DE60034779T2 - Insulin-like growth factor-binding protein-4 protease - Google PatentsThe invention relates to the use of schwangerschaftsassoziiertem plasma protein A as a marker for focal growth states in non-pregnant patients. Primobolan gep opiniones cleavage of the six known insulin-like growth factor to the anbindenden proteins IGFBP is a powerful means of rapid structure and function modification of peak anabolic protein fusion unterschied important growth-regulating proteins. It is further described that insulin-like growth factor IGF -I and -II are about 7,5 kDa polypeptides capable of acting in autocrine, paracrine and peak anabolic protein fusion unterschied manner in order on the growth of both normal and to stimulate of transformed cells. Prak present the object underlying the invention is to provide a method for screening or diagnosing growth-promoting states. This object is solved by the features of claim.
FÜR MANN - Luxury Power Nutrition
The invention relates to the use of schwangerschaftsassoziiertem plasma protein A as a marker for focal growth states in non-pregnant patients. Proteolytic cleavage of the six known insulin-like growth factor to the anbindenden proteins IGFBP is a powerful means of rapid structure and function modification of these important growth-regulating proteins.
It is further described that insulin-like growth factor IGF -I and -II are about 7,5 kDa polypeptides capable of acting in autocrine, paracrine and endocrine manner in order on the growth of both normal and to stimulate of transformed cells. The present the object underlying the invention is to provide a method for screening or diagnosing growth-promoting states.
This object is solved by the features of claim. Advantageous embodiments of the invention are described by the features of claims 2 to 10 degrees. In one embodiment, the invention provides methods for screening out on a growth-promoting or growth-inhibiting state in a non-pregnant patient. The biological sample may be selected from the group consisting of blood, urine, pleural fluid, oral rinses, tissue biopsies, and follicular fluid.
An increase in PAPP-A levels in the non-pregnant patient indicates the presence of a growth-promoting state, whereas a decrease in the PAPP-A level indicating the presence of a growth-inhibiting state. The growth-promoting state can be, for example, restenosis, atherosclerosis, or ovulation. PAPP-A protein can be immunologically determined, for example by at least one monoclonal antibody.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials that are similar or equal to those described herein can be used to practice the invention, suitable methods and materials are described below. In addition, used the materials, procedures and examples are illustrative and are not intended to be limiting.
Other features and advantages of the invention will become apparent from the following detailed description and from the claims. HFcm was precleared with a 1: Since there is a substantial difference in the product sizes between PCR product 1 and 2, contamination with genomic DNA in the mRNA preparation can be easily determined.
A and B represent the 5'- and 3'-competitive PCR primers. Shaded boxes represent the part of the cDNA, which is deleted to generate the IS, either by excision of a restriction fragment or by primer mediated deletion. Pufferkomponenten, dNTP, und Primer eluieren zuerst. Buffer components, dNTP, and primers elute first. The vertical arrow indicates the change of scale 8 fold increase in sensitivity. Standardabweichungen werden als Fehler-Balken gezeigt. Standard deviations are shown as error bars.
The number of samples for each tissue is indicated above the columns. Fractions at fraction 24 fractions were pooled and PAPP-A antigen was purified by heparin chromatography and the gel path 1 loaded. Insulin-like growth factor IGF are essential polypeptides with strong anabolic and mitogenic action both in vivo and in vitro. Incubation of IGFBP-4 in the humanfibroblast-conditioned medium HFcm under cell-free conditions results non-reduced about 24 kDa, about 32 kDa reduced for the cleavage of IGFBP-4 in the central portion of the molecule generating distinct fragments of about 18 and about 14 kDa.
A similar IGF-dependent IGFBP-4 proteolysis has been described for example, in cultures of normal human osteoblasts, vascular smooth muscle cells, endometrial stromal cells, decidual cells, and granulosa cells, as well as in ovarian follicular fluid of.
PAPP-A has been described as a large placental glycoprotein present in the serum of pregnant women in increasing concentrations throughout pregnancy.
The cDNA sequence of PAPP-A indicates that the serum form is derived from a pre-proprotein with a putative residue signal peptide, a pro-part of 58 residues, and an residue circulating mature polypeptide. PAPP-A, which has not formed a complex with proMBP has not been isolated from pregnancy serum, but it may as described herein, be isolated from conditioned media from human fibroblasts, human osteoblasts, smooth muscle cells of human coronary artery and from mammalian cells transfected with PAPP-A cDNA were transfected.
As used herein, "growth-promoting" in one or more increases in the number of cells, an increase in the size of the cells or an increase in differentiated cell function.
Not restrict de Examples of growth-promoting states include atherosclerosis, restenosis, fibrosis, wound healing, IGF dependent growth of cancer cells and ovulation, including a Follikularentwicklung.
Suitable biological samples include, for example, blood, urine, pleural fluid, an oral rinses, tissue biopsies such as skin, bone, or blood vessel plaque, and follicular fluid. Blood is a particularly useful biological sample. PAPP-A protein can be detected immunologically, for example. Alternatively, immunohistochemical standard methods can be used to detect PAPP-A protein, using such antibodies.
Siehe zum Beispiel Qin et al. See, for example, Qin et al, Clin, , 43 The measurement of PAPP-A without complex formation in pregnancy serum has diagnostic value potential. In general, PAPP-A, which forms a complex with proMBP, be prepared in various ways, including recombinantly, or can be purified from a biological sample, and used to immunize animals. In general, nucleic acid constructs include a regulatory sequence operably linked to a PAPP-A nucleic acid sequence.
Regulatory sequences typically do not encode a gene product, but instead affect the expression of the nucleic acid sequence. Generally, these fusion proteins are soluble and can easily be purified from produced by lysing cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites such that the cloned target gene product from the GST moiety can be released. Mammalian cell lines with stable expression of PAPP-A can be produced by expression vectors are used with the appropriate control elements and a selectable marker. For example, the eukaryotic expression vector pCDNA. After the introduction of the expression vector by electroporation, DEAE dextran, or other suitable method, stable cell lines are selected.
Bei einem Expressionssystem, das pCNA3. In an expression system using pCNA3. The precipitated product was a dimer devoid of proMBP. Alternatively, PAPP-A can be transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysase of use. In eukaryotic host cells, a number of expression systems can be used viral based for expression of PAPP-A. A nucleic acid encoding PAPP-A can be cloned, for example, a baculoviral vector and then used to transfect insect cells.
Alternatively, the nucleic acid encoding PAPP-A into a SV40, retroviral vector or viral vector on Vak zinenbasis can be introduced and used to infect host cells. PAPP-A kann, wie hierin beschrieben, gereinigt werden. PAPP-A, as described herein, to be cleaned. After elution of bound proteins with a stepwise decreasing pH gradient, the pH 5.
Bound proteins can be eluted with a tri-salt solution, then by N-acetylglucosamine. Host animals include rabbits, chickens, mice, guinea pigs and rats. Various adjuvants that can be used to enhance the immunological response depend on the host species, and include the Freund's adjuvant complete and incomplete constantly a mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin , pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol nude.
Polyclonal antibodies are heterogeneous populations of antibody molecules that are contained in the sera of the immunized animals. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be prepared using a PAPP-A polypeptide and standard hybridoma technology.
In particular, monoclonal antibodies can be obtained by a process which provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler, G. Antibody fragments that have specific binding affinity for PAPP-A polypeptide can be prepared by known methods. These fragments include, for example F ab ' 2 fragments produced by pepsin digestion of the antibody molecule, and Fab fragments produced by reducing the disulfide bridges of F ab' may be prepared two fragments, but are not limited thereto.
Alternatively, Fab expression libraries can be built. Siehe zum Beispiel Huse et al. See, for example, Huse et al, Science, In general, PCR refers amplification of a target nucleic acid sequence information is used by the ends of the respective region or beyond it in order to make oligonucleotide primer whose sequence is identical to opposite strands of the template to be amplified, or similar to.
Primers are typically 14 to 40 nucleotides in length, but can be up to hundreds of nucleotides in length ranging from 10 nucleotides. A Laboratory Manual, herausgegeben von Dieffenbach, C. A Laboratory Manual, edited by Dieffenbach, C. Nucleic acids also can be amplified by ligase chain reaction, strand displacement by, by autonomous replication sequence, by amplification of based on the nucleic acid sequence. Siehe zum Beispiel Lewis, R.
Thus, the specific abundance is a measure of the mRNA level of the gene, normalized against a measure of the total mRNA in the sample. An internal reference is used, such as amplification of 28S rRNA with limiting primer concentration. Dieses Verfahren gestattet die Quantifizierung nach unten bis auf zirka Kopien der Zielsequenz.
This method allows quantitation down to approximately copies of the target sequence. Alternatively, testing different tissues for the presence of specific mRNA can be carried out routinely by RNA blotting ting procedures such as Northern or dot blotting. PAPP-A was detected in the placenta and in placental non-woven fabrics, such as kidney, heart, adrenal cortex, adrenal medulla, testes, small intestine and stomach. PAPP-A was detected in low levels in non-placental tissues.
Therefore, the mRNA specific abundances which are calculated here for a number of tissues, low, when compared to the levels in placenta. All previous investigations were carried out on the basis of polyclonal antisera, and a number of reports have been published describing the polyspecificity and heterogeneity of different preparations of these antisera. Ein ermittelbar markiertes Substrat kann zum Beispiel beim Vorliegen der biologischen Probe unter geeigneten Bedingungen inkubiert werden, und proteolytische Produkte werden dann ermittelt.
A detectable labeled substrate can be incubated, for example in the presence of the biological sample under suitable conditions, and proteolytic products are then determined. Typischerweise wird das Substrat radioaktiv mit Isotopen markiert, wie zum Beispiel I oder 32 P, oder es wird nicht-radioaktiv markiert mit Biotin, Digoxygenin oder einem Fluorphor. Typically, the substrate is radioactively labeled with isotopes such as I or 32 P, or is non-radioactively labeled with biotin, digoxygenin, or a fluorophore.
Proteolytic cleavage products can also be detected by immunoblotting. After washing away unbound protein of the biological sample, the activity of PAPP-A with a synthetic substrate of low molecular weight can be measured, which releases a colored product which can be spectrophotometrically determined.
The substrate is specifically labeled, i. After proteolytic cleavage of the substrate labeled fragments in the liquid phase are released and can be determined. The determination of bound IGFBP-4 can be achieved using standard ELISA methodology then by a peroxidase-conjugated monoclonal antibody is used, for example, that recognizes the other tag. IGFBP-4 can also be immobilized and detected using monoclonal antibodies are employed that recognize the N-terminal or the C-terminal.
The proteolytic activity resulting in a decreased signal, dependent on the extent of proteinase activity and time of incubation. The invention also provides methods for the identification of agents prepared that alter the protease activity of PAPP-A. As used herein, "agents" refers to a biological macromolecule such as an oligonucleotide or a peptide, a chemical compound, a mixture of chemical compounds, or a extract isolated from a bacterial agent, a herbal substance, a fungus or an animal material.
Inhibitory agents are identified by incubating an isolated PAPP-A polypeptide, an activator of protease activity and a substrate of PAPP-A with the active ingredient and whether the proteolysis of the substrate is inhibited determining.