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WO2000066787A2 - Genes et proteines hepatiques pouvant etre regules par l'hormone de croissance, utilisations associees - Google Patents

Genes et proteines hepatiques pouvant etre regules par l'hormone de croissance, utilisations associees Download PDF

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Publication number
WO2000066787A2
WO2000066787A2 PCT/US2000/012366 US0012366W WO0066787A2 WO 2000066787 A2 WO2000066787 A2 WO 2000066787A2 US 0012366 W US0012366 W US 0012366W WO 0066787 A2 WO0066787 A2 WO 0066787A2
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gene
liver
expression
level
activity
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WO2000066787A3 (fr
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John Joseph Kopchick
Jean Tiong
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Ohio University
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Ohio University
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Priority to CA002370134A priority Critical patent/CA2370134A1/fr
Priority to US09/959,716 priority patent/US6858389B1/en
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Anticipated expiration legal-status Critical
Publication of WO2000066787A3 publication Critical patent/WO2000066787A3/fr
Priority to US11/032,232 priority patent/US7309572B2/en
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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
    • C07K14/46—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
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • This invention relates to the diagnosis of abnormal GH activity or general pathological activity in the liver.
  • the growth hormones are vertebrate proteins with about 191 amino acid residues, the number varying from species to species. There are four cysteine residues, and two disulfide bridges.
  • the 3D-structure of porcine GH is known; it is composed of four major antiparallel alpha-helices, at residues 7-34, 75-87, 106-127 and 152-183.
  • the 3D structure of the hGH.hGH receptor complex is also known. Each molecule of hGH binds two molecules of the receptor. hGH binds to two binding sites on hGH receptor. Helix 4, the loop residues 54-74, and, to a lesser extent, helix 1, mediate binding to binding site 1. Helix 3 mediates binding to binding site 2.
  • GH is synthesized and secreted by the somatotrophic and somatomammotrophic cells of the lateral anterior pituitary.
  • the control of GH production and secretion is complex, but is mainly under the influence of growth hormone releasing hormone (GHRH) and somatostatin, which stimulate and inhibit it, respectively.
  • GHRH growth hormone releasing hormone
  • somatostatin somatostatin
  • the shifting balance between these regulatory agents is responsible for the pulsatile nature of GH secretion, with normal human concentrations ranging from a baseline value ⁇ 1 ⁇ g/L to peaks of 25-50 ⁇ g/L.
  • Glucocorticoids and thyroid hormones, and various carbohydrates, amino acids, fatty acids and other biomolecules are also known to directly or indirectly regulate GH secretion.
  • GH is secreted at night, during deep sleep, but some is secreted in response to exercise and other forms of physical stress. About 500 ⁇ g/m2 body surface area are secreted by women, and 350 by men. GH secretion rates are highest in adolescents and lowest in the elderly. GH has a plasma half life of about 20-25 min. and is cleared at a rate of 100-150 ml/m2 body surface area.
  • GH Besides affecting skeletal growth, can also influence other organ systems, in particular, the liver and kidney.
  • the kidney In the kidney, it has been associated with glomerulosclerosis and nephropathy. (Diabetic glumerosclerosis and nephropathy has been attributed to a GH effect.)
  • the liver In the liver, it has been shown to cause an increase in liver size, as a consequence of both hyperplasia and hepatocyte hypertrophy. The hepatocellular lesions associated with high GH levels progress with age. See Quaife, et al, Endocrinol . , 124: 49 (1989); Sharp, et al . , Lab.
  • agents useful for this purpose are those which are competitive binding antagonists of GH . It was discovered that certain mutants of the third alpha helix of GH are useful for this purpose. Kopchick, USP 5,350,836. In order to determine whether it is appropriate to initiate or terminate use GH antagonists or other GH- inhibiting drugs, it is important to be able to monitor GH activity.
  • GH Activi ty The most straightforward marker of GH activity is the serum level of GH per se. For humans, the mean GH concentration (ug/L) in blood is preadolescent 4.6 early adolescent 4.8 late adolescent 13.8 adult 1.8
  • ISS idiopathic short stature
  • IDDM insulin dependent diabetes mellitus
  • IGF-1 insulin-like growth factor-1
  • proinsulin insulin-like growth factor-1
  • IGF-1 levels have been used by clinicians to confirm suspected cases of acromegaly.
  • mice transgenic for IGF-1 do not develop the same abnormalities as mice transgenic for GH, in particular, they do not develop similar liver and kidney abnormalities. See Quafe, supra, and Yang, et al . , Lab. Invest., 68:62-70 (1993) .
  • Applicants have identified certain genes whose expression in liver cells is elevated as a result of higher than normal GH levels. In contrast, Applicants were unable to identify similarly GH-regulated genes in kidney cells.
  • nucleic acid binding agents to bind messenger RNA transcripts produced by the transcription of any of these genes (or to bind the corresponding complementary DNAs synthesized in vitro)
  • a protein binding agent to bind a protein encoded by any of these genes
  • transgenic mammals especially mice, rats and rabbits, which overproduce these proteins may be useful as animal models of liver pathologies.
  • agents which inhibit expression of these proteins i.e., antisense nucleic acids
  • the binding of these proteins to their receptors by binding either the protein or the receptor
  • interferon- ⁇ / ⁇ receptor IFNR ⁇
  • CBG corticosteroid binding globulin
  • ⁇ - fetoprotein ⁇ - fetoprotein
  • cytochrome P450 fetuin
  • Ahsg fetuin
  • 3 ⁇ HSD 3- ⁇ - hydroxysteroid
  • PON-3 paraoxonase-3
  • rab8 interacting protein coagulation factor V.
  • liver pathologies include:
  • Liver cirrhosis hepatic disease of various etiology
  • liver disease such as -Alcoholic liver disease -portal hypertension
  • Liver tumor hepatic disease of various etiology
  • transgenic mammals e.g. mice
  • transgenic mammals that overexpress the cDNA in a liver-specific manner (using a liver-specific promoter like the albumin or PEPCK promoter) , and determine if these transgenic mammals develop liver histopathologies, or other signs of aging (GH transgenic mice die prematurely of liver and kidney disease) .
  • transgenic mammals in which expression of these genes is knocked out can be examined to determine if they provide any protection to the liver against any of the agents known to cause liver pathology, e.g., viral infection (esp. hepatitis), alcoholism, hepatoxic drugs, tumors, etc. if so, then an agent interfering with the expression or activity of the gene product would have therapeutic value.
  • agents known to cause liver pathology e.g., viral infection (esp. hepatitis), alcoholism, hepatoxic drugs, tumors, etc. if so, then an agent interfering with the expression or activity of the gene product would
  • the proteins of interest include both secreted and mtracellular proteins.
  • They can also be used as indicators of a pathophysiological state. Also, they may be "peptide hormones". Thus, they could have diagnostic or therapeutic value. Depending upon the scenario, recombinant agonists or antagonists may emerge from these molecules .
  • Intracellular proteins could regulate the intrinsic biological functions of certain cells. These proteins could be potential drug targets in that one may design molecules to activate or inhibit them.
  • 3- ⁇ -Hydroxysteroid Dehydro ⁇ enase (3- ⁇ -HSD)- Isomerase and Dehydrogenase that plays an important role in all aspects of steroid production. It is present in many different isofor s which indicates multiple functionality. It acts in the liver as a key enzyme in the cholesterol biosynthetic pathway and as a transporter of bile acids [Marscall HU, et al . Hepa tology 2000 Apr; 31 ( 4 ): 990-6] It has also been reported that GH administration to cultured cells stimulated the activity of 3- ⁇ -HSD. [Gregoraszczuk EL, et al.
  • Rab8 interacting protein- Rab proteins are small GTP binding proteins involved in vesicular transport during endocytosis and exocytosis. They are distant relatives of the ras family of oncogenes, but are not oncogenic themselves. Rab ⁇ ip shows similarity to the GC kinase, a serine/threonine kinase that has recently been identified in stress activated human lymphoid tissue. It is thought that Rab ⁇ ip may have a role in modulation of secretion in response to stress stimuli. [Ren M, et al . Proc Na tl Acad Sci USA 1996 May 14;93(10) :5151-5]
  • PON3 Paraoxonase 3
  • HDL high density lipoprotein
  • LDL low density lipoprotein
  • Interferon / ⁇ receptor (IFN ⁇ R)- Interferons are antiviral, antiproliferative, immune responsive cytokines. Recombinant forms have been in use for the treatment of various malignancies. Serum levels of soluble IFN ⁇ R have been found to be elevated in patients with chronic hepatitis C. [Mizukoshi E, et al . Hepa tology 1999 Nov; 30 (5) : 1325-31] It is thought that resistance to IFN therapy in patients with chronic hepatitis C may be due to low levels of hepatic IFN ⁇ R. [Yatsuhashi H, J Hepa tol 1999 Jun; 30 ( 6) : 995-1003] . Thus any method by which this IFN "binding protein" would be increased could be beneficial. Since the soluble version of this has been found, and it is secreted, it could be used as a diagnostic marker.
  • GHR Growth Hormone Receptor
  • Cytochrome P450- The cytochromes are an extensive family of Heme containing electron transport molecules found in liver microsomes. They convert a wide range of substrates to forms that are more easily excreted by the cell, some of which may be carcinogenic. The cytochromes are also involved in steroid and prostaglandin biosynthesis.
  • Proteosome subunit Z- A component of the multicatalytic Proteinase complex found in the eukaryotic cytosol and nucleus that is responsible for ubiquitin dependent protein degradation. It has recently been reported that GHR internalization requires proteosome action and active ubiquitin conjugation system, [van Kerkhof P, J Biol Chem 2000 Jan 21; 275 (3) : 1575-80] . Any substance that could control ubiquitination could be of value.
  • CBG Corticosteroid Binding Globulin
  • Coagulation Factor V- Coagulation factors are a group of protease enzymes and cofactors involved in clotting. Their activation is triggered by tissue injury and phospholipoprotein release, which ultimately leads to the production of thrombin. Again, any substance that could up or down regulated blood clotting could be of value.
  • Gene expression may be said to be specific to a particular tissue if the average ratio of the specific mRNA to total mRNA for the cells of that tissue is at least 10% higher than the average ratio is for the cells of some second tissue. Absolute specificity is not required. Hence, a gene may be said to be expressed specifically in more than one tissue.
  • the markers of the present invention are, singly or in combination, more specific to the target tissue than are serum GH or IGF-1 levels, or than GH mRNA or IGF-1 mRNA levels in the target tissue.
  • sequences are to be aligned using a local alignment program with matches scored +5, mismatches scored -4, the first null of a gap scored -12, and each additional null of the same gap scored -2.
  • Percentage identity is the number of identities expressed as a percentage of the length of the overlap, including internal gaps .
  • the in vitro assays of the present invention may be applied to any suitable analyte-containing sample, and may be qualitative or quantitative in nature.
  • the assay target is a messenger RNA transcribed from a gene which, in liver cells, has increased transcriptional activity if serum GH levels are increased.
  • This messenger RNA may be a full length transcript of the gene, or merely a partial transcript. In the latter case, it must be sufficiently long so that it is possible to achieve specific binding, e.g., by nucleic acid hybridization.
  • the messenger RNA is extracted from liver cells by conventional means.
  • the assay target may be a complementary DNA synthesized in vitro from the messenger RNA as previously described.
  • the term "gene" or “target sequence” will be used to refer to the messenger RNA or complementary DNA corresponding to the induced gene, and to the coding gene proper.
  • the assay target is a protein encoded by said gene and expressed at higher levels in response to elevated GH levels. If the protein is secreted, the assay may be performed on serum. If the protein is not secreted, then liver cells will be obtained from the subject and lysed to expose the cytoplasmic contents. In either embodiment, one or more purification steps may be employed prior to the practice of the assay in order to enrich the sample for the assay target.
  • alpha-fetoprotein fetuin alpha-fetoprotein fetuin
  • genes of particular interest are those encoding the above proteins. These genes were identified, as described in Example 1, on the basis of the identity or similarity of mouse cDNAs obtained by subtractive hybridization methods to known mouse genes or cDNAs (or, in the case of the S-2 hydroxyacid oxidase, to a known rat gene) . The mouse sequencens were transferred onto a nylon membrane.
  • the membrane may then be used in a hybridization reaction with a suitable probe, which may be a synthetic probe directed against a gene already known to be a marker, or which may be a cDNA probe prepared directly from subtractive hybridization, wherein the fragment encoding the gene of interest, that is enriched in GH-overproducing subjects, will be labeled, preferably either radioactively with 32 P or non-radiactively with DIG (Digoxigenin) .
  • a negative control such as one composed of RNA sample from liver of normal subjects, may be resolved side by side with the patients' sample, Detection of this gene or protein could therefore indicate the presence of liver problem.
  • DNAs are also of interest. These are identified below as clones 5 and 45.
  • the proteins encoded by the ORFs embedded in these DNAs are also of interest.
  • the sample may be of any biological fluid or tissue which is reasonably expected to contain the messenger RNA transcribed from one of the above genes, or a protein expressed from one of the above genes.
  • the sample may be of liver tissue or interstitial fluid, or of a systemic fluid into which liver proteins are secreted.
  • a non-invasive sample collection will involve the use of urine samples from human subjects. Blood samples will also be obtained in order to obtained plasma or serum from which secreted proteins can be evaluated. Liver aspirates can also be obtained to detect for the presence of genes and proteins of interest. The most invasive method would involve obtaining liver biopsies.
  • the preferred binding reagent is a complementary nucleic acid.
  • the nucleic acid binding agent may also be a peptide or protein.
  • a peptide phage library may be screened for peptides which bind the nucleic acid assay target.
  • a DNA binding protein may be randomly mutagenized in the region of its DNA recognition site, and the mutants screened for the ability to specifically bind the target. Or the hypervariable regions of antibodies may be mutagenized and the antibody mutants displayed on phage.
  • the preferred binding reagent is an antibody, or a specifically binding fragment of an antibody.
  • the antibody may be monoclonal or polyclonal. It can be obtained by first immunizing a mammal with the protein target, and recovering either polyclonal antiserum, or immunocytes for later fusion to obtain hybridomas, or by constructing an antibody phage library and screening the antibodies for binding to the target.
  • the binding reagent may also be a binding molecule other than an antibody, such as a receptor fragment, an oligopeptide, or a nucleic acid. A suitable oligopeptide or nucleic acid may be identified by screening a suitable random library.
  • the assay may be a binding assay, in which one step involves the binding of a diagnostic reagent to the analyte, or a reaction assay, which involves the reaction of a reagent with the analyte.
  • the reagents used in a binding assay may be classified as to the nature of their interaction with analyte: (1) analyte analogues, or (2) analyte binding molecules (ABM) . They may be labeled or insolubilized.
  • the assay may look for a direct reaction between the analyte and a reagent which is reactive with the analyte, or if the analyte is an enzyme or enzyme inhibitor, for a reaction catalyzed or inhibited by the analyte.
  • the reagent may be a reactant, a catalyst, or an inhibitor for the reaction.
  • An assay may involve a cascade of steps in which the product of one step acts as the target for the next step. These steps may be binding steps, reaction steps, or a combination thereof.
  • SPS Signal Producing System
  • the assay In order to detect the presence, or measure the amount, of an analyte, the assay must provide for a signal producing system (SPS) in which there is a detectable difference in the signal produced, depending on whether the analyte is present or absent (or, in a quantitative assay, on the amount of the analyte) .
  • SPS signal producing system
  • the detectable signal may be one which is visually detectable, or one detectable only with instruments. Possible signals include production of colored or luminescent products, alteration of the characteristics (including amplitude or polarization) of absorption or emission of radiation by an assay component or product, and precipitation or agglutination of a component or product.
  • signal is intended to include the discontinuance of an existing signal, or a change in the rate of change of an observable parameter, rather than a change in its absolute value.
  • the signal may be monitored manually or automatically.
  • the signal is often a product of the reaction.
  • a binding assay it is normally provided by a label borne by a labeled reagent.
  • a label may be, e.g., a radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound, an agglutmable particle.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography .
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H, 32 P, 125 I, 131 I, 35 S, 14 C, and, preferably, 125 I .
  • the label may also be a fluorophore.
  • the fluorescently labeled reagent When the fluorescently labeled reagent is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamme, phycoerythnn, phycocyanm, allophycocyanm, o- phthaldehyde and fluorescamme .
  • fluorescence-emitting metals such as 125 Eu, or others of the lanthanide series, may be incorporated into a diagnostic reagent using such metal chelatmg groups as diethylenetriammepentaacetic acid (DTPA) of ethylenediamme-tetraacetic acid (EDTA) .
  • DTPA diethylenetriammepentaacetic acid
  • EDTA ethylenediamme-tetraacetic acid
  • the label may also be a chemilummescent compound.
  • the presence of the chemilummescently labeled reagent is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemilummescent labeling compounds are lum ol, lsolummo, theromatic ac ⁇ dmium ester, lmidazole, acridinium salt and oxalate ester.
  • Biolummescence is a type of chemilummescence found biological systems in which a catalytic protein increases the efficiency of the chemilummescent reaction. The presence of a biolummescent protein is determined by detecting the presence of luminescence.
  • Important biolummescent compounds for purposes of labeling are luciferin, luciferase and aequonn.
  • Enzyme labels such as horseradish peroxidase and alkaline phosphatase, are preferred.
  • the signal producing system must also include a substrate for the enzyme. If the enzymatic reaction product is not itself detectable, the SPS will include one or more additional reactants so that a detectable product appears.
  • An enzyme analyte may act as its own label if an enzyme inhibitor is used as a diagnostic reagent.
  • a label may be conjugated, directly or indirectly (e.g., through a labeled anti-ABM antibody), covalently (e.g., with SPDP) or noncovalently, to the ABM, to produce a diagnostic reagent.
  • the ABM may be conjugated to a solid phase support to form a solid phase (“capture") diagnostic reagent.
  • Suitable supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to its target.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Binding assays may be divided into two basic types, heterogeneous and homogeneous.
  • heterogeneous assays the interaction between the affinity molecule and the analyte does not affect the label, hence, to determine the amount or presence of analyte, bound label must be separated from free label.
  • homogeneous assays the interaction does affect the activity of the label, and therefore analyte levels can be deduced without the need for a separation step.
  • the ABM is insolubilized by coupling it to a macromolecular support, and analyte in the sample is allowed to compete with a known quantity of a labeled or specifically labelable analyte analogue.
  • the "analyte analogue” is a molecule capable of competing with analyte for binding to the ABM, and the term is intended to include analyte itself. It may be labeled already, or it may be labeled subsequently by specifically binding the label to a moiety differentiating the analyte analogue from analyte.
  • the solid and liquid phases are separated, and the labeled analyte analogue in one phase is quantified. The higher the level of analyte analogue in the solid phase, i.e., sticking to the ABM, the lower the level of analyte in the sample.
  • both an insolubilized ABM, and a labeled ABM are employed.
  • the analyte is captured by the insolubilized ABM and is tagged by the labeled ABM, forming a ternary complex.
  • the reagents may be added to the sample in either order, or simultaneously.
  • the ABMs may be the same or different.
  • the amount of labeled ABM in the ternary complex is directly proportional to the amount of analyte in the sample.
  • the two embodiments described above are both heterogeneous assays. However, homogeneous assays are conceivable.
  • the key is that the label be affected by whether or not the complex is formed.
  • PCR can be performed using primers specific for the genes of interest. This would amplify the genes of interest. Primers may be designed to anneal to any site within the open reading frames of the genes of interest. Resolution of the fragments by electrophoresis on agarose gel may be used to determine the presence of the genes. PCR product may be quantitated by densitometry in order to estimate the concentration of the genes in the samples. Detection of genes of interest may also be done by Northern blot analysis on liver biopsies . Tissue sample from patients may be obtained and the total RNA extracted using RNAStat 60. The total RNA sample may then be resolved on denaturing gel by electrophoresis and then transferred onto a nylon membrane.
  • the membrane may then be used m hybridization with a suitable probe, which may be a synthetic probe directed against a gene already known to be a marker, or which may be a cDNA probe prepared directly from subtractive hybridization, wherein the fragment encoding the gene of interest, that is enriched m GH-overproducmg subjects, will be labeled, preferably either radioactively with 32 P or non-radiactively with DIG (Digoxigenm) .
  • a negative control such as one composed of RNA sample from liver of normal subjects, may be resolved side by side with the patients' sample, to determine quantitatively whether there is a significant increase in the level of gene expression. Elevation of the messenger RNA transcript from this gene would imply that liver damage might have occurred.
  • the DNA sequences of the present invention may be used either as hybridization probes per se, or as primers for PCR.
  • a nucleic acid reagent may be used either as a probe, or as a primer.
  • probe use only one reagent is needed, and it may hybridize to all or just a part of the target nucleic acid.
  • more than one probe may be used to increase specificity.
  • primer- based assay two primers are needed. These hybridize the non-overlappmg, separated segments of the target sequence. One primer hybridizes to the plus strand, and the other to the minus strand.
  • the target nucleic acid region starting at one primer binding site and ending at the other primer binding site, along both strands is amplified, including the intervening segment to which the primers do not hybridize.
  • the primer thus will not correspond to the entire target, but rather each primer will correspond to one end of the target s equence .
  • hybridizations may be carried out on filters or in solutions. Typical filters are nitrocellulose, nylon, and chemically-activated papers.
  • the probe may be double stranded or single stranded, however, the double stranded nucleic acid will be denatured for binding .
  • a hybridization assay whether primer- or probe-based, must be sufficiently sensitive and specific to be diagnostically useful.
  • sensitivity is affected by the amount and specific activity of the probe, the amount of the target nucleic acid, the detectability of the label, the rate of hybridization, and the duration of the hybridization.
  • the hybridization rate is maximized at a Ti (incubation temperature) of 20-25°C. below Tm for DNA.-DNA hybrids and 10-15°C. below Tm for DNA:RNA hybrids. It is also maximized by an ionic strength of about 1.5M Na + .
  • the rate is directly proportional to duplex length and inversely proportional to the degree of mismatching.
  • sensitivity is not usually a major issue because of the extreme amplification of the signal .
  • Hybrid stability is a function of duplex length, base composition, ionic strength, mismatching, and destabilizing agents (if any) .
  • the Tm of a perfect hybrid may be estimated.
  • DNA hybrids as
  • Tm 81.5°C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L and for DNA: RNA hybrids, as
  • Tm 79.8°C + 18.5 (log M) + 0.58 (%GC) - 11.8 (%GC) 2 - 0.56(% form) - 820/L
  • Tm length hybrid in base pairs Tm is reduced by 0.5-1.5°C for each 1% mismatching.
  • Tm may also be estimated by the method of Tinoco et al . , developed originally for the determination of the stability of a proposed secondary structure of an RNA. Tm may also be determined experimentally. Filter hybridization is typically carried out at 68°C, and at high ionic strength (e.g., 5 - 6 x SSC) , which is nonstringent , and followed by one or more washes of increasing stringency, the last was being of the ultimately desired stringency. The equations for Tm can be used to estimate the appropriate Ti for the final wash, or the Tm of the perfect duplex can be determined experimentally and Ti then adjusted accordingly.
  • ionic strength e.g., 5 - 6 x SSC
  • mouse cDNA was used to probe a mouse liver cDNA library, and could be used to probe nonmurine liver cDNA libraries, it would be expected that there would be some sequence divergence between cognate mouse and nonmouse DNAs, possibly as much as 25-50%.
  • the mouse (or rat) gene may be used as a probe. In this case, more moderate stringency hybridization conditions should be used.
  • the nonhuman gene may be modified to obey a more human set of codon preferences .
  • the mouse (or rat) gene may be used once as a probe to isolate the human gene, and the human gene then used for diagnostic work. If a partial human cDNA is obtained, it may be used to isolate a larger human cDNA, and the process repeated as needed until the complete human cDNA is obtained.
  • the Ti should be reduced further, by about 0.5-1.5°C, e.g., 1°C, for each expected 1% divergence in sequence.
  • the degree of divergence may be estimated from the known divergence of the most closely related pairs of known genes from the two species .
  • hybridization conditions set forth in the Examples may be used as a starting point, and then made more or less stringent as the situation merits.
  • the probe is preferably at least 15, more preferably at least 20, still more preferably at least 50, and most preferably at least 100 bases (or base pairs) long.
  • the probe is not complementary to the entire gene, it targets a region low in allelic variation.
  • the primer is preferably at least 18-30 bases m length. Longer primers do no harm, shorter primers may sacrifice specificity.
  • the distance between the primers may be as long as 10 kb, but is preferably less than 3kb, and of course should taken into account the length of the target sequence (which is likely to be shorter for mRNA or cDNA than for genomic DNA) .
  • primers Preferably, primers have similar GC content, minimal secondary structure, and low complementa ⁇ ly to each other, particularly in the 3' region. For theoretical analysis of probe design considerations, see Lathe, et al., J. Mol . Biol., 183:1-12 (1985) .
  • ELISA can be done on blood plasma or serum from patients using antibodies specific to the protein of interest. Samples will be incubated with primary antibodies on plates. This primary antibody is specific to the protein of interest.
  • Another method that can be conducted will involve the use of chemical or enzymatic reactions in which the protein of interest will act as a substrate (or, if the protein is an enzyme, as a catalyst) to cause a reaction that lead to the production of colored solution or emission of fluorescence.
  • Spectrometric analysis can be done in order to determine the concentration of the proteins m the sample.
  • Western blot analysis can also be done on the plasma/serum, liver aspira te , liver biopsies or urine samples . This would involve resolving the proteins on an electrophoretic gel, such as an SDS PAGE gel, and transferring the resolved proteins onto a nitrocellulose or other suitable membrane.
  • the proteins are incubated with a target binding molecule, such as an antibody.
  • This binding reagent may be labeled or not. If it is unlabeled, then one would also employ a secondary, labeled molecule which binds to the binding reagent.
  • a secondary, labeled molecule which binds to the binding reagent.
  • immunoprecipitation can be conducted. This typically will involve addition of a monoclonal antibody against the protein of interest to samples, then allowing the Ig-protein complex to precipitate after the addition of an affinity bead (ie antihuman Ig sepharose bead) .
  • affinity bead ie antihuman Ig sepharose bead
  • the immunoprecipitates will undergo several washings prior to transfer onto a nitrocellulose membrane.
  • the Western blot analysis can be perform using another antibody against the primary antibody used.
  • the assay may be used to predict the clinical state of the liver if the level of GH activity remains unchanged.
  • a scheme for the diagnostic interpretation of the level of the target in question is determined in a conventional manner by monitoring the level of GH, the level of the target, and the liver condition in a suitable number of patients, and correlating the level of the target at an earlier time point with the simultaneous or subsequent liver tissue state.
  • This correlation is then used to predict the future clinical state of the liver in new patients with high GH levels.
  • the diagnosis may be based on a single marker, or upon a combination of markers, which may include, besides the markers mentioned above, the level of GH or of IGF-1.
  • a suitable combination may be identified by any suitable technique, such as multiple regression, factor analysis, or a neural network using the scaled levels of the markers as inputs and the current or subsequent liver state as an output .
  • Radio-labelled ABM which are not rapidly degraded in blood may be administered to the human or animal subject. Administration is typically by injection, e.g., intravenous or arterial or other means of administration in a quantity sufficient to permit subsequent dynamic and/or static imaging using suitable radio-detecting devices.
  • the dosage is the smallest amount capable of providing a diagnostically effective image, and may be determined by means conventional in the art, using known radio-imaging agents as a guide.
  • the imaging is carried out on the whole body of the subject, or on that portion of the body or organ relevant to the condition or disease under study.
  • the amount of radio-labelled ABM accumulated at a given point in time in relevant target organs can then be quantified.
  • a particularly suitable radio-detecting device is a scintillation camera, such as a gamma camera.
  • a scintillation camera is a stationary device that can be used to image distribution of radio-labelled ABM.
  • the detection device in the camera senses the radioactive decay, the distribution of which can be recorded.
  • Data produced by the imaging system can be digitized.
  • the digitized information can be analyzed over time discontinuously or continuously.
  • the digitized data can be processed to produce images, called frames, of the pattern of uptake of the radio- labelled ABM in the target organ at a discrete point in time.
  • quantitative data is obtained by observing changes in distributions of radioactive decay in target organs over time. In other words, a time-activity analysis of the data will illustrate uptake through clearance of the radio-labelled binding protein by the target organs with time.
  • the radioisotope must be selected with a view to obtaining good quality resolution upon imaging, should be safe for diagnostic use in humans and animals, and should preferably have a short physical half-life so as to decrease the amount of radiation received by the body.
  • the radioisotope used should preferably be pharmacologically inert, and, in the quantities administered, should not have any substantial physiological effect.
  • the ABM may be radio-labelled with different isotopes of iodine, for example 123 I, 125 I, or 131 I (see for example, U.S. Patent 4,609,725).
  • radio-labeling must, however be monitored, since it will affect the calculations made based on the imaging results (i.e. a diiodinated ABM will result in twice the radiation count of a similar monoiodinated ABM over the same time frame) .
  • radioisotopes other than 125 I for labelling in order to decrease the total dosimetry exposure of the human body and to optimize the detectability of the labelled molecule (though this radioisotope can be used if circumstances require) . Ready availability for clinical use is also a factor. Accordingly, for human applications, preferred radio-labels are for example, 99m Tc, 67 Ga, 68 Ga, 90 Y, n ⁇ In, 113m In, 123 I, 186 Re, 188 Re or 211 At .
  • the radio-labelled ABM may be prepared by various methods. These include radio-halogenation by the chloramine - T method or the lactoperoxidase method and subsequent purification by HPLC (high pressure liquid chromatography) , for example as described by J. Gutkowska et al in "Endocrinology and Metabolism Clinics of America: (1987) 1_6 (1):183. Other known method of radio-labelling can be used, such as IODOBEADSTM. There are a number of different methods of delivering the radio-labelled ABM to the end-user. It may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal.
  • hGH Human growth hormone
  • TM transgenic mice
  • bGH bovine GH
  • WT wild type mice
  • Our work aimed to create a library of liver GH inducible- genes in liver and to identify genes that are associated with the progression of liver disease that may eventually be use to diagnose pathologic liver in humans as observed on patients with acromegaly, liver cirrhosis, and viral infections causing hepatitis.
  • the method employed to determine the GH-inducible genes in bGH TM involves subtractive hybridization using Clontech's PCR-Select cDNA Subtraction kit. This method requires that mRNAs be isolated first and then converted into cDNAs. The mRNAs from liver of 60 days old bGH TM and WT mice were isolated by passing through oligo-dT columns (Invitrogen ' s Fastract 2.0) total RNAs prepared by RNAStat 60. Conversion of mRNAs to cDNAs involves the use of AMV reverse transcriptase (Clontech) .
  • the primer used for the first strand cDNA synthesis is 5' TTTTGTACAAGCTT 3' (SEQ ID NO: 6) which binds to polyA tail of the mRNA. This primer introduces a unique restriction site Rsa 1 downstream of polyA tail.
  • the second strand cDNA synthesis involves the use of an enzyme cocktail composed of RNase H, DNA polymerase and ligase enzymes.
  • Adaptor 1 5 ' -CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3 ' (SEQ ID NO:l)
  • Adaptor 2R 5 ' -CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3 ' (SEQ ID NO:3)
  • Isolation of differentially expressed genes from GH TM is achieved by performing two hybridization steps.
  • the first hybridization step involved mixing each of the adapter ligated testers with excess of drivers. This resulted in annealing of identical ss cDNA fragments common to both the tester and driver.
  • Differentially expressed sequences from GH TM that did not form hybrids with the driver sequences underwent a second hybridization step. This step involved mixing two reaction products from the first hybridization in the presence of more drive cDNA. This resulted in the formation of new hybrids between adaptor ligated ss cDNAs from GH TM .
  • primer sites for PCR primer 1(5'- CTAATACGACTCACTATAGGGC-3' , bases 1-22 of SEQ ID NO : 1 ) were generated.
  • Subtraction is achieved by preventing the tester-driver hybrid sequences from being amplified during PCR amplification while hybrids between testers with adaptor 1 and adaptor 2R can.
  • those cDNA fragments that undergo PCR amplification correspond to differentially expressed GH TM.
  • PCR primer 1 was used in the first PCR amplification at 94 °C for 25 sec followed by 27 cycles at three different temperatures of 94°C for 10 sec, 66°C for 30 sec, and 72°C for 1.5 mm.
  • nested PCR primer 1 (5'- TCGAGCGGCCGCCCGGGCAGGT-3' , bases 23-44 of SEQ ID NO:l) and nested PCR primer 2R (5 ' -AGCGTGGTCGCGGCCGAGGT-3 ' , bases 23-42 of SEQ ID NO: 3) were added to the first PCR amplified reaction mixture.
  • the second PCR amplification step was conducted at 10-12 cycles of amplification at 94 °C for 10 sec, 68°C for 30 sec and 72°C for 1.5 mm. to further enrich the differentially expressed sequence from GH TM.
  • the integrity of the products from each manipulation was determined by gel electrophoresis of an aliquot of the reaction mixtures.
  • the differentially expressed sequences obtained by subtractive hybridization were subcloned directly into PCR II cloning vector.
  • the pool of partial cDNA fragments was ligated into a pCR ⁇ 2.1 expression vector using the TA Cloning® Kit from Invitrogen®. The ligation mixture was subsequently transformed into Library Efficiency DH5 ⁇ TM Competent Cells from Life Technologies. Ampicill resistant colonies were propagated and plasmid DNA was extracted and purified using an alkaline lysis m iprep protocol (Birnhoim, H.C. 1983). The purified plas id DNAs containing different partial cDNA fragments were then sequenced using S labeled dNTPs and the T7 SequenaseTM version 2.0 DNA polymerase from Amersham Life Science Products.
  • the sequencing primer 5' TACTCAAGCTATGCATCAAG 3' (SEQ ID NO: 5), hybridized to the pCR® 2.1 expression vector in the multiple cloning site -60 bases 5' of the partial cDNA insert.
  • the sequence data was analyzed and matched against known sequences using BLAST (Basic Local Alignment Search Tools), available through the National Centers for Biotechnology Information (NCBI) internet database.
  • BLAST Basic Local Alignment Search Tools
  • NCBI National Centers for Biotechnology Information
  • GH-inducible genes in the liver of GH TM are mouse ⁇ -fetoprotein, fetuin, 3- ⁇ -Hydroxysteroid, rab8-interacting protein, paraoxonase-3, interferon ⁇ / ⁇ receptor (IFNR ⁇ ), proteasome z-subunit, corticosteroid binding globulin (CBG) , growth hormone receptor, cytochrome P450IIIA, cytochrome P450, and coagulation factor V, and rat S-2-hydroxyacid oxidase. It follows that the cognate human genes may be used as probes for observing GH-regulated expression of those genes in the liver, which genes are presumed to be regulated in a similar manner.
  • the fragments were purified using the Qiaex® II Agarose Gel Extraction Kit from Qiagen®. The purified fragments were then labeled using the Random Primed DNA Labeling Kit from Boehr ger Mannheim. The membrane bound RNA was then hybridized with ⁇ P labeled DNA probes specific for the aforementioned partial cDNA sequences (see previous page) . Preliminary results indicate that IFNR ⁇ and CBG mRNA are expressed livers of GH TM and not in NT littermates.
  • Non-radioactive DIG-labeled probe for Northern blot was constructed by amplification of the target sequence in the first PCR step followed incorporation of d ⁇ gox ⁇ genm-11-UTP or DIG-UTP (Roche) on the antisense strand during the second PCR.
  • probe for used in the confirmation of differential gene expression in GH transgenic mice versus non-transgenic mice fragments from subtractive hybridization that were subcloned into pCR2.1 cloning vector were PCR amplified using primers pCR 2.1A (5 1 ATTACGCCAAGCTTGGTACCG 3') and pCR IIB (5' CCCTCTAGATGCATGCTC 3') .
  • DIG-UTP incorporation is accomplished using primer pCR 2.1A or pCRIIB m the second PCR step.
  • T3 (5' AATTAACCCTCACTAAAGGG 3') and mKS (5' CCTCGAGGTCGACGGTATC 3') primers were used for the first PCR amplification step and mKS primer for the second DIG-UTP incorporation step.
  • a cDNA library was constructed from the liver of growth hormone (GH) trangenic mice.
  • the cDNA that was used in the construction of the cDNA library was prepared from mRNAs, which was obtained from total RNA isolated from the liver of GH transgenic mice.
  • the cDNA prepared was then used to produce the lambda zap (Stratagene) cDNA library.
  • the titer of the amplified library was 10 9 pfu/ml and the recombination efficiency determined to be 75%. Screening of the cDNA library for novel genes was done by probe hybridization of the nitrocellulose plaque lifts.
  • the probe used in the screening was prepared by PCR amplification of gene fragment, which previously was identified by subtractive hybridization as differentially expressed in GH transgenic mice and not in wild type mice. After screening of approximately 2.5 X 10 5 plaques, five plaques that hybridized with the probe were purified and then the pBluescript plasmids, which contain the cDNA inserts, were excised out of the lambda zap vector utilizing helper phages following the manufacturer's protocol. The cDNA sequence of the insert was determined by "walking" through the sequence starting with T3 and KS primers complementary to sequences in the plasmid vector.
  • Protein motif search utilizing PROSITE database indicate that the protein corresponding to the longest open reading frame in cDNA sequence of Clone 5 possess the following motifs: N-glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, and amidation sites. The protein appears to have a signal peptide but no transmembrane region found. Thus, this protein encoded by the longest open reading from in Clone 5 could be cytoplasmic in location.
  • the DNA sequence of Clone 45 has several ORFs; the longest, corresponding to 1029 bases (SEQ ID NO: 9) which encodes a protein of 342 amino acids (SEQ ID NO:10) . All ORFs are set forth in Table 3B.
  • Protein motif search utilizing PROSITE database indicates that the protein corresponding to the longest open reading frame in cDNA sequence of Clone 45 possess the following motifs: N-glycosylation, protein kinase C phosphorylation, casein kinase II phosphorylation, and amidation sites, as well as a Myc-type helix-loop-helix dimerization domain.
  • the protein appears to have signal peptide at the N-terminal and transmembrane region close to the N-terminal. This could indicate that the protein encoded by longest open reading frame in Clone 45 is membrane bound and/or secreted.
  • N-glycosylatio post-translational modification of proteins involving attachment of carbohydrate residues.
  • Phosphorylation sites site of attachment of phosphate group. Reversible phosphorylation-dephosphorylation of protein is associated with regulation of activity of the protein. Some proteins are activated when phosphorylated and inactivated when unphosphorylated, or vice versa. N-myristoylation: (usually at the N-termmus) this protein modification involves addition of myristoyl group which is believed to cause some of the attached proteins to be loosely associated with membranes. Some my ⁇ stoylated proteins are not associated with membranes to any significant extent. Some of my ⁇ stoylated proteins such as protein kinases and phosphatases have important roles m modulating cellular metabolism.
  • Amidation is usually seen on carboxy-termmus of peptide hormones. Enzymes involve in amidation reaction are usually found in secretory granules.
  • HHLH dimerization domains are usually present in proteins that interact with DNA. Myc proteins are involve in growth regulation.
  • RNA Total RNA will be extracted from liver biopsy using 10 mL RNAStat60 per gram of liver tissue.
  • IX MOPS, formaldehyde, formamide and ethidium bromide will be added, heat denatured at 60 °C then loaded on a formamide containing denaturing 1% agarose gel.
  • the RNA will then be resolved by electrophoresis at 50V for about 2- 2 M. h . After electrophoresis, the gel will be washed twice briefly with deionized water; then once with 0.05N NaOH, with 0.1M T ⁇ s at pH 7.5, and with 10X SSC at washing times of at least 30 mm in each case.
  • the resolved RNA after electrophoresis will be transferred onto a nylon membrane by upward gradient adsorption using 10X SSC as transfer buffer.
  • the RNA on the membrane will be UV crosslinked at 120 mJ, after which the RNA blots will be ready for hybridization.
  • Northern blot hybridization using digoxigenin (DIG)- labeled probe will be conducted to determine whether the genes of interest are present in liver RNA blots.
  • the probes to be used for hybridization will be prepared from pCR2 clones, which contain as inserts the fragments isolated by subtractive hybridization of liver genes from GH mice versus WT mice.
  • the sequence homology of the fragments to that of the human genes range from about 74% to 94%, which were obtained using the default parameters of Blast 2.0 sequence alignment version blastn 2.0.8.
  • the DIG-labelled probe preparation will require PCR amplification of the inserts in pCR2 clones using Taq polymerase as polymerization enzyme and pCR 2.1A and pCR 2B as primers.
  • the conditions for PCR amplification will be 95°C for 2 min.; 55 cycles at three temperature conditions of 95°C forl5 sec, 58°C for 20 sec, and 72 °C for 45 sec; then 72°C for 7 min.
  • the amplified double-stranded cDNA fragment will undergo a second PCR amplification using a single primer, pCR 2.1A, in the presence of DIG labeled dNTPs to produce a single stranded DIG-labeled PCR product which will serve as the probe for RNA blot hybridization.
  • concentrations of the DIG labeled probe will be determined by comparing the signals produced by the probe to that of control DIG-labeled DNA upon exposure to radiographic film.
  • the concentration of DIG-labeled probe to be used for hybridization will be 50ng/mL of DIG Easy Hyb solution (Boehringer-Mannheim) .
  • DIG Easy Hyb solution Boehringer-Mannheim
  • the RNA blots will be prehybridized in DIG Easy Hyb solution at 42 °C for 30-60 min.
  • the RNA blots will undergo hybridization using the probes prepared form the different pCR 2 clones. Hybridization will be done at 42 °C for at least 8 hours.
  • Posthybridization washings of the membrane will then be performed at room temperature for 5min using a solution of 2X SSC and 0.1% SDS; and twice at 60 °C for 15 min. using a solution of 0.5X SSC and 0.1%SDS.
  • the RNA blots will then be incubated with DIG antibody, which is conjugated to alkaline phosphatase. This antibody recognizes the DIG labeled hybrids in the RNA blot.
  • CSPD Boehringer- Mannheim
  • CSPD which is a chemiluminescent substrate for alkaline phosphatase, will be use to achieve detection of the RNA of interest in the blot. The presence of bands that is specific to the liver genes of interest could be diagnostic of liver damage.
  • the 32 P-labeled probe will be prepared by first isolating the cDNA fragments that were inserted into the pCR 2 vector by performing EcoRI restriction enzyme digestion. The fragments will be purified though a Qiaex R agarose gel extraction column (Qiagen) . A 25ng of the purified fragment will serve as a template for the production of single- stranded 32 P-labeled probe using Random Primed DNA Labeling kit (Boehringer-Mannheim) . The unincorporated dNTPs will be separated from the radiolabeled fragments using STE Select D G-25 column. The purified radiolabeld probe will then be quantified to determine the activity of the probe per ug of the DNA template. A good labeling of the template would have a specific activity range of 10 8 -10 9 cpm/ug of the template DNA.
  • prehybridization of the RNA blots will be performed by incubating the membrane in prehybridization solution made up of 50% formamide, 1% SDS, 1M NaCl, and 10% Dextran sulfate for 1 hour at 42 °C .
  • Hybridization of the RNA blot with the 32 P-labeled probes prepared will follow after prehybridization. This will be conducted at 42 °C for at least 8 hours. Washing of the blots will be conducted once with 2X SSC at room temp for 5 mm. and then with 2X SSC, 0.1% SDS at 56 °C which could last for about 5 minutes to an hour depending on the intensity of the radiactive signal. Radiographic exposure of the blots will determine whether the genes of interest are present.
  • Table 1 shows the sequence of each clone, and its BLASTN alignment to the known mouse (or rat) gene as found in a sequence databank, to which it appears to be most closely related.
  • the known genes are as follows:
  • mice alpha-fetoprotein (A mouse alpha-fetoprotein (WLAC #2) (B mouse fetuin (WLAC #6) (C mouse 3- ⁇ -hydroxysteroid (WLAC #7) (D mouse rab8 interacting protein (WLAC #13) (E mouse paraoxonase-3 (WLAC #14) (F rat S-2-hydroxyacid oxidase (WLAC #21) (G mouse interferon ⁇ / ⁇ receptor (WLAC #26) (H mouse growth hormone receptor (WLAC #29) (I mouse cytochrome P450IIIA (WLAC #20, #36) (J mouse cytochrome P450 (WLAC #39) (K mouse proteasome z-subunit (WLAC#37) ( mouse corticosteroid binding globulin ((WLAC #3, 34, 35, 52 (M mouse coagulation factor V (WLAC #56)
  • Table 2 (A) full-length single stranded nucleotide sequence of clone 5 and (B) ORFs 1-16 corresponding to clone 5.
  • WLAC #26 SEQUENCE GGCCACACTGAGATCTTAAACAACGCCAGCTCCTCCAGTTAGTGTCCCTTTCTC CATGGTTCAGTGACTTCTGGTCAGAAG
  • MASLNVQVAASVAGSRVKPQSSSTVASRDMAGLQFS Note: First six sequences are ORFs starting from met to stop codon, the next two sequences were also identified as ORFs from the beginning of the sequence to the stop codon and the last ORF is the sequence starting from met to the end of the sequence. ORF analysis conducted using GeneRunner version 3.05 software by Hastings Software, Inc.

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Abstract

On décrit des gènes et des protéines hépatiques pouvant être régulés par l'hormone de croissance. On peut les utiliser en tant que marqueurs de diagnostic pour les maladies du foie.
PCT/US2000/012366 1999-05-05 2000-05-05 Genes et proteines hepatiques pouvant etre regules par l'hormone de croissance, utilisations associees Ceased WO2000066787A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003038124A1 (fr) * 2001-10-31 2003-05-08 Institut National De La Recherche Agronomique - Inra - Utilisation du gene de la cbg comme marqueur genetique de l'hypercortisolemie et des pathologies associees
WO2005082398A3 (fr) * 2004-02-26 2006-01-26 Univ Ohio Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de façon differentielle dans les cellules musculaires

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GR850899B (fr) * 1984-04-12 1985-11-25 Gen Hospital Corp

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003038124A1 (fr) * 2001-10-31 2003-05-08 Institut National De La Recherche Agronomique - Inra - Utilisation du gene de la cbg comme marqueur genetique de l'hypercortisolemie et des pathologies associees
FR2831890A1 (fr) * 2001-10-31 2003-05-09 Agronomique Inst Nat Rech Utilisation du gene de la cbg comme marqueur genetique de l'hypercortisolemie et des pathologies associees
WO2005082398A3 (fr) * 2004-02-26 2006-01-26 Univ Ohio Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de façon differentielle dans les cellules musculaires

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