[go: up one dir, main page]

WO1998035016A1 - Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection - Google Patents

Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection Download PDF

Info

Publication number
WO1998035016A1
WO1998035016A1 PCT/US1998/002494 US9802494W WO9835016A1 WO 1998035016 A1 WO1998035016 A1 WO 1998035016A1 US 9802494 W US9802494 W US 9802494W WO 9835016 A1 WO9835016 A1 WO 9835016A1
Authority
WO
WIPO (PCT)
Prior art keywords
pyk2
leu
glu
lys
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.)
Ceased
Application number
PCT/US1998/002494
Other languages
English (en)
Inventor
Le T. Duong
Gideon A. Rodan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Priority to JP53498698A priority Critical patent/JP2002513284A/ja
Priority to CA002277028A priority patent/CA2277028A1/fr
Priority to EP98905026A priority patent/EP0998553A4/fr
Publication of WO1998035016A1 publication Critical patent/WO1998035016A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • PROTEIN TYROSINE KINASE 2 PYK2
  • NUCLEIC ACIDS NUCLEIC ACIDS
  • This invention is directed to nucleic acids encoding protein tyrosine kinase 2 (PYK2), to murine PYK2, to methods of making this protein using the nucleic acids, and to assays for inhibitors of PYK2.
  • PYK2 protein tyrosine kinase 2
  • PYK2 Protein tyrosine kinase 2
  • CAK ⁇ Cell Adhesion Kinase ⁇
  • RAFTK Adhesion Focal Tyrosine Kinase
  • PYK2 was first cloned from human brain as a Grb-2 binding protein, and has also been cloned from rat and human brain libraries. There have been conflicting reports as to its cellular expression. In one study, abundant PYK2 transcripts were found in brain and lower levels were detected in the kidney. In another report, PYK2 expression was also found to be most abundant in rat brain, but its transcripts could also be detected in kidney, spleen, lung, intestine and epididymis. PYK2 transcripts were also detected in rat fibroblast 3Y1 and WFB cell lines, as well as in the human T cell leukemia Jurkat line.
  • nucleic acids substantially free from associated nucleic acids, which encode murine protein tryosine kinase 2 (PYK2).
  • PYK2 murine protein tryosine kinase 2
  • the nucleic acid which encodes PYK2 is a DNA.
  • Murine PYK2 cDNA is set forth in Figure 1 (SEQ ID NO:5).
  • FIG. 6 Yet another aspect of this invention is murine PYK2 which is free from associated murine proteins.
  • FIG. 6 One murine PYK2 is set forth in Figure 1 (SEQ ID NO:6).
  • Another aspect of this invention is a method of making PYK2 by introducing nucleic acids into a cell, the nucleic acids comprising nucleic acids which encode PYK2, under conditions which transcription and translation of PYK2 occur. It is preferred that the nucleic acids be present in a vector, such as a plasmid or baculovirus vector. It is also preferred that the nucleic acids be under the control of transcriptional control elements, such as promoters and optionally enhancers. Such control elements are well known in the art.
  • Host cells which express PYK2 are also part of this invention.
  • Preferred host cells include mammalian cells, insect cells, yeast and bacterial cells such as E. coli. Cell lines which permanently (rather than transiently) express murine PYK2 are also another aspect of this invention.
  • the recombinant PYK2 which is made using the cloning process of this invention may be used in assays in order to further characterize the biological function of PYK2 and to identify compounds such as agonists and antagonists which modulate its activity.
  • a further aspect of this invention is an assay for the identification of compounds which modulate the activitiy of PYK2, and particularly inhibitors of PYK2 activity.
  • This assay comprises the steps of: contacting recombinant PYK2 with a tyrosine substrate in the presence of radiolabeled ATP and a putative activity-modifying compound, and measuring the amount of radiolabeled tyrosine which is formed; and optionally comparing the amount of radiolabeled tyrosine formed in the presence of the putative activity-modifying compound with that formed in the absence of the putative activity-modifying compound.
  • Integrins are the major family of cell surface receptors that mediate adhesive interactions, either to adjacent cells or to the extracellular matrix. Integrin signalling is mediated through the focal adhesion kinase (FAK) family of proteins. PYK2 is a member of the FAK family, and is involved in integrin-mediated signal transduction pathways in megakaryocytes, brain tissue and hematopoietic cells. Modulators of PYK2 would therefore be potential therapeutic agents for modulating platelet levels.
  • FAK focal adhesion kinase
  • Figure 1 is the cDNA sequence of mouse PYK2 and the deduced protein sequence. Intron sequences are in lower case letters. The exon sequence is capitalized. The boxed sequence of the deduced protein indicates the kinase domain. The circled prolines of the deduced protein indicate the proline rich domain.
  • PYK2 means protein tyrosine kinase 2, allelic variations of protein tyrosine kinase 2, and mutations or fragments thereof which retain at least about 85%, and preferably at least about 90% of the biological activity of native PYK2.
  • Native PYK2 means the protein tyrosine kinase which is naturally occuring in an organism.
  • Substantially free from associated nucleic acids means that in a sample, there is less than about 5% (by weight) nucleic acids present which are other than nucleic acids encoding PYK2.
  • Substantially free from associated murine proteins means that in a sample, there is less than about 5% (by weight) protein which is other than murine PYK2.
  • FAM focal adhesion kinase.
  • Heterologous PYK2 nucleic acid means that the nucleic acid was introduced to the cell, without regard as to whether the nucleic acid is from the same species as the cell; alternatively it refers to nucleic acids encoding PYK2 in a cell whose ancestor had PYK2 introduced into the cell.
  • Heterologous PYK2 protein means that the PYK2 was encoded by a heterologous nucleic acid.
  • FAK proteins which are involved in cell adhesion processes, were not detected in a number of macrophage cell lines and it was therefore hypothesized that another cell adhesion-dependent kinase, homologous to FAK, may assume its function in these cells.
  • PYK2 was recently identified as another member of the FAK family and its expression was detected in spleen, thymus, lung and peripheral blood leukocytes (Avraham, et al., 1995 supra; Sasaki, et al., 1995 supra).
  • specific probes were generated for PYK2 and FAK which were used to examine the expression of PYK2 and FAK in mouse tissues. As previously reported, for other species, PYK2 is highly expressed in brain and spleen, and at lower levels in kidney, lung and liver and has a more restricted tissue distribution than FAK.
  • the full length cDNA was cloned from a mouse spleen cDNA library.
  • the deduced amino acid sequence of the full length clone was found to be identical to the recently published amino acid sequence of the mouse RAFTK (Avraham, et al., 1995, supra).
  • PYK2 and FAK cDNAs were subsequently transfected into human embryonic kidney (HEK) 293 cells.
  • HEK human embryonic kidney
  • Macrophages were induced by thioglycolate injection into the peritoneal cavities of adult BALB/c mice. After 4 days, cells were collected, washed and cultured in RPMI 1640 medium containing 10% FBS. After 3h at 37°C, the cultures were washed extensively to remove non-adherent cells and cultured overnight before samples were prepared for immunoprecipitation. Bone marrow derived macrophages were prepared as described by Li and Chen, 1995 J. Leuk. Biol. 57:484- 490, which is hereby incorporated by reference.
  • Non adherent cells were cultured in RPMI completed medium in the presence of human macrophage colony-stimulating factor (MCS-F, 250 units/ml, Genetics Institute, Cambridge, MA).
  • MCS-F human macrophage colony-stimulating factor
  • Differentiated macrophages were prepared for immunoprecipitation after 5 days in culture.
  • Bone marrow derived osteoclast-like cells were prepared as described by Wesolowski, et al., 1995 Exp. Cell Res. 219:679-686, which is hereby incorporated by reference. After collagenase-dispase treatment, mononucleated tartrate resistant phosphatase positive cells were released from the tissue culture plate using 30 nM echistatin (Merck Res. Labs., West Point, PA). Freshly isolated osteoclast-like cells were immediately solubilized in immunoprecipitation buffer.
  • a specific probe for FAK (700bp) was generated using the following primers: 5'- CAGCA CACAA TCCTG GAGGA G (SEQ. ID. NO:3) and 3'- GCTGA AGCTT GACAC CCTCA T (SEQ. ID. NO. 4) with cDNAs of mouse osteoblastic MB 1.8 cells as template (Wesolowski, et al., 1995, supra). These probes were confirmed by sequencing analysis. PYK2 cDNA fragments were cloned from a mouse spleen ⁇ -ZAP II cDNA library (Stratagene, La Jolla, CA) using the specific PYK2 probe.
  • Full length PYK2 cDNA were constructed by ligation of two overlapping clones at the Vspl site.
  • the amino acid sequence of the isolated PYK2 cDNA clone was identical to the previously published mouse RAFTK sequence (Avraham, et al., 1995 supra).
  • Full length FAK cDNA was generated by PCR according to the published sequence as described in Hanks, et al., 1992 Proc. Natl. Acad. Sci. USA. 89:8487-8491.
  • Both PYK2 and FAK cDNAs were subcloned into pCDNA3 plasmid (InVitrogen, San Diego, CA) and transfected into human embryonic kidney (HEK) 293 cells (ATCC, Rockland, MD) by electroporation at 200V, 960 ⁇ F using a GenePulser (Biorad Labs, Richmond, CA).
  • HEK 293 cells were subsequently subjected to G418 selection (800 ⁇ g/ml, Gibco BRL) and clones were picked after 3 weeks in selection medium.
  • Expression of PYK2 and FAK in HEK293 cells were confirmed by Northern analysis using the respective probes, and Western blots were performed using anti-PYK2 antibodies.
  • Mouse multiple tissue Northern blot was purchased from Clonetech (Palo Alto, CA) and hybridization of the Northern blot using probes specific for PYK2, FAK and glyceraldehde 3-phosphate dehydrogenase (GAPDH) were performed as described previously (Wesolowski, et al., 1995, supra).
  • the PYK2 C-terminal domain (from Methionine residue 685 to end) was amplified by PCR using the mouse PYK2 as template. Amplified product was cloned into plasmid pGEX-4T (Pharmacia Biotech., Piscataway, NJ) and transformed in E.coli XLl-Blue (Stratagene). Expression of GST-PYK2 C-terminal fragment was induced using 0.5 mM IPTG, purified and cleaved from GST with thrombin, essentially according to the instructions of the manufacturer (Pharmacia).
  • the purified C-terminal fragment of mouse PYK2 was used to immunize two rabbits (Research Genetics, Huntsville, AL) and the titers of both antisera were initially determined by ELISA using the recombinant C-terminal fragment of PYK2. Specificity of the immune sera was subsequently determined by Western blot by comparison to the preimmune sera. Polyclonal antibodies were then affinity purified by passing the combined fractions of both antisera through an affinity column, which was constructed using the same purified antigen cross linked to CNBr-activated Sepharose 4B according to the instructions of the manufacturer (Pharmacia).
  • the antibodies were eluted from the column using 0.2 M Glycine, pH 2.5 and lmM EGTA and the eluted fraction was then dialyzed against PBS containning 0.02% azide.
  • Anti-PYK2 antibodies were stored at -70°C at a concentration of 0.5mg/ml.
  • IC-21 cells were solubilized in TNE lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, lmM EDTA, 10% glycerol, 50 mM NaF, 1 mM sodium vanadate and protease inhibitors as described above.
  • PYK2 were immunoprecipitated from the clarified lysates, half of the sample was subjected to immunoblotting with anti PYK2 antibodies, as described above, and the other half was washed 2 times with the same lysis buffer, and with kinase assay buffer (IX) containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MnCl2 and 1 mM dithiothreitol.
  • IX kinase assay buffer
  • kinase assay buffer containing 5 ⁇ Ci [ ⁇ -32p] ATP (3000 Ci/mmol, Amersham), 10 mM ATP, 0.1% BSA and 100 ⁇ g of poly (Glu,Tyr) (molar ratio 4:1; Sigma) was added to the beads and incubated for 10 min at 30°C (Howell and Cooper, 1995 Mol. Cell. Biol. 14:5402-5411).
  • the reaction mixtures (25 ⁇ l) were added to 25 ⁇ l of 30% trichloroacetic acid (TCA) and 0.1 M sodium pyrophosphate, followed by incubation at 4°C for 15 min.
  • TCA trichloroacetic acid
  • Multiscreen-FC filter plate (Millipore, Marlborough, MA), washed with ice cold 15% TCA (3X), allowed to dry and incorporation of 32p into the substrate was counted on a Packard top count microplate scintillation counter (Packard, Meriden, CT). Each assay were performed as triplicate. The specific activity was determined by comparing the radioactive counts with immunoblot signals.
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 5 :
  • AGTGTACCCC TAACGGCCAA GATGGCTTTC TGCATGGACA TTTGAGAGCC AGTATTCCTC 3540
  • CTTTTCTTAC GTCTCCTTTT TCTCCTCCCC
  • CTTTTCACAT CTGCTCCCCT CCTCTCTCAT 3900
  • Val Glu Lys Glu Asp Val Arg lie Leu Lys Val Cys Phe Tyr Ser Asn

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des acides nucléiques codant la protéine tyrosine-kinase 2 (PYK2), la PYK2 murine, des procédés de fabrication de ladite protéine au moyen desdits acides nucléiques et des méthodes de détection des inhibiteurs de la PYK2.
PCT/US1998/002494 1997-02-11 1998-02-09 Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection Ceased WO1998035016A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP53498698A JP2002513284A (ja) 1997-02-11 1998-02-09 タンパク質チロシンキナーゼ２（ｐｙｋ２）、核酸及びアッセイ
CA002277028A CA2277028A1 (fr) 1997-02-11 1998-02-09 Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection
EP98905026A EP0998553A4 (fr) 1997-02-11 1998-02-09 Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3756197P 1997-02-11 1997-02-11
US60/037,561 1997-02-11

Publications (1)

Publication Number Publication Date
WO1998035016A1 true WO1998035016A1 (fr) 1998-08-13

Family

ID=21895004

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/002494 Ceased WO1998035016A1 (fr) 1997-02-11 1998-02-09 Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection

Country Status (4)

Country Link
EP (1) EP0998553A4 (fr)
JP (1) JP2002513284A (fr)
CA (1) CA2277028A1 (fr)
WO (1) WO1998035016A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1009395A4 (fr) * 1997-02-18 2000-11-08 Lxr Biotechnology Inc Systeme d'expression de promoteur de bak

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538858A (en) * 1994-04-08 1996-07-23 Pierce Chemical Company Rapid assay for radioactive determination of protein kinase activity
US5573944A (en) * 1994-07-22 1996-11-12 President And Fellows Of Harvard College Yeast cell expressing heterologous receptor kinase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538858A (en) * 1994-04-08 1996-07-23 Pierce Chemical Company Rapid assay for radioactive determination of protein kinase activity
US5573944A (en) * 1994-07-22 1996-11-12 President And Fellows Of Harvard College Yeast cell expressing heterologous receptor kinase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AVRAHAM S., ET AL.: "IDENTIFICATION AND CHARACTERIZATION OF A NOVEL RELATED ADHESION FOCAL TYROSINE KINASE (RAFTK) FROM MEGAKARYOCYTES AND BRAIN.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 270., no. 46., 17 November 1995 (1995-11-17), US, pages 27742 - 27751., XP002912212, ISSN: 0021-9258, DOI: 10.1074/jbc.270.46.27742 *
See also references of EP0998553A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1009395A4 (fr) * 1997-02-18 2000-11-08 Lxr Biotechnology Inc Systeme d'expression de promoteur de bak
US6436639B1 (en) 1997-02-18 2002-08-20 Tanox, Inc. Bak promoter expression system

Also Published As

Publication number Publication date
EP0998553A4 (fr) 2002-09-18
JP2002513284A (ja) 2002-05-08
EP0998553A1 (fr) 2000-05-10
CA2277028A1 (fr) 1998-08-13

Similar Documents

Publication Publication Date Title
JP3561268B2 (ja) レセプターチロシンキナーゼ標的タンパク質のｃＤＮＡクローニング方法及びｈＧＲＢタンパク質
Oehrl et al. The germinal center kinase (GCK)-related protein kinases HPK1 and KHS are candidates for highly selective signal transducers of Crk family adapter proteins
US5821082A (en) Anti-proliferation domain of a human Bcl-2 and DNA encoding the same
EP0536350A1 (fr) Identification d'un nouveau gene humain recepteur de tyrosine kinase
Matsushita et al. Identification and characterization of a novel SH3-domain binding protein, Sab, which preferentially associates with Bruton's tyrosine kinase (BtK)
WO1998039410A1 (fr) IλB $i(KINASES)
US5610276A (en) Polypeptides and antibodies derived from GAP associated protein, p62
EP0935467A1 (fr) Composes inhibiteurs de l'interaction entre proteines transductrices de signaux et le domaine glgf (pdz/dhr), et utilisation de tels composes
WO2000052173A2 (fr) Homologues de sphingosine kinase humaine clones
US6187552B1 (en) Method for identifying inhibitors of JAK2/cytokine receptor binding
AU713937B2 (en) Peptides capable of binding to the gap protein sh3 domain, nucleotide sequences coding therefor, and preparation and use thereof
WO1998035056A1 (fr) Identification d'inhibiteurs de la proteine tyrosine-kinase 2
WO1993023539A1 (fr) ADNc DE LA PROTEINE 1 ASSOCIEE AU RETINOBLASTOME
WO1998043087A9 (fr) Procede d'identification d'inhibiteurs de la liaison proteine jak2/recepteur de cytokine
US6255074B1 (en) Abl-interactor protein
US6824971B1 (en) Methods of inhibiting or enhancing the TGFβ-SMAD signaling pathway
EP0998553A1 (fr) Proteine tyrosine-kinase 2 (pyk2), acides nucleiques et methode de detection
US6310181B1 (en) Adaptor protein FRS2 and related products and methods
Xu et al. Identification of Rgl3 as a potential binding partner for Rap-family small G-proteins and profilin II
EP0803571B1 (fr) Protéine TAB1 et ADN codant pour celle-ci
US5716782A (en) Nucleic acid encoding a signal mediator protein that induces cellular morphological alterations
US7256256B1 (en) CDK4 binding peptide
US6753413B1 (en) P35NCK5A binding proteins
AU731570B2 (en) Tyrosine phosphorylated cleavage furrow-associated proteins (PSTPIPs)
US20030143582A1 (en) Human CDC4 modulates cyclin E degradation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2277028

Country of ref document: CA

Ref country code: CA

Ref document number: 2277028

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1998905026

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1998905026

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1998905026

Country of ref document: EP