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EP0929667A1 - Marqueurs de l'hypertension - Google Patents

Marqueurs de l'hypertension

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Publication number
EP0929667A1
EP0929667A1 EP97942823A EP97942823A EP0929667A1 EP 0929667 A1 EP0929667 A1 EP 0929667A1 EP 97942823 A EP97942823 A EP 97942823A EP 97942823 A EP97942823 A EP 97942823A EP 0929667 A1 EP0929667 A1 EP 0929667A1
Authority
EP
European Patent Office
Prior art keywords
pro gly
seq
gly leu
ala
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97942823A
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German (de)
English (en)
Inventor
Michael Mulvany
Nilesh Samani
Stephen John Fey
Peter Mose Larsen
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Individual
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Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0929667A1 publication Critical patent/EP0929667A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • hypertension is in itself a very widespread disease which, in addition to having a lowering effect on the life quality, also directly leads to death of the patient.
  • Hypertension may in fact be the cause of many different cardiac/vascular diseases (such as cardiac arrest, cerebral haemorrhage, and thrombi) .
  • cardiac/vascular diseases such as cardiac arrest, cerebral haemorrhage, and thrombi
  • Krieger and Dzau 1991.
  • the part of the vascular and venous system which regulates the blood pressure consists of the small resistance vessels and thus, these very vessels were used in the present studies - other tissues and possibly organs (e.g. the kidneys) could also have been used.
  • various physiologi- cal parameters were measured in the animals (e.g. blood pressure, body temperature, Ca 2+ /Na + /Mg 2+ concentrations) in order to ensure that the rats complied with the requirements in question.
  • proteome analysis studies were carried out on each of the isolated vessels from the rats, again to ensure that they were all optimum vessels.
  • ACE-1 has been associated with a very large number of cardiac/vascular diseases, and in particular with the development of hypertension.
  • the reason for this interconnection may be seen in Fig. 8 where ACE-1 first of all takes an active part in synthesizing a vasopres- sor and, secondly, contributes to the destruction of a vasodilator (bradykinin) .
  • ACE-1 has a double negative influence on hypertension.
  • the especially interesting feature of the present findings is that it is 100% certain that the protein #325 in question is not identical with ACE-1 [the amino acid sequence is different, molecular weight and pi are not the same (neither are they identical with degradation products of ACE-1)] .
  • the present inventors are therefore of the opinion that they have identified at least one protein/gene which may take over the regulation of the blood pressure in hypertensive individuals, e.g. as a replacement of ACE-1, but which cannot be regulated in the same way.
  • the inventors have identified a number of other proteins which are involved, directly or indirectly, in the process leading to or sustaining hypertension.
  • the further analysis of the protein # 325 and its possible relationship to the 52 kD protein described by M ⁇ ns et al . is outlined in Fig. 10.
  • proteins identified by MALDI tof MS have been related to human diseases and thus their function have been shown (in other contexts) to be crucial to the health of the individual, but none of the proteins have been associated to hypertension.
  • the present findings may be used in connection with the diagnosis of a genetic predisposition for hypertension which will enable the individual to take appropriate actions with respect to his or her life style in order to minimize the potential adverse effects of the genetic predisposition.
  • the present invention will be useful in the treatment of hypertension which is a disease for which there is currently no effective treatment.
  • Such gene transfer can take place e.g. by use of a retroviral vector or by use of DNA liposomes.
  • the present invention thus relates to a test animal having a nucleotide sequence of the invention which may be used for an effective screening of novel medicaments for the treatment of hypertension.
  • the invention relates to a method using pro- teome analysis involving the separation of proteins obtained from biological materials by 2DGE; the computer analysis of the resulting images; the statistical analysis of the data to select the proteins which play a role, directly or indirectly in the studied biological problem; and the identification and characterisation of the selected proteins by microsequencing or by mass spectrometry .
  • the invention relates to a method for determining at least one marker protein which is indicative of a high likelihood of having hypertension or a genetic predisposition for having hypertension, the method comprising performing a 2D gel electrophoresis of a sample from at least one mammal having hypertension and a 2D gel electrophoresis of a sample from at least one mammal which does not have hypertension, analyzing and comparing the two 2D gel electrophoresis patterns by computer and determining at least one marker protein which is expressed in a significantly different amount in the hyper- tensive mammal.
  • the detection of any combination of more than one of the markers would be expected to make the analysis an even more reliable indicator for hypertension. Combinations of two markers would thus be preferred and three or more markers would be strongly pre- ferred.
  • Example 1 this approach is exemplified with reference to rats. It is evident that it will be possible to perform similar experiments with respect to other mammals, including human beings .
  • the invention thus relates to a method of detecting for an individual mammal a high likelihood of having hypertension or a genetic predisposition for having hypertension, the method comprising determining in a biological sample from the individual mammal the presence of at least one marker protein which is indicative of a high likelihood of having hypertension or a genetic predisposition for having hypertension.
  • the mammal may e.g. be a rat, mouse, rabbit, human or bovine species.
  • any biological sample that contains or is suspected of containing at least one marker of the invention can be analyzed.
  • biological samples include, but are not limited to, blood, serum, plas- ma, tissue biopsy, organ biopsy, synovial fluid, urine, bile fluid, cerebrospinal fluid, saliva, mucosal secretion, effusion, and sweat.
  • one or more of the markers may be present in soluble or solubilized form in such samples, or may be present in cells isolated with such samples. In the latter instance, the markers may be biosyn- thesized after isolation of the sample, thus providing an opportunity for biosynthetic labelling.
  • the marker protein is selected from the group consisting of the following proteins listed in Table 1 below:
  • modified forms of a native protein may for example be glycosylated, phosphorylated, acetylated, methylated, or lipidified forms.
  • peptide comprises a) short peptides with a length of at least two amino acid residues and at most 10 amino acid residues; b) oligopeptides (11-100 amino acid residues) as well as c) proteins (greater than 100 amino acid residues) , the functional entity comprising at least one peptide, oligopep- tide, or polypeptide. Any of the above may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups.
  • the definition of peptides also comprises native forms of peptides/proteins in animals including humans as well as recombinant proteins or peptides in any type of expression vectors transforming any kind of host, and also chemically synthesized peptides.
  • One specific embodiment of the invention relates to a peptide which is not present or present in a low amount in 2D gel electrophoresis of proteins from resistance artery biopsies from hypertensive rats and which is present in a significantly higher amount in 2D gel electrophoresis of proteins from resistance artery biopsies from normotensive rats.
  • This embodiment comprises the marker proteins marked with "-" in Table 1.
  • Another embodiment of the invention relates to a peptide which is present in 2D gel electrophoresis of proteins from resistance artery biopsies from hypertensive rats and which is not present or present in a significantly lower amount in 2D gel electrophoresis of proteins from resistance artery biopsies from normotensive rats.
  • This embodiment comprises the marker proteins marked with "+" in Table 1.
  • the invention relates to a peptide which has a molecular weight of about 57.5 kD and an isoelectric point of about 6.39, the peptide comprising the following partial amino acid sequence Arg Ser Ala Pro Gly Leu Asn Ser Gly X X Pro Ala Glu Glu Val (SEQ ID N0:1) .
  • the invention relates to a peptide which has a molecular weight of about 57.5 kD and an isoelectric point of about 6.39, the peptide comprising the following partial amino acid sequence (Met) Leu His Glu Leu Glu Lys Ala Tyr (Arg) Phe (Lys) (SEQ ID NO: 2) .
  • the invention relates to peptides which contain both partial sequences.
  • a third embodiment of the invention is a peptide which has a molecular weight of about 17.4 kD and an isoelectric point of about 4.49, the peptide comprising the following partial amino acid sequence X Pro Thr Glu Ala Ala X (SEQ ID NO: 3) .
  • a fourth embodiment of the invention relates to a peptide which has a molecular weight of about 14.9 kD and an isoelectric point of about 4.65, the peptide comprising the following partial amino acid sequence Met Gin Ala Glu Met Ser Pro Ala Phe X Ser Tyr X X Gin (SEQ ID NO: 4) .
  • the invention further relates to a peptide which has a degree of homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%, with an amino acid sequence selected from the group consisting of sequences SEQ ID N0:1, SEQ ID N0:2, SEQ ID NO: 3 and SEQ ID NO: 4 irrespective of any modifications of said amino acids.
  • modified amino acids such as phos- phorylated, acetylated, amidated, methylated, glycosylated or lipidated derivatives of an amino acid should thus be considered to be the same as the amino acid without any such modification.
  • Such peptides may be derived from similar pro- teins from other species, e.g. other mammals such as mouse, rabbit, guinea pig, pig, cow or human or may be entirely or predominantly of synthetic origin.
  • the degree of homology required may be lower, such as when a consecutive string of 6, 7, 8, 9 or 10 amino acids are selected from the amino acid sequences shown in SEQ ID N0:1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID N0:4. Then the degree of homology may be at least 62.5%, such as at least 66%, or at least 70%. Under certain circumstances, it is advantageous that the degree of homology is even higher such as at least 83% or at least 90%.
  • the invention relates to a peptide selected from the group consisting of
  • proteins which are capable, after trypsin digestion, of producing mass spectra peaks when analysed by mass spectrometry which could correspond to at least 3 or more preferably 6 of the peaks detected on the spectra from individual proteins (shown in Figures 12 - 13) are considered as homologous to or identical with the 29 hypertension marker proteins described in this patent application and thus within the scope of the present invention.
  • Some of these mass spectra when compared to the database information publicly available (in manner described in the note to Table 1) corresponded to known proteins. Even though they are known, none had previously been associated with a potential role in the development of hypertension and so this association is novel.
  • These proteins are listed in Table 1 and the accession numbers for the human and rat proteins are given. Thus, the entire sequence of these proteins (including the corresponding proteins from other species) showing levels of homology as defined above is thus included in the scope of this invention with respect to their relationship to hypertension in a manner similar to that of the sequences obtained by microsequencing.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as a gene encoding a marker protein, i.e. a marker gene, may be used in the practice of the present invention.
  • a marker gene i.e. a marker gene
  • these include, but are not limited to, allelic genes, homolo- gous genes from other species, and nucleotide sequences comprising all or portions of marker genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • the marker derivatives of the in- vention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a marker protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight determined by polyacryl- amide gel electrophoresis.
  • the invention comprises any peptide which comprises a sequence which differs from an amino acid sequence as defined above in that at least one amino acid has been deleted, substituted or modified or at least one additional amino acid has been inserted so as to result in an amino acid sequence which encodes a peptide which is a hypertension marker protein useful in the method of the invention.
  • Nucleic acid fragments comprising a nucleotide sequence which codes for a peptide according to the invention as well as nucleic acid fragments which hybridize with these nucleic acid fragments or a part thereof under stringent hybridization conditions, e.g. 5 mM monovalent ions (O.lxSSC), neutral pH and 65 °C are important aspects of the invention.
  • stringent hybridization conditions e.g. 5 mM monovalent ions (O.lxSSC), neutral pH and 65 °C are important aspects of the invention.
  • the term "highly stringent”, when used in conjunction with hybridization conditions, is as defined in the art, i.e. 5-10°C under the melting point T m , cf. Sambrook et al , 1989, pages 11.45- 11.49.
  • nucleic acid is meant a polynucleotide of high molecular weight which can occur as either DNA or RNA and may be either single- stranded or double-stranded.
  • PCR polymerase chain reaction
  • nucleic acid fragment (s) for detecting the presence of a peptide according to invention.
  • nucleic acid fragments which code for a peptide of the invention and fragments which hybridize with nucleic acid fragments coding for a peptide of the invention or a part thereof under stringent or highly stringent hybridization conditions are considered useful in a PCR-DNA analysis method of detecting whet- her an individual has a high likelihood of having hypertension or a genetic predisposition therefor.
  • the length of the primer is preferably at least 12, more preferably at least 13 or 15 and often about 18 nucleotides.
  • the first primer is a DNA sequence of 15-25 nu- cleotides which differs from any subsequence of the proteins from the other species in at least 4 nucleotides per 20 nucleotides of the primer, preferably the differences being in 5 nucleotides per 20 nucleotides.
  • a combination of different primers comprising, for each of marker proteins to be detected, a type of primer unique to the protein and differing from any subsequences of similar normal proteins from the same or other species in at least 4 nucleotides per 20 nu- cleotides of primers.
  • the PCR kit should include a set of 2 primers for each marker peptide to be detected, e.g. a kit having 3 sets of 2 primers in appropriate amounts together with other PCR reagents.
  • the primer may be labelled, such as with radioactive labels, fluorescent dyes, and biotin, or labelled nucleotide triphosphates (e.g. labelled with thymidine) can be included in the PCR reaction to label the PCR amplification product.
  • labelled nucleotide triphosphates e.g. labelled with thymidine
  • the PCR primers used according to the invention may be prepared using well-known methods. Thus, they may be prepared by oligonucleotide synthesis, or they may be prepared by frag- mentation of a larger nucleotide sequence using suitable restriction enzymes.
  • the labelling of the primers can be performed by methods well-known per se.
  • the PCR reagents can be included in suitable PCR kits.
  • the invention relates to a binding means which specifically binds to a peptide shown in Table 1 or a peptide or nucleic acid fragment as described above.
  • the invention relates to an antibody which specifically binds to a peptide of the invention or an antigen- binding fragment thereof, i.e. a polyclonal antibody, a monoclonal antibody, chimeric antibody, single chain antibody fragment, Fab and Fab' fragments, and an Fab expression library.
  • both monoclonal and polyclonal anti- bodies will be useful in providing the basis for one or more assays to detect peptides and marker proteins.
  • Antibodies which are directed against epitopes that are specific for the marker proteins will be most useful as cross reaction will be minimized therewith.
  • immunoassays are contemplated as including various types of enzyme linked immunoassays (ELISAS) , immunoblot techniques, and the like, known in the art.
  • EISAS enzyme linked immunoassays
  • utility is not limited to such assays, and useful embodiments include RIAs and other nonen- zyme linked antibody binding assays or procedures.
  • An important embodiment of the invention is a test kit for detecting whether an individual mammal has a high likelihood of having hypertension or a genetic predisposition for having hypertension, comprising:
  • binding means which specifically binds to at least one marker protein shown in Table 1 or at least one peptide or nucleic acid fragment described above,
  • testing for and the detection of one marker protein may be sufficient, it is likely that testing for and the detection of more than one marker protein (or its nucleotide sequence (e.g. DNA, cDNA or RNA) will be much more valuable for the identification and characterisation of hypertension.
  • more than one marker protein or its nucleotide sequence (e.g. DNA, cDNA or RNA) will be much more valuable for the identification and characterisation of hypertension.
  • test kit for detecting whether an individual mammal has a high likelihood of having hypertension or a genetic predisposition for having hypertension, comprising:
  • a method of determining the presence of a marker protein in a mammal comprising administering a binding means of the invention to the mammal or incubating the sample with the binding means, and detecting the presence of bound marker protein resulting from the administration or incubation, forms part of the invention.
  • the invention relates to a method for determining the effect of a substance, the method comprising using a mammal which has been established to be an individual having a high likelihood of having hypertension or a genetic predisposition for having hypertension by use of the method of the invention, the method comprising administering the substance to the individual and determining the effect of the substance e.g. on the blood pressure of the individual.
  • a pharmaceutical composition which comprises a substance which is capable of regul- ating the expression of a nucleic acid as defined above, at least one marker protein shown in Table 1 or a peptide as defined above or the activity of at least one marker protein shown in Table 1 or a peptide and/or a nucleic acid as defined above.
  • the invention comprises a pharma- ceutical composition comprising at least one marker protein shown in Table 1 or a peptide, a nucleic acid fragment or a binding means as defined above and the use of a such nucleic acid fragments for the treatment or prophylaxis of hypertension (antisense and sense therapy) .
  • Resistance arteries were collected by microsurgery from the mesenteric bed, washed in Hank's buffer, and placed into 100 ⁇ l of labelling medium [DMEM without methionine, containing 10% dialysed human AB serum and 1 mCi/ml [ 35 S] -methionine (Amersham SJ204)] and incubated for 20 hours at 37°C in a 5% C0 2 /95% air humidified atmosphere.
  • the biopsies were homogenized on ice in microhomogenizers using 100 ⁇ l of RNAse/DNAse buffer containing 25 ⁇ g/ml RNAse A (Cooper, Catalog No.
  • Polyacrylamide gels (3.5%, 185 x 1.55 mm, 8 M urea, 2% NP-40) were prepared containing: for the IEF gels 6% AMPHOLINES ® pH range 3.5-10, 2% AMPHOLINES ® 5-7, and 2% SERVALYTES ® 5-7 (Serva) ; and for the NEPHGE gels 3.3% AMPHOLINES ® 7-9, 3.3% AMPHOLINES ® 8-9.5, and 0.5% AMPHOLINES ® 3.5-10. In each case, the actual mixture of ampholytes was calibrated for each batch.
  • Phosphoric acid H 3 P0 4 ; 10 mM was used as the anode buffer and degassed NaOH (20 mM) was used as the cathode buffer.
  • IEF gels were prefo- cused at 1200 V, 133 ⁇ A (limiting values) per tube until the limiting voltage was reached.
  • Half a million TCA precipitable counts were applied to both the IEF and NEPHGE gels and samples were overlayed with 8 M urea, 0.8% AMPHOLINES ® (pH 5- 7), and 0.2% AMPHOLINES ® (pH 3.5-10).
  • Electrophoresis was carried out at 1200 V, 133 ⁇ A (limiting) per gel: for 18 hours for IEF gels; and for 4 hours after the gels reached the limiting voltage for the NEPHGE gels. The gels were then extruded using compressed air and equilibrated for 6 minutes before being frozen at -70°C.
  • the gels were quickly thawed in a water bath (80°C) , incubated for 2 minutes at room temperature, loaded and run on 12.5% polyacrylamide gels (200 x 200 mm, 1 mm) at 20°C.
  • the second dimension gels were fixed for 45 minutes in 45% methanol, 7.5% acetic acid, treated for 45 minutes with AMPLIFY ® (Amersham) to enhance fluorographic detection, dried, and exposed to X-ray film (AGFA Curix RP2) at -70°C for 5 days.
  • the developed images were compared either visually or with an image processing computer (Biolmage programme version 4.5 M, Millipore) .
  • Images were captured by either scanning the autoradiographs, fluorographs or dried silver stained gels using a Truvel scanner (Bio Image) or by importing the TIFF files from the fluorescence imager or phosphor imager (AGFA ADC) .
  • the images were edited and matched using the Bio Image software (version 6.1) on a Sun Ultrasparc workstation.
  • the developed X-ray film was positioned over the dried gel using the crosses and used to locate the pro- teins of interest.
  • a stencil -mask was made to relocate these proteins once the X-ray film was removed and the proteins were cut directly from the gel, taking as little backing filter paper with the spot as possible.
  • the gel piece was then dried in a vacuum centrifuge until it became white on the surface and then the gel was allowed to reswell in 50 mM NH 4 HC0 3 , 5 mM CaCl 2 , 12.5 ng/ ⁇ l trypsin (Boehringer Mannheim, sequencing grade) at 4°C for 45 mins . The supernatant was then replaced with 5-10 ⁇ l of the same buffer lacking trypsin and left to digest overnight at 37°C.
  • Mass spectra were recorded on a Bruker Reflex (Bruker- Fran- zen, Bremen, Germany) or a Voyager Elite (PerSeptive Bio- systems, Framingham, Massachusetts, USA) mass spectrometer, both operated in delayed extraction reflector mode. Samples were prepared using cyano- 4 -hydroxy cinnamic acid as matrix. When appropriate, nitrocellulose was mixed with the matrix. The criteria for choice of matrix and the sample preparation procedures are described in detail elsewhere [10] .
  • Nanoelectrospray mass spectra were obtained on a Finnigan TSQ 70 triple quadrupole mass spectrometer (Finnigan Corporation, San Jose, California, USA) or a Bruker Esquire ion trap mass spectrometer (Bruker-Franzen, Bremen, Germany) both equipped with nanoelectrospray sources. Sample preparation was performed as described above.
  • hypertension markers # 847 An example for one of the hypertension markers # 847 is shown in Fig. 6.
  • Four examples of the resulting 2D Gel of [ 35 S] - methionine labelled resistance artery are shown. Comparison of these patterns clearly illustrates, by differences in protein spot intensity, a number of very significant changes in protein expression: proteins that are expressed in WKY rats or normotensive F2-rats are expressed at non-detectable or low detectable levels in SHR rats and hypertensive F2-rats (indicated by arrows in Fig. 6) .
  • a number of marker proteins identified on the IEF gels were analyzed further by microsequencing.
  • the proteins were elec- troeluted using a 0.1% SDS, 27.5 mM Tris-HCl, 95.9 mM glyci- ne, pH 8.3 buffer and recovered on PVDF membrane.
  • the mem- brane was washed and saturated with 0.2% polyvinylpyrrolido- ne.
  • the membrane-associated protein was the trypsin digested with 0.5 ⁇ g trypsin for 24 hours at 37°C in 100 ⁇ l of 100 mM Tris-HCl, pH 8.5, 10% acetonitrile .
  • the peptides were separated by reverse phase-HPLC using a C-18 column with a tri- fluoroacetic acid-acetonitrile gradient. Peaks were detected at 214 nm and manually collected and treated with POLYBRENE ® (Applied Biosystems) . Sequencing of the peptides was carried out in a pulsed- liquid phase sequencer (Applied Biosystems, model 477A) equipped with an on-line phenylthiohydantoin amino acid derivative analyzer (Applied Biosystems, model
  • the programme GCG Genetics Computer Group, Wisconsin
  • the programme GCG was used to search the SWISS -PROT protein sequence database (40,292 sequences, 14,147,368 symbols) with the FASTA subroutine, and using the EMBL genebank (257,716 sequences, 263,275,079 symbols) using the subroutine tFASTA so that the amino acid sequences were translated to base sequences and these were used to search all entered sequences using a word size of 2.
  • the first partial sequence (SEQ ID NO:l) for protein 305 showed high similarity with ACE-1.
  • a second partial sequence from this spot (SEQ ID NO: 2) did not demonstrated any significant homology or similarity with ACE-1.
  • Both proteins appear to be novel proteins, i.e., proteins that have not been sequenced previously.
  • the sequence data therefore suggest that at least one of the polypeptides which have been detected corresponds to a new variant of ACE, having a different amino acid sequence, a different size, and a different isoelectric point.
  • Circumstantial evidence for the importance of this or these molecules can be obtained by careful examination of the literature .
  • Fig. 1 shows the genetic relationships between WKY and SHR rats.
  • WKY and SHR rats were crossed, yielding an FI generation.
  • These Fl rats were then crossed with each other in order to obtain an F2 generation, and the rats of this generation were used for the studies.
  • this F2 gene- ration showed a larger range of the blood pressures of the rats than the parental generation.
  • Fig. 2 shows the blood pressure distribution among WKY and SHR rats and the F2 rats.
  • the mean blood pressure (mm Hg) is shown on the abscissa, and the different groups are shown on the ordinate.
  • Fig. 3 shows raw data with respect to protein expression in normal and hypertensive rats.
  • the numbers in the column headings relate to the individual rats.
  • the abbreviation AVG stands for average; STDS stands for standard deviation; and t-value is from the Student's t-test.
  • Fig. 4 shows the 10 most significant correlations (by Student's t-test) of blood pressure with protein expression in the F2 generation (middle panel) .
  • the same 10 proteins are selected from the parental generation (right-hand panel) and the Figure shows that four of these (proteins 847, 803, 325 and 862) were found to be highly correlated also in the parental generation.
  • %IOD in this case is the average %I0D for each protein from the whole group.
  • Std is the standard deviation for each protein from the said group.
  • P is the normal probability value for the comparison between the F2 and the parental generation for each protein.
  • NS means not significant .
  • Fig. 5 shows the two-dimensional gel of [ 35 S] -methionine labelled resistance arteries. All the proteins whose expression is significantly correlated to blood pressure are indicated with circles with a cross (if the protein is downregu- lated in the hypertensive relative to the normotensive) or without a cross (if the protein is upregulated in the hypertensive relative to the normotensive) .
  • Fig. 6 shows a detailed picture of protein # 847 from the regions of the gels from the normotensive and hypertensive rats from the F2 and the parental generations.
  • Fig. 7 shows the amino acid sequences of the hypertension marker #325 compared with the sequence of ACE from various species . Note that some amino acids are completely conserved and some are less well conserved.
  • Fig. 8 shows the functions of the angiotensin- converting enzyme (ACE) .
  • Angiotensinogen is cut by the protease renin to the inactive peptide angiotensin I.
  • ACE cuts angiotensin I to angiotensin II (which is a potent vasopressor) .
  • ACE can inactivate the active vasodilator bradykinin by proteolysis and so ACE has a double effect.
  • Fig. 9 shows an analysis of protein # 325 and its possible relationship to the 52 kD protein described by M ⁇ ns et al . and Danilov et al .
  • Fig. 10 shows a strategy as to how to go further with the analysis of the protein.
  • Fig. 11 shows the strategy followed in obtaining the data described in Example 1.
  • Fig. 12 shows the MALDI tof MS analysis of marker protein 27,
  • Fig. 13 shows the MALDI tof MS analysis of marker protein 785.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne une méthode de détection chez un mammifère, un risque élevé d'hypertension ou une prédisposition génétique à l'hypertension. Ladite méthode consiste à déterminer la présence dans un échantillon biologique prélevé sur ledit mammifère d'au moins une protéine marqueuse signalant un risque élevé d'hypertension ou une prédisposition génétique à l'hypertension, ladite protéine étant choisie dans le groupe constitué des protéines répertoriées dans le tableau 1. L'invention se rapporte aussi à des nouvelles protéines, des fragments d'acide nucléique et leur utilisation pour la détection du risque élevé d'hypertension chez un mammifère ou de la prédisposition génétique à l'hypertension ainsi que sur des trousses d'essai prévues à cet effet.
EP97942823A 1996-10-04 1997-10-06 Marqueurs de l'hypertension Withdrawn EP0929667A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DK109596 1996-10-04
DK109596 1996-10-04
US2884996P 1996-10-23 1996-10-23
US28849P 1996-10-23
PCT/DK1997/000429 WO1998015623A1 (fr) 1996-10-04 1997-10-06 Marqueurs de l'hypertension

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EP0929667A1 true EP0929667A1 (fr) 1999-07-21

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EP97942823A Withdrawn EP0929667A1 (fr) 1996-10-04 1997-10-06 Marqueurs de l'hypertension

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EP (1) EP0929667A1 (fr)
JP (1) JP2001510328A (fr)
AU (1) AU740021B2 (fr)
CA (1) CA2267637A1 (fr)
NZ (1) NZ335258A (fr)
WO (1) WO1998015623A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9800378D0 (en) * 1998-01-08 1998-03-04 Univ Liverpool Proteome analysis
RU2240629C2 (ru) * 2001-11-23 2004-11-20 Эл Джи Электроникс Инк. Способ изготовления сетчатого экрана безэлектродной осветительной установки (варианты)
BRPI0206903B8 (pt) * 2002-12-20 2021-07-27 Fund De Amparo A Pesquisa Do Estado De Sao Paulo Fapesp método de prognóstico de uma predisposição para o desenvolvimento de hipertensão e lesões no coração, sistema nervoso, sistema vascular ou rins, e uso da isoforma de 90 kda da enzima conversora de angiotensina i no referido método

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Publication number Priority date Publication date Assignee Title
FR2636968B1 (fr) * 1988-09-27 1992-01-03 Inst Nat Sante Rech Med Acide nucleique codant pour l'enzyme de conversion de l'angiotensine (eca) humaine, et ses applications, notamment pour le diagnostic in vitro de l'hypertension arterielle
US5510264A (en) * 1993-09-28 1996-04-23 Insight Biotech Inc. Antibodies which bind meningitis related homologous antigenic sequences

Non-Patent Citations (1)

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Title
See references of WO9815623A1 *

Also Published As

Publication number Publication date
JP2001510328A (ja) 2001-07-31
WO1998015623A1 (fr) 1998-04-16
CA2267637A1 (fr) 1998-04-16
AU740021B2 (en) 2001-10-25
AU4451997A (en) 1998-05-05
NZ335258A (en) 2002-06-28

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