assessing the effect of the compound upon the response provided by ob-protein upon an ob-protein activated STAT DNA response element coupled to a reporter gene; the response element and the reporter being expressed in an ob-protein responsive cell line; a kit of parts adapted for use in such method and a compound when identified by such method.
Description
The present invention relates to a kind of novel method, more specifically say the method for the compound of the physiological action that relates to a kind of detection simulation, strengthens or suppress obesity albumen (ob protein).
Obesity albumen (being leptine) is to regulate a kind of secreting hormone (Zhang etc., Nature, 1994,372,425) of body weight and energy balance as the signal from fatty tissue to other organs.Tool is inferred obesity albumen also work (Cioffi etc., NatureMedicine, 1996,2 (5), 585) in hematopoiesis and reproductive function.Containing one and comprise four α spirals, form a branch of high and low of having up-up-down-down) protein molecule of the core of topological framework comprises a family of cytokine and somatomedin.The protein of this family cause known activation cause the membrane receptor homotype of the kinase cascade reaction of genetic transcription-and abnormal shape-oligomerization close reaction.This family receptors that is closed reacting activation by oligomerization is divided into two classes; Promptly as the epidermal growth factor subclass, they have complete tyrosine kinase activity (A.Ullrich at its intracellular functional domain; J.Schlessinger, Cell, 1990,61,203-212), and as IL4 and erythropoietin class, they lack this activity and mediate its reaction (J.N.Ihle etc., TIBS by a kind of bonded protein tyrosine kinase pathway, 1994,19,222-227).Two kinds of receptor subtypes are activated by cytokine all, but 4 helix bundle proteins only activate non-complete Tyrosylprotein kinase hypotype.This non-whole protein tyrosine kinase receptor generally works by the approach relevant with transcriptional activation agent (STAT) albumen with the signal transducer of Janus kinases (JAK) and these receptors bind.In the activation process,, stat protein transcribes thereby being attached on the DNA response element controlling gene.The oligonucleotide sequence that comprises the DNA regulatory element of general sequence TT (N) nAA has been accredited as STAT response element (Seidel etc., Proc.Nat.Acad.Sci.USA, 1995,92,3041).When signaling molecule such as cytokine were reacted, these elements combined with stat protein.
The discovery that we have described us in common unsettled United Kingdom's number of patent application 9509164.1 is that obesity albumen is feature with the four-helix bundle tertiary structure.We think that obesity albumen and the membrane-bound receptor that activates the reaction of JAK-STAT kinase cascade interact and be configured for detecting simulation thus, strengthen or the basis of the analytical system of the compound of the proteic physiological action of inhibition obesity now.This detection is in that to select compound to be subjected to treatment body weight, energy balance, hematopoiesis, reproduction and other aspect disease of " obesity albumen " mediation useful.This detects selecting compound particularly useful with obesity, apocleisis, cachexia and diabetes diseases associated with treatment.
Therefore, the invention provides a kind of method that detects simulation, strengthens or suppress the compound of obesity albumen physiological action, this method comprises:
(a), assess the effect of this compound pair and reporter gene link coupled obesity albumen activation signal transducer and transcriptional activation agent (STAT) DNA response element at the compound of simulation obesity albumen physiological action; Or
(b) to strengthening or suppress the compound of the proteic physiological action of obesity, assess this compound to provide by obesity albumen, for the effect of the reaction of reporter gene link coupled obesity albumen activated STATDNA response element;
Response element and reporter gene are expressed in obesity albumen test clone.
This response element is fit to and reporter gene, best and minimal promoter coupling.
A kind of suitable response element is nucleic acid molecule formula TT (N)
nAA, wherein N is that any Nucleotide and n are 4,5 or 6.
By with the protein mediated cell of the obesity of its acceptor interaction in the preferred response element of incident selectively activate.This selective reaction element can be by with some response elements-when reporter gene construct transfection obesity reacting cells is, detect to be subjected to obesity albumen and to determine with respect to the relative activation of other cytokines.
The preferred reaction element is nucleic acid molecule formula TT (N)
nAA, wherein N is that any Nucleotide and n are 5.
Further the response element that is fit to is TTCCCGGAA.
Further the response element that is fit to is the promoter region that is subjected to the gene of obesity albumen adjusting, and it is that the STAT interaction is needed.This gene depends on the concrete therepic use that detects the compound of selecting by this.
The reporter gene that is fit to is Photinus pyralis LUC or E.C. 2.3.1.28.
The promotor that is fit to is minimal promoter such as herpes simplex virus thymidine kinase or SV40 promotor.
An example of " the obesity reaction " clone is liver or liver cancer deutero-clone.Liver or hepatoma cell line obtain from American Type Culture Collection (ATCC) or EurpeanCollection of Animal Cell Cultures (ECACC).An example of this class clone is that Hep G2 (human hepatocellular carcinoma) obtains from ATCC (HB8065).This Hep G2 clone is open and the proposition claim by United States Patent (USP) 4393133.
Further " the obesity reaction " clone example is rat-1 inoblast (Kroder etc., Exp.Clin.Endocrinol.Diabetes, 1996,104 (suppl2), 66).Rat 1 fibroblast obtains from ATTCC (CRL-2210).
Available permutations is identified other reacting cells systems in conjunction with detecting.Though in conjunction with may not being the persistent forms of function of receptors, it is to pass the signal along to cytoplasmic form.Can identify the persistent forms of function of receptors by PCR or Northern engram analysis (for example people's obesity albumen: Tartaglia etc., Cell, 1995,83,1263).Cellular activity detection reaction cell when finally existing with different concns by monitoring leptin.The possible method of identifying candidate clone or monitoring these cellular activities comprises following method:
1. little physiological measurements instrument (Microphysiometer): this method detects the subtle change of the pH that the biological chemistry variation is caused in the cell.Thereby obesity albumen test cell can experience the minor alteration that the biological chemistry variation causes the extracellular acidification rate during irriate, and acidification rate can detect by the little physiological measurements instrument of siloxanes.This little physiological measurements instrument method for biosensor is by McConnell, and Science summarizes in 1992,257,1906.
2. electrophoretic mobility displacement calibrating (EMSA): the nuclear extract of the cell that in the future personal obesity albumen was handled with contain miscellaneous or single-minded STAT response element dna sequence dna and mix mutually.Extract from the cell that obesity albumen is reacted can cause the displacement of SATA response element oligonucleotide gel.
Referring to: book " recombinant DNA ", second edition, Watson etc., 1992,158 pages; Lamb etc., Blood, 1994,83,2063;
3. protein phosphorylation is examined and determine method of masurement: can examine and determine the coupling of receptor activation reaction by intracellular protein tyrosine phosphorylation and end reaction by the antibody that uses the identification phosphorylated tyrosine.More specifically say, because the leptin acceptor can stimulate the tyrosine phosphorylation of JAK/STAT approach, the method that this method provides a kind of leptin of detection reacting cells to be.Specificity JAK/STAT antibody can use the leptin to detect in the leptin reacting cells system to activate with tyrosine phosphorylation antibody side by side.The inhibition and the stimulation of protein phosphorylation may take place.Particularly, in cross expressing rat-1 inoblast of (overpress) insulin receptor, demonstrated by obesity albumen produce to the restraining effect (Kroder etc. in the phosphorylation process of the insulin stimulating of plain acceptor in skin island and IRS-1,1996, Exp.Clin.Endocrinol.Diabetes, 104, suppl 2, p66).
4. displacement combination: with radiolabeled leptin, for example [
125I]-leptin is after temperature is bathed clone together, can combine with specificity by adding cold leptin research with respect to non-specificity bonded leptin.Specificity/non-single-minded this clone of the prompting of combination at high proportion may contain the leptin acceptor.
5. by using the protein function form of antibodies selective detection obesity protein receptor, the proteinic function persistent forms of best detection.
6. pass through Northen, the mRNA functional form of RT-PCR or " slit engram " analyzing and testing obesity protein receptor, the function persistent forms of best detection mRNA.
Preferred knownly relate to control those seek the clone of the special disease state aspect of compound just for it.
Cell that obtains from liver, brain or pancreas tissue and inoblast are particularly useful to being suitable for examining and determine at " obesity reaction " cell of the compound of obesity and diabetes.Some zone of brain is weight management and the energy balance center of regulating the proteic effect of obesity.Liver is controlled the metabolic process of many adjustment fat and sugar levels.Contain suitable endogenous JAK from those, stat protein and other specific region deutero-cells for the organ of the necessary intracellular protein of mediation leptin function are paid the utmost attention to.
In the carrier that participates in energy transfection obesity reacting cells system that described response element, reporter gene and preferred promoter is suitable.
The carrier that is fit to is that commercially available carrier such as pGL-2 are basic luciferase carrier (Promega).
The configuration that is fit to of this carrier is the STATDNA response element of promotor and reporter gene upstream.A kind of configuration that is more suitable for of described carrier is the STAT DNA response element that repeats (2-10) in the series multiple of thymidine kinase promoter and luciferase reporter gene upstream.
The carrier that makes up contains a reporter gene for example Photinus pyralis LUC or E.C. 2.3.1.28, and for example herpes simplex virus thymidine kinase or SV40 promotor link to each other with minimal promoter.The dna fragmentation of STAT response element can be inserted into described carrier with the suitable restriction enzyme sites that is arranged in described minimal promoter upstream.
On demand response element, reporter gene and promotor are attached in the carrier with conventional expression technology, for example the dna fragmentation of response element can be inserted into described carrier with the suitable restriction enzyme sites that is arranged in the minimal promoter upstream.
According to Blood such as Lamb, 1994,8,2963 and Seidel etc., Proc.Nat.Acad.SciUSA can make up STAT response element-luciferase reporter gene system described in 1995,92,3041.
Adopt for example calcium phosphate method (Graham and Van Der Eb, Virology, 1973,52,456) STAT response element-minimal promoter-luciferase reporter gene construct transfection obesity reacting cells of standard method.For revising the difference of transfection efficiency, this cell of reference plasmid co-transfection of available expression betagalactosidase activity.In transfection after date (12-24 hour), this cell of compound treatment of usefulness different concns is harvested cell and molten born of the same parents then.The uciferase activity of calibrating lysate and if suitable words calibrating beta galactosidase enzyme activity.In assessment with measure with respect to the enhancing of the proteic luciferase reaction of independent use obesity and in this compound, can identify toughener or antagonistic activity by pre-adding or the obesity albumen that adds proper concn altogether when reducing.The existing standard method of identifying uciferase activity is Ow etc. for example, " Science, 1986,234,856 and Wet etc., Mol.Cell Biol.1987,7,725 and several commercial reagent box.
But by producing stable clone with reporter gene construct and selective marker transfection " obesity reacting cells system ".But the conventional selective marker of using is used to produce stable clone as at recombinant DNA, the description in the second edition, 1992,216 pages of J.D.Watson etc.Can the transfectional cell series that these are stable be used to produce simulation, strengthen or the high yield calibrating of the compound of the proteic physiological action of blocking-up obesity.
When examining and determine with method disclosed herein, the present invention also provides simulation, strengthens or suppress the compound of the proteic physiological action of obesity.
The present invention also provides a cover test kit that is fit to use in the method disclosed herein.
' compound of simulation obesity albumen physiological action ' that uses in this article is meant can play when lacking described obesity albumen stimulates the obesity protein receptor so that the compound of basic identical effect in proteic physiological role of obesity or activation this receptor downstream reaction (behind the acceptor) to be provided.
' the strengthening the compound of the proteic physiological action of obesity ' of using in this article is meant the compound that strengthens obesity albumen potential and/or maximum physiological action.
' compound that suppresses the proteic physiological action of obesity ' that use in this article is meant the compound that reduces or block the proteic physiological action of obesity basically.
Following embodiment explains the present invention
Embodiment
General method:
With the reporter plasmid transfection obesity reacting cells that contains series multiple copy STAT response element, this response element is positioned at minimal promoter for example herpes simplex virus thymidine kinase and luciferase gene reporter gene upstream, this plasmid construction is with standard method calcium phosphate method (Grahamand Van Der Eb for example, Virology, 1973,53,456).For revising the difference of transfection efficiency, this cell of reference plasmid co-transfection of available expression-galactosidase activity.Collect and molten born of the same parents then with the compound treatment cell of different concns (12-24 hour) after transfection for some time.The uciferase activity of calibrating lysate, and if suitable time calibrating beta galactosidase enzyme activity.In this compound, can identify toughener or antagonistic activity by pre-adding or the obesity albumen that adds proper concn altogether in assessment and under measuring with respect to the condition of the enhancing of the proteic luciferase reaction of independent use obesity and reduction.The existing standard method of identifying uciferase activity is Ow etc. for example, " Science, 1986,234,856 and de Wet etc., 1987,7,725 and several commercial reagent box.
Embodiment
Adopt for example calcium phosphate method (Graham and Van Der Eb of standard method, Virology, 1973,52,456), with reporter plasmid, promptly contain a pGL2 who inserts oligonucleotide for luciferase carrier (promega) transfection on basis from liver cancer deutero-clone, this oligonucleotide is corresponding to the STAT response element of herpes simplex virus thymidine kinase (35 to+10) minimal promoter upstream, the quadruple tandem repetitive sequence of TTCCCGGAA.For revising the difference of transfection efficiency, this cell of reference plasmid co-transfection of available expression galactosidase activity.Collect and molten born of the same parents then with the compound treatment cell of different concns (12-24 hour) after transfection for some time.If the uciferase activity of calibrating lysate is suitable time calibrating beta galactosidase enzyme activity.In assessment with measure with respect to the enhancing of the proteic luciferase reaction of independent use obesity and when reducing, in this compound, can identify toughener or antagonistic activity by pre-adding or the obesity albumen that adds proper concn altogether.The standard method of existing calibrating uciferase activity is Ow etc. for example, " Science, 1986,234,856 and de Wet etc., Mol.cell biol, 1987,7,725 and several commercial reagent box.
Claims (11)
1. method that detects simulation, strengthens or suppress the compound of the proteic physiological action of obesity, this method comprises:
(a) at the compound of simulation obesity albumen physiological action, assess this compound to obesity albumen activated signal transducer and with the effect of reporter gene link coupled transcriptional activation agent (STAT) DNA response element; Or
(b) to strengthening or suppress the compound of the proteic physiological action of obesity, assess this compound to provide by obesity albumen, for the effect of the reaction of reporter gene link coupled obesity albumen activated STATDNA response element;
Response element and reporter gene are expressed in obesity albumen test clone.
2. one kind meets and the process of claim 1 wherein response element and promoter gene coupling, preferably minimal promoter.
3. method that meets claim 2, wherein response element is formula TT (N)
nThe Nucleotide of AA, wherein N is that arbitrary Nucleotide and n are 4,5 or 6, preferably 5.
4. method that meets claim 2, wherein said response element is a formula TTCCCGGAA Nucleotide.
5. one kind meets and the process of claim 1 wherein that reporter gene is Photinus pyralis LUC or E.C. 2.3.1.28.
6. one kind meets and the process of claim 1 wherein that described promotor is herpes simplex virus thymidine kinase or SV40 promotor.
7. one kind meets and the process of claim 1 wherein that described obesity reacting cells system is liver or liver cancer deutero-clone.
8. one kind meets and the process of claim 1 wherein described response element, reporter gene and promotor participated in can the carrier of transfection obesity reacting cells system in.
9. method that meets claim 8, wherein said carrier are that pGL2 is the luciferase carrier (Promega) on basis.
10. method that meets claim 8 or claim 9, the configuration of wherein said carrier is such, promptly STAT DNA response element is in the upstream of promotor and reporter gene.
11. be adapted at detecting simulation, strengthen or suppress the cover reagent that uses in the method for compound of obesity albumen physiological action,
Described method comprises:
(a) at the compound of simulation obesity albumen physiological action, assess this compound to obesity albumen activation signal transducer and with the effect of reporter gene link coupled transcriptional activation agent (STAT) DNA response element; Or
(b) to strengthening or suppress the compound of the proteic physiological action of obesity, assess this compound to provide by obesity albumen, for the effect of the reaction of reporter gene link coupled obesity albumen activated STATDNA response element;
Response element and reporter gene are expressed in obesity albumen test clone.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9510857.7A GB9510857D0 (en) | 1995-05-30 | 1995-05-30 | Novel assay |
| GB9510857.7 | 1995-05-30 | ||
| GBGB9511602.6A GB9511602D0 (en) | 1995-06-08 | 1995-06-08 | Novel assay |
| GB9511602.6 | 1995-06-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1192248A true CN1192248A (en) | 1998-09-02 |
Family
ID=26307107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96195983A Pending CN1192248A (en) | 1995-05-30 | 1996-05-28 | Method for the detection of compounds that modulate the effects of the obese protein |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0832284A1 (en) |
| JP (1) | JPH11505725A (en) |
| KR (1) | KR19990022134A (en) |
| CN (1) | CN1192248A (en) |
| AU (1) | AU715215B2 (en) |
| BR (1) | BR9608686A (en) |
| CA (1) | CA2222409A1 (en) |
| CZ (1) | CZ379097A3 (en) |
| HU (1) | HUP9801744A3 (en) |
| MX (1) | MX9709360A (en) |
| NO (1) | NO975504D0 (en) |
| NZ (1) | NZ309777A (en) |
| PL (1) | PL323638A1 (en) |
| TR (1) | TR199701469T1 (en) |
| WO (1) | WO1996038586A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001968A (en) | 1994-08-17 | 1999-12-14 | The Rockefeller University | OB polypeptides, modified forms and compositions |
| US6429290B1 (en) | 1994-08-17 | 2002-08-06 | The Rockefeller University | OB polypeptides, modified forms and derivatives |
| US6007998A (en) * | 1996-04-22 | 1999-12-28 | Merck & Co., Inc. | Leptin assay |
| JP2000511406A (en) * | 1996-04-22 | 2000-09-05 | メルク エンド カンパニー インコーポレーテッド | Leptin assay |
| US6001816A (en) * | 1996-06-20 | 1999-12-14 | Merck & Co., Inc. | Gene therapy for leptin deficiency |
| WO1998020158A1 (en) * | 1996-11-01 | 1998-05-14 | Smithkline Beecham Plc | Method for the detection of compounds that modulate the effects of the obese (ob) protein |
| IT1293533B1 (en) * | 1997-07-14 | 1999-03-01 | Angeletti P Ist Richerche Bio | METHOD FOR THE SELECTION OF MOLECULES ABLE TO MIMIC, INHIBIT OR ENHANCE THE EFFECTS OF THE INTERACTION BETWEEN LEPTIN AND CELLS THAT |
| WO1999023493A1 (en) * | 1997-10-31 | 1999-05-14 | The Rockefeller University | Methods of identifying agents that modulate leptin activity |
| JP2002506004A (en) * | 1998-02-11 | 2002-02-26 | ベス イスラエル ディーコネス メディカル センター | Methods and compositions for modulating leptin activity |
| GB9814199D0 (en) * | 1998-06-30 | 1998-08-26 | Univ Buckingham | Novel method |
| GB9828087D0 (en) * | 1998-12-18 | 1999-02-17 | Smithkline Beecham Plc | Novel method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU7795094A (en) * | 1993-09-15 | 1995-04-03 | New York University | Dna sequence which binds transcriptional regulatory proteins activated in response to various cytokines and uses thereof |
| JPH09500026A (en) * | 1994-04-14 | 1997-01-07 | リガンド・ファーマシューティカルズ・インコーポレーテッド | Cytokine-responsive DNA spacer regulatory element and method of use thereof |
-
1996
- 1996-05-28 JP JP8536176A patent/JPH11505725A/en active Pending
- 1996-05-28 HU HU9801744A patent/HUP9801744A3/en unknown
- 1996-05-28 AU AU60023/96A patent/AU715215B2/en not_active Ceased
- 1996-05-28 CZ CZ973790A patent/CZ379097A3/en unknown
- 1996-05-28 PL PL96323638A patent/PL323638A1/en unknown
- 1996-05-28 MX MX9709360A patent/MX9709360A/en unknown
- 1996-05-28 CA CA002222409A patent/CA2222409A1/en not_active Abandoned
- 1996-05-28 EP EP96917454A patent/EP0832284A1/en not_active Withdrawn
- 1996-05-28 BR BR9608686A patent/BR9608686A/en active Search and Examination
- 1996-05-28 WO PCT/EP1996/002291 patent/WO1996038586A1/en not_active Application Discontinuation
- 1996-05-28 NZ NZ309777A patent/NZ309777A/en unknown
- 1996-05-28 TR TR97/01469T patent/TR199701469T1/en unknown
- 1996-05-28 KR KR1019970708613A patent/KR19990022134A/en not_active Application Discontinuation
- 1996-05-28 CN CN96195983A patent/CN1192248A/en active Pending
-
1997
- 1997-11-28 NO NO975504A patent/NO975504D0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| MX9709360A (en) | 1998-02-28 |
| AU6002396A (en) | 1996-12-18 |
| CZ379097A3 (en) | 1998-05-13 |
| HUP9801744A2 (en) | 1998-10-28 |
| TR199701469T1 (en) | 1998-03-21 |
| HUP9801744A3 (en) | 2000-06-28 |
| KR19990022134A (en) | 1999-03-25 |
| NZ309777A (en) | 2000-01-28 |
| WO1996038586A1 (en) | 1996-12-05 |
| CA2222409A1 (en) | 1996-12-05 |
| BR9608686A (en) | 1999-07-06 |
| NO975504L (en) | 1997-11-28 |
| PL323638A1 (en) | 1998-04-14 |
| EP0832284A1 (en) | 1998-04-01 |
| AU715215B2 (en) | 2000-01-20 |
| JPH11505725A (en) | 1999-05-25 |
| NO975504D0 (en) | 1997-11-28 |
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