Classifications

• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
  • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the invention relates to a nucleic acid molecule which comprises a nucleic acid sequence coding for a chemokine, a neuropeptide precursor or at least one neuropeptide, and to host cells containing this nucleic acid molecule.
• the invention further relates to a polypeptide molecule which acts as a chemokine or neuropeptide or contains at least one neuropeptide, as well as fragments thereof which contain at least one neuropeptide and a method for producing the polypeptide molecule or a fragment thereof.
• the invention relates to antibodies, detection methods and test kits and pharmaceutical compositions.
• SDF-1 ⁇ (stromal cell derived factor 1 ⁇ ) and its isoform SDF-1 ß, which results from alternative splicing, were originally cloned from a cell line from stromal cells of the bone marrow of the mouse (Tashiro et al, 1993). Based on the established homology of the deduced amino acid sequence to the sequences of interleukin 8 (32%) and the macrophage inflammatory protein 1 ⁇ (32%) and the presence of four characteristic cysteine residues, SDF-1 ⁇ and SDF-1 ß were determined assigned to the group of CXC ( ⁇ ) chemokines.
• the CXC ( ⁇ ) chemokines are a subset of the intercrine cytokine family, which consists of various distantly related inflammatory cytokines.
• the cDNA sequences for SDF-1 ⁇ and SDF-1 ⁇ of the mouse and of the human being show a high degree of homology to one another and arise from alternative splicing of a single gene.
• SDF-1 The biological function of SDF-1 was examined using the human SDF-1.
• SDF-1 ⁇ is required for the maturation of B cells, has a T-lymphotropic effect and induces cell death in the neuronal cell line hNT.
• SDF-1 is a natural ligand of the CXCR4 (LESTR / Fusin) chemokine receptor of T cells, which is a binding cofactor of T-lymphotropic HIV-1 strains.
• SDF-1 ⁇ and -1ß show a growth arrest-specific expression pattern in fibroblasts and hepatocytes both in vitro and in vivo. Mice in which the SDF-1 gene has been inactivated show a reduced formation of B cells, a defect in the ventricular septum and defects in cell migration in the Cerebellum, and die shortly after birth. SDF-1 could play an important role in nerve regeneration.
• the present invention is based on the object of providing new means which are targeted for the diagnosis and / or treatment of diseases which are associated with a defect in the SDF-1 factor or its receptor (CXCR4).
• nucleic acid molecule comprising:
• nucleic acid sequence coding for a chemokine, a neuropeptide precursor or at least one neuropeptide which is selected from the following sequences:
• polypeptide as used in the description below includes peptides or proteins composed of 7 or more amino acids.
• chemokine stands for a member of a family of relatively small proteins that are divided into four subgroups based on the characteristic arrangement of cysteine groups: C, CC, CXC and CX3C. The chemokines bind to specific receptors (Rollins, BJ, 1997). In connection with the present invention, the term “chemokine” refers in particular to members of the CXC chemokine family.
• the chemokine is preferably a polypeptide molecule which, in a ca-imaging experiment under the conditions described in Koller et al (2001), shows a 1.5-10-fold increase in the intracellular calcium concentration in primary astrocytes and / or elicits neurons from the central nervous system of rats or humans.
• Neuropeptides Biologically active and physiologically important signaling molecules with regulatory and modulatory functions in the nervous system are referred to as "neuropeptides".
• the functional areas include neurotransmission, receptor modulation, changes in the electrophysiological properties of cell membranes and metabolic processes. Neuropeptides are synthesized by neurons and mostly released at the synapse (Siegel et al., 1989).
• Neuropeptide precursor is understood to mean a precursor protein which is converted into an active neuropeptide by proteolytic cleavage.
• the nucleic acid sequence contained in the nucleic acid molecule according to the invention can be a genomic DNA, cDNA or synthetic DNA, synthetic DNA sequences also being understood to mean those which contain modified internucleoside bonds.
• the expression “at least 60% identical” refers to identity at the DNA level, which can be determined according to known methods, for example computer-aided sequence comparisons (Altschul et al., 1990).
• identity denotes the degree of relationship between two or more nucleic acid molecules, which is determined by the correspondence between the sequences.
• the percentage of “identity” results from the percentage of identical regions in two or more sequences taking into account Gaps or other sequence peculiarities.
• the identity of related nucleic acid molecules can be determined using known methods. As a rule, special computer programs with algorithms that take account of the special requirements are used. Preferred methods for determining identity initially produce the greatest agreement between the sequences examined.
• Computer programs for determining the identity between two sequences include the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 387, 1984); Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul et al., 1990), however, are not limited to these.
• the BLASTX program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbuch, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul et al., 1990).
• NCBI National Center for Biotechnology Information
• the well-known Smith Waterman algorithm can also be used to determine identity.
• Preferred parameters for nucleic acid sequence comparison include the following:
• the GAP program is also suitable for use with the above parameters.
• the above parameters are the default parameters for nucleic acid sequence comparisons.
• gap opening penalties can be used.
• the selection will depend on the comparison to be carried out and also on whether the comparison is carried out between pairs of sequences, GAP or Best Fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred.
• One with the 60% match identified above algorithm is referred to as 60% identity in the context of this application. The same applies to higher degrees of identity.
• sequence which hybridizes with the counter strand of the sequence specified in (a) indicates a sequence which hybridizes under stringent conditions with the counter strand of the sequence specified in (a).
• the hybridizations can be carried out at 42 ° C. with a hybridization solution consisting of 5 ⁇ SSPE, 5 ⁇ Denhardt's, 0.1% SDS, 100 ⁇ g / ml salmon sperm DNA, 30-50% formamide (Sambrook et al., 1989) , A wash step is a twice repeated 10-15 minute wash in 2 x SSPE, 0.1% SDS at 42 ° C, followed by a repeated 20 minute wash in 0.2 x SSPE, 0.1% SDS at 50 ° C.
• SSC can be used instead of SSPE for the washing solutions.
• the nucleic acid molecule according to the invention represents a new member of the SDF family of chemokines, which is referred to below as SDF-1 ⁇ .
• SDF-1 ⁇ SDF family of chemokines
• the SDF-1 ⁇ nucleic acid sequence consists of the complete nucleic acid sequence of SDF-1 ß and an additional sequence of 2572 nucleotides, which follows the codon 89 downstream of SDF-1 ß in the same reading frame. This insert results in an amino acid sequence with 119 amino acids and a theoretical molecular weight of 13.6 Kd for the new SDF-1 ⁇ polypeptide, the sequence at the carboxy terminus being extended by 30 amino acids compared to the known SDF-1 ⁇ sequence is. It is believed that SDF-1 ⁇ is generated by inserting a new alternative exon purple between the known exons III and IV (cf. Shirozu et al., 1995).
• 5 groups of two basic amino acids can form recognition patterns for a membrane-bound protease of the Golgi system and the secretion vesicles.
• Proteolytic cleavage at these sites gives rise to five short peptides (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10) and a shortened protein (SEQ ID NO: 7), two of which are peptides and one polypeptide (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) have a carboxy-terminal glycine residue.
• Peptides with a glycine residue at the carboxy terminus are potential substrates for the peptidyl-amidating monooxygenase (PAM), which catalyzes the carboxy-terminal cleavage of carboxylate and thereby produces ⁇ -amidated carboxy terminus (CONH 2 ), which are characteristic of neuropeptides (see overview by Eipper et al., 1992).
• PAM peptidyl-amidating monooxygenase
• the rat SDF-1 ⁇ transcript encodes a protein with 93 amino acids (see SEQ ID NO: 17) and a theoretical molecular weight of 10.5 Kd, the first 19 amino acids being a signal sequence for secreted Represent proteins.
• the first 17 amino acids of the mature protein, which are found in all isoforms, are required for binding to the CXCR4 receptor (cf. Loetscher et al. 1998; Doranz et al., 1999).
• Two basic amino acids (Lys 89 and Arg 90) in the carboxy-terminal region provide a recognition pattern for the proteolytic cleavage, which results in a pentapeptide (Lys 89-Met 93, SEQ ID NO: 18) and a truncated protein.
• the nucleic acid molecule according to the invention comprises a nucleic acid sequence which is at least 80%, preferably at least 90%, particularly preferably at least 95% identical to the nucleic acid sequence according to SEQ ID NO: 1.
• Nucleic acid molecules which comprise a nucleic acid sequence according to SEQ ID NO: 3 or a nucleic acid sequence coding for a polypeptide with an amino acid sequence according to SEQ ID NO: 4 are particularly preferred.
• the nucleic acid molecule according to the invention can further comprise a promoter suitable for expression, the coding nucleic acid sequence being under the control of the promoter.
• a "promoter suitable for expression”, as used here, denotes a DNA fragment through which the initiation point and the initiation frequency of the transcription (RNA synthesis) of a nucleic acid sequence which is under the control of the promoter element and which is a chemokine, a neuropeptide precursor or at least one neuropeptide is coded and fixed in the host organism.
• the choice of the promoter depends on the expression system used for expression. In general, constitutive promoters are preferred, but inducible promoters, such as the metallothionein promoter, are also possible.
• Potential promoters include the FMD, MOX, TPS1, PMA1 and DyAS promoters from Hansenula polymo ⁇ ha, the AOX1 and G / AP7 promoters from Pichia pastoris, the ADH1, PDC1, GAP1 and C / JP7 promoters from S. cerevisiae, the AXDH and> AWSß4 promoters from Arxula adeninovorans and the NDK1 and CPC2 promoters from Sordaria macrospora.
• the nucleic acid molecule according to the invention can furthermore comprise sequences of a vector which enable the nucleic acid molecule to replicate in a host cell and / or to integrate the nucleic acid molecule into the genome of a host cell.
• a vector which enable the nucleic acid molecule to replicate in a host cell and / or to integrate the nucleic acid molecule into the genome of a host cell.
• Numerous cloning and expression vectors are known in the prior art, cf. Recombinant Gene Expression Protocols, Meth. Mol. Biol. Vol. 62, Humana Press, New Jersey, USA.
• the vector used must contain an origin of replication and possibly other regulatory regions.
• the vector can consist of bacteriophages such as ⁇ derivatives, adenoviruses, vaccinia viruses, baculoviruses, SV40 virus, retroviruses; Plasmids such as Ti plasmids from Agrobacterium tumefaciens, YAC vectors and BAC vectors can be selected.
• bacteriophages such as ⁇ derivatives, adenoviruses, vaccinia viruses, baculoviruses, SV40 virus, retroviruses
• Plasmids such as Ti plasmids from Agrobacterium tumefaciens, YAC vectors and BAC vectors can be selected.
• the present invention furthermore relates to a host cell which contains at least one nucleic acid molecule according to the invention, the host cell being a prokaryotic or eukaryotic cell which is suitable for expressing the nucleic acid molecule and, if appropriate, for processing the resulting polypeptide molecule.
• a prokaryotic or eukaryotic cell which is suitable for expressing the nucleic acid molecule and, if appropriate, for processing the resulting polypeptide molecule.
• Host cells can, for example, from prokaryotic cells such as E. coli or B. subtilis, or from eukaryotic cells such as fungal cells, plant cells, insect cells and mammalian cells, e.g. B. CHO cells, COS cells or HeLa cells, and derivatives thereof.
• the eukaryotic host cell is preferably the yeast Saccharomyces cerevisiae, the methylotrophic yeast Hansenula polymorpha, the dimorphic yeast Arxula adeninivorans or the filamentous fungus Sordaria macrospora.
• the invention further provides a polypeptide molecule comprising an amino acid sequence selected from the following sequences:
• the term "at least 85% identical” refers to amino acid sequence level agreement according to known methods, e.g. of computer-aided sequence comparisons (Altschul et al., 1990) can be determined.
• identity here denotes the degree of relationship between two or more polypeptide molecules, which is determined by the agreement between the sequences, whereby agreement means both identical agreement and conservative amino acid exchange.
• the percentage of the “identity” results from the percentage of matching areas in two or more sequences, taking into account gaps or other sequence peculiarities.
• the term "conservative amino acid exchange” refers to an exchange of an amino acid residue for another amino acid residue, the exchange being intended to exert as little influence as possible on the (spatial) structure of the polypeptide molecule.
• a basic distinction is made between four physico-chemical groups into which the naturally occurring amino acids are divided.
• the group of basic amino acids includes arginine, lysine and histidine.
• the group of acidic amino acids includes glutamic acid and aspartic acid.
• the uncharged / polar amino acids include glutamine, asparagine, serine, threonine and tyrosine.
• the non-polar amino acids include phenylalanine, tryptophan, cysteine, glycine, alanine, valine, methionine, isoleucine, leucine and proline.
• a conservative amino acid exchange means in In this context, the replacement of a given amino acid by an amino acid that belongs to the same physico-chemical group.
• the identity of related polypeptide molecules can be determined using known methods. Preferred methods for determining identity initially produce the greatest agreement between the sequences examined.
• Computer programs for determining identity between two amino acid sequences include the GCG program package, including GAP (Devereux et al., 1984; Genetics Computer Group University of Wisconsin, Madison, (WI)); BLASTP, BLASTN and FASTA (Altschul et al., 1990), however, are not limited to these.
• the BLAST X program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbuch, Altschul, S. et al., NCB NLM NIH Bethesda MD 20894; Altschul et al., 1990).
• NCBI National Center for Biotechnology Information
• the well-known Smith Waterman algorithm can also be used to determine sequence identity.
• Preferred parameters for sequence comparison include the following:
• the GAP program is also suitable for use with the above parameters.
• the above parameters are the default parameters for amino acid sequence comparisons, with gaps at the ends not reducing the identity value. In the case of very short sequences compared to the reference sequence, it may also be necessary to increase the expected value to up to 100,000 and, if necessary, to reduce the word size to up to 2.
• gap opening penalties can be used. The selection will depend on the comparison to be made and whether the comparison between sequence pairs, where GAP or Best Fit are preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred.
• a 85% match determined with the above-mentioned algorithm is referred to as 85% identity in the context of this application. The same applies to higher degrees of identity.
• the polypeptide molecule according to the invention comprises a sequence which is at least 90%, preferably at least 95% identical to the amino acid sequence given above under (i), (ii), (iii) or (iv) , Polypeptide molecules which comprise an amino acid sequence according to SEQ ID NO: 12 or SEQ ID NO: 13 are particularly preferred.
• the polypeptide molecule according to the invention comprises the amino acid sequences according to SEQ ID NO: 5, SEQ ID NO: 6 and / or SEQ ID NO: 7.
• the invention provides a fusion protein which comprises at least one polypeptide according to the invention.
• Fragments of the polypeptide molecules according to the invention which comprise at least one neuropeptide, are also encompassed by the invention. Fragments are preferred which comprise at least one of the amino acid sequences according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and / or SEQ ID NO: 10. Fragments with the amino acid sequence according to SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 are particularly preferred.
• the fragments of the polypeptide molecules according to the invention can also be modified, for example by glycosylation, phosphorylation, acetylation or amidation.
• the polypeptide molecule, fusion protein or fragment according to the invention is preferably a polypeptide molecule which, in a Ca imaging experiment under the conditions described in Koller et al (2001), shows a 1.5 to 10-fold increase in the intracellular Calcium concentration in primary astrocytes and / or neurons from the central nervous system of rats or humans.
• the invention further relates to a method for producing a polypeptide molecule according to the invention and / or a fragment thereof, which comprises cultivating a host cell according to the invention under conditions suitable for expression and eventual processing and optionally purifying the expressed polypeptide molecule or fragment.
• polypeptide molecules and fragments thereof according to the invention can also be obtained by chemical and enzymatic synthesis, such as, for example, Merrifield synthesis, and / or fragment condensation. Combinations of chemical, enzymatic and recombinant manufacturing processes are also possible.
• the invention further relates to an antibody which is specific for a polypeptide molecule according to the invention and / or a fragment thereof.
• specific antibodies are obtained by immunizing laboratory animals, such as. B. mice or rabbits, with the polypeptide molecules according to the invention and / or fragments, which are preferably coupled to suitable high-molecular carrier molecules (often proteins), available. Immunization can be facilitated by adding suitable adjuvants known in the art. Monoclonal antibodies are usually obtainable by fusing spleen cells, which have been removed from an immunized mouse, with tumor cells and selecting the hybridomas formed in the process. Those hybridomas that efficiently secrete specific antibodies can be determined by screening the supernatant.
• antibodies can be produced recombinantly; in the production of recombinant antibodies, the mRNA is isolated from hybridoma cells or B lymphocytes, which acts as the basis for the synthesis of the corresponding cDNA and is amplified by PCR. After ligation into a suitable vector and introduction into a suitable host cell culture, the antibody can be obtained from the cell culture supernatants or the cell lysates. Recombinant antibodies allow "humanization" of the antibody and are therefore less immunogenic. The relevant methods are known in the prior art.
• the invention provides an in vitro method which involves contacting the sample with a reagent specific for the polypeptide molecule and / or a fragment thereof and detecting of binding.
• the invention further provides a test kit for detecting a polypeptide and / or a fragment thereof, which comprises at least one reagent specific for the polypeptide and / or fragment according to the invention.
• specific reagents are antibodies, antibody fragments, e.g. B. Fab or F (ab) 2 fragments or antibody derivatives, with antibodies being particularly preferred.
• the antibodies, antibody fragments, e.g. B. Fab or F (ab) 2 fragments or antibody derivatives can be of monoclonal or polyclonal origin.
• the invention further provides an in vitro method for detecting a nucleic acid encoding a polypeptide molecule according to the invention in a biological sample, which:
• nucleic acid molecule according to the invention and / or fragment thereof, the nucleic acid molecule and / or the fragment bearing a detectable label
• the invention further relates to pharmaceutical compositions which contain at least one polypeptide molecule according to the invention and / or a fragment thereof and / or a pharmaceutically acceptable salt of such a polypeptide molecule or fragment.
• These pharmaceutical compositions can contain a pharmacologically acceptable carrier and / or diluent. Suitable carriers and / or diluents are known in the prior art.
• Pharmaceutical compositions which are suitable for intravenous, subcutaneous or intramuscular administration are preferred.
• the composition also contains at least one antibody in addition to one or more polypeptide molecules according to the invention or fragments thereof.
• these pharmaceutical compositions can be used for the therapy of demyelinating or neurodegenerative diseases or of developmental disorders of the nervous system.
• Another application of the pharmaceutical compositions according to the invention is for prevention or Treatment of HIV infection and especially HIV encephalopathy in humans.
• the invention also encompasses the use of the pharmaceutical compositions according to the invention for the therapy of diseases of the haematopoietic system, the immune system and the cardiovascular system.
• compositions which contain at least one reagent specific for a polypeptide molecule according to the invention and / or a fragment thereof are also encompassed by the invention.
• the reagent is preferably an antibody.
• Such pharmaceutical compositions can be used for the diagnosis or therapy of demyelinating or neurodegenerative diseases or of developmental disorders of the nervous system. Another application relates to the diagnosis or treatment of HIV infection and in particular HIV encephalopathy in humans.
• FIG. 1 shows the strategy based on RT-PCR for cloning SDF-1 ⁇ and SDF-1 ⁇ cDNA from rats.
• 5 'and 3' UTR areas are represented by lines, coding areas by boxes. Small arrows show the position and orientation of the primers.
• Identical or homologous sequences are represented by identical graphic elements.
• the last four codons of the coding region of SDF-1 ⁇ are shown as a black box.
• the dashed lines mark an insert of 2572 nucleotides in SDF-1 ⁇ .
• the 30 carboxy-terminal codons of SDF-1 ⁇ are shown as hatched boxes.
• FIG. 2 shows the result of a Northern blot test for the detection of SDF-1 ⁇ and SDF-1 ⁇ transcripts in the sciatic nerve of adult rats.
• the filters were (A) shared with a radioactively labeled, 626 nucleotide long PCR fragment of the NT-1-15 cDNA, which corresponds to nucleotides 743-1368 of SDF-1 ⁇ and part of the SDF-1 ⁇ and SDF-1 ⁇ 3 'UTR sequence, (B) with a 190 nucleotide long PCR fragment of the coding region common to all SDF-1 isoforms, which corresponds to nucleotides 49-239 in SEQ ID NO: 16, (C) with a The 1702 nucleotide fragment, which corresponds to nucleotides 625-2327 of the SDF-1 ⁇ cDNA and hybridizes from the SDF-1 ⁇ -specific insert by digestion of the PCR product obtained with the primers GAS2 and MMSE2 with PvuW.
• FIG. 3 shows the nucleic acid sequences and the amino acid sequences derived therefrom of the SDF-1 ⁇ and SDF-1 ⁇ cDNA from rats.
• the SDF-1 ⁇ -specific insert is highlighted by a frame.
• the nucleic acid sequence of the common signal peptide is underlined.
• the numbering of the nucleotides (left) and the amino acids (right) corresponds to the sequence of SDF-1 ⁇ . (93) indicates the last amino acid of SDF-1 ß.
• FIG. 4 shows a comparison of the amino acid sequences of the SDF-1 proteins from mouse and rat.
• the dots stand for identical amino acids.
• the 19 amino acid signal peptide is framed.
• FIG. 5 shows the result of various Northern blot experiments for the detection of SDF-1 ⁇ and SDF-1 ⁇ transcripts in different tissues (A) and developmental stages (B).
• A Northem blot filter with total RNA from sciatic nerve (SN), brain (Br), lung (Lu), heart (HE), muscle (Mu), testes (Te), liver (Li), kidney ( Ki), spleen (Sp) and thymus (Th) of rats were hybridized with a radioactively labeled cDNA probe from the SDF-1 ⁇ and SDF-1 ⁇ common 3'-UTR region.
• B Detection of SDF-1 ⁇ and SDF-1 ⁇ mRNA in the brain of rats during development.
• C Detection of SDF- ⁇ - and SDF-1 ⁇ -mRNA in the sciatic nerve of rats in the course of development.
• Northern blot filters with total RNA obtained from nerves of 1, 4, 7, 14 and 21 day old rats (P1-21) and from adult rats (Ad) were analyzed with an SDF-1 ⁇ / ⁇ Probe hybridized as in (A).
• the arrowhead in the upper part shows the position of the 28S ribosomal RNA; the lower parts show Northern blot filters stained with methylene blue.
• FIG. 6 shows the result of in s / fu hybridization experiments for the cellular localization of SDF-1 ⁇ -mRNA in the brain of adult rats.
• the sections were made with a digoxigenin-UTP-labeled, 596 nucleotide long antisense transcript, which consists of a subclone of the common 5'-UTR and coding sequence (A, D, E) and of all SDF-1 mRNA isoforms a subclone of the SDF-1 ⁇ -specific insert (B, E, H).
• Hybridization with sense transcripts (C, F, I) served as a negative control.
• A, B, C Corpus callosum with "pearl necklace” -like arrangement of labeled oligodendrocytes and strongly labeled neurons in the bilateral layers of the indusium griseum dorsale of the corpus callosum.
• D, E, F Strong hybridization signals are observed in Purkinje and granular cells of the cerebellum and weak signals in the outer layer.
• G, H, I Very strong hybridization signals are detected in pyramidal and granular neurons of the hippocampus. Line: 10 ⁇ m.
• FIG. 7 shows the result of in s / fw hybridization experiments for the cellular localization of SDF-1 ⁇ -mRNA in the neocortex of rats using the same probes as in FIG. 6.
• A frontolateral
• B mediolateral
• the neurons in all neocortex layers (I-VI) are strongly labeled with both the antisense probe (A) common to all SDF-1 mRNA isoforms and the SDF-1 ⁇ -specific probe (B).
• FIG. 8 shows the result of in s / fu hybridization experiments for the cellular localization of SDF-1 ⁇ -mRNA in the sciatic nerve of adult rats.
• the sections were hybridized with digoxigenin-UTP-labeled RNA probes in sense and antisense orientation, which (a) from the SDF-1 ⁇ and SDF-1 ⁇ common 3'-UTR region (AC, E, F), (b) from the 5'-UTR and coding regions (G) common to all SDF-1 isoforms and from the SDF-1 ⁇ -specific insert (H, I).
• A, B, C The longitudinal section shows several spindle-shaped Schwann cells near the axons.
• (D) Cross-section immunostained with an antibody against the S100 protein (a marker for Schwann cells).
• E The hybridization signals in the case of a cross section adjacent to the cross section in (D) show semicircular labeled Schwann cells surrounding the axons, which occur at the same locations as the S100 immunopositive cells (arrowheads in D, E).
• G, H Neighboring cross sections marked with one of the two antisense transcripts, which show numerous semicircular Schwann cells (see arrowheads) and the wall of a blood vessel in the upper right corner.
• C, F, I Hybridization with sense-oriented transcripts served as a negative control. Lines in A, C, G-l: 100 ⁇ m, in D-F: 10 ⁇ m.
• FIG. 9 shows the coding region of the nucleic acid sequence of rat SDF-1 ⁇ and the amino acid sequence derived therefrom.
• FIG. 10 shows the coding region of the nucleic acid sequence of human SDF-1 ⁇ and the amino acid sequence derived therefrom.
• FIG. 11 shows a comparison of the coding regions of the nucleic acid sequence of human and rat SDF-1 ⁇ . "hum”: human sequence; "rat”: rat.
• FIG. 12 shows a comparison of the amino acid sequences of human and rat SDF-1 ⁇ derived from the nucleic acid sequences in FIG. 11.
• "hum” human sequence
• "rat” rat.
• FIG. 13 schematically shows the hSDF-1 ⁇ and hSDF-1 ⁇ -H6 constructs built into the plasmid PCRII-TOP ⁇ (Invitrogen, Groningen, NL) and the constructs M-mhSDF-1 ⁇ -H6, hSDF-1 ⁇ -H6 and MF ⁇ - mhSDF-1 ⁇ -H6 in plasmid pFPMT121.
• FIG. 14 shows the restriction map of the 439 bps long DNA fragment with the coding region of the hSDF-1 ⁇ gene.
• FIG. 15 shows the restriction map of the 457 bps long DNA fragment with the coding region of the hSDF-1 ⁇ gene and the His tag.
• FIG. 16 shows the restriction map of the expression plasmid pFPMT-M-mhSDF-1 ⁇ -H6.
• Figure 17 shows the restriction map of the expression plasmid pFPMT-hSDF-1 ⁇ -H6.
• FIG. 18 schematically shows the strategy for generating the expression plasmid pFPMT-MF ⁇ -mhSDF-1 ⁇ -H6.
• the arrows marked "P” represent PCR primers.
• FIG. 19 shows the restriction map of the expression plasmid pFPMT-MF ⁇ -mhSDF-1 ⁇ -H6.
• FIG. 20 shows the result of a Western blot test for the detection of the expression products in cell extracts from H. polymorpha (A) with the SDF-1-specific antibody SDF-1 (C19) (Santa Cruz Biotechnology, USA) and (B) with a His-Tag-specific antibody (RGS-His Antibody, Mouse IgG1, Qiagen, Hilden, FRG).
• the traces in (A) contain: (1) Sea Blue Prestained Standard, (2) M-mhSDF-1 ⁇ -H6, (3) M-mhSDF-1 ⁇ -H6 treated with PNGaseF, (4) hSDF-1 ⁇ -H6, (5) hSDF-1 ⁇ -H6 treated with PNGaseF, (6) MF ⁇ -mhSDF-1 ⁇ -H6, (7) MF ⁇ -mhSDF-1 ⁇ -H6 treated with PNGaseF and (8) cell extract without SDF-1 ⁇ .
• the traces in (B) contain: (1) cell extract without SDF-1 ⁇ , (2) Sea Blue Prestained Standard, (3) M-mhSDF-1 ⁇ -H6, (4) M-mhSDF-1 ⁇ -H6 treated with PNGaseF, (5) hSDF-1 ⁇ -H6, (6) hSDF-1 ⁇ -H6 treated with PNGaseF, (7) MF ⁇ -mhSDF-1 ⁇ -H6 and (8) MF ⁇ -mhSDF-1 ⁇ -H6 treated with PNGaseF.
• FIG. 21 shows a comparison of the effect of SDF-1 ⁇ and SDF-1 ⁇ on the Ca concentration in astrocytes.
• A 50 nM SDF-1 ⁇ ;
• B 35 ⁇ g yeast cell extract with recombinant SDF-1 ⁇ (M-mhSDF-1 ⁇ -H6);
• C 22.4 ⁇ g control extract;
• D quantitative evaluation of the intracellular calcium increase in SDF-1 ⁇ and the control extract based on the calcium increase caused by SDF-1 ⁇ .
• FIG. 22 shows the result of a Ca imaging experiment in astrocytes for SDF-1 ⁇ without (A) and with (B) preincubation with antibody against CXCR4.
• FIG. 23 shows the result of a Ca imaging experiment in astrocytes for SDF-1 ⁇ without (A) and with (B) preincubation with antibody against CXCR4.
• FIG. 24 shows the result of a Ca imaging experiment in cortex neurons for SDF-1 ⁇ without (A) and with (B) preincubation with antibody against CXCR4.
• FIG. 25 shows the result of a Ca imaging experiment in astrocytes for the C-terminal basic peptide of SDF-1 ⁇ (30 amino acids) without (A) and with (B) preincubation with antibody against CXCR4.
• FIG. 26 shows the result of a Ca imaging experiment in astrocytes for: (A) peptide 2 (KKEKIG; SEQ ID NO: 6) and (B) peptide 3 (KKKRQ; SEQ ID NO: 8).
• RNA from rat tissue was isolated by the phenol guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). The frozen tissue samples were homogenized twice at 2500 rpm with a Polytron (Brinkmann, Westbury, NY) for 45 seconds. Poly (A) + RNA was isolated by oligo (dT) cellulose chromatography (Sambrook et al., 1998).
• cDNA was generated with the TimeSaver cDNA synthesis kit (Pharmacia-LKB, Piscataway, NJ). The cDNA was linked using the Gigapack II packaging extract (Strategege) with EcoRI pre-cut ⁇ -ZAP II phage particles by ligation. The titration of the cDNA library obtained revealed a complexity of approximately 0.5 x 10 6 .
• the cDNA library was screened by standard methods (Sambrook et al., 1989) with a radioactively labeled cDNA fragment from the untranslated 3 'region of rSDF-1ß (nucleotides 743-1368).
• oligonucleotides were synthesized using a GeneAssembler Plus synthesizer (Pharmacia, Piscataway, NJ).
• MMSE2 5'-ACGCCATGGACGCCAAGGTCG-3 '(SEQ ID NO: 19) corresponds to nucleotides 49-69 of the rSDF-1 ⁇ cDNA.
• GAS2 5'-ACTGTAAGGAAGACCCTCTCTCACC-3 '(SEQ ID NO: 20) corresponds to nucleotides 2327-2303 of SDF-1 ⁇ .
• GAS3 5'-GTTGAGACTATGCATCGACTCCAAC-3 '(SEQ ID NO: 21) corresponds to nucleotides 2576-2552 of SDF-1 ⁇ .
• the reverse transcription was carried out with 1-5 ⁇ g total RNA and reverse transcriptase superscript (Gibco, Gaithersburg) according to the manufacturer's instructions.
• the first strand of cDNA was digested with RNase H (Boehringer Mannheim) and then 1/10 of the volume was used as a template for the PCR amplification with Amplitaq polymerase (Perkin Elmer) or Pfu polymerase (Stratagene, La Jolla) (for SDF -1 ⁇ ) is used.
• NT-1-15 When identifying genes that are differentially expressed after a nerve lesion, the cDNA clone NT-1-15 with 2174 nucleotides was isolated from a cDNA library that was generated from rat sciatic nerve. Analysis of the sequence of NT-1-15 clones showed that this clone had 86% homology to the untranslated 3 'region (UTR) of the SDF-1 ⁇ cDNA from mouse (cf. Tashiro et al., 1993) having. In Northern blot experiments under stringent washing conditions, NT-1-15 hybridized with two transcripts from the sciatic nerve of adult rats (FIG. 2). While the smaller transcript with approximately 3 Kb corresponded to the size of the SDF-1 ß-mRNA, the longer transcript with 5.5 Kb was unknown. This transcript was called SDF-1 ⁇ .
• cDNA probes from the SDF-1 ⁇ and - ⁇ common 3'-UTR area (FIG. 2A) or from the 5'-area of the coding region common to all SDF isoforms (FIG. 2B) hybridized with both 3 Kb long (SDF-1 ⁇ ) as well as with the 5.5 Kb long (SDF-1 ⁇ ) transcript, while a cDNA probe, which was specific for the 2.5 Kb long insert, only with the 5.5 Kb SDF-1 ⁇ transcript hybridized (Fig. 2C). No SDF-1 ⁇ -mRNA with a length of 1.5 Kb could be detected in the sciatic nerve of rats.
• Both strands of rat SDF-1 ⁇ cDNA were sequenced.
• SDF-1 ⁇ the new insert with a length of 2572 nucleotides and the flanking regions with the known SDF-1 ⁇ sequences were also sequenced twice.
• the deduced amino acid sequence for SDF-1 ⁇ gives a peptide of 93 amino acids with a theoretical molecular weight of 10.5 Kd.
• the first 19 amino acids represent a signal peptide for secreted proteins.
• the deduced amino acid sequence for SDF-1 ß contains the first 89 amino acid residues of SDF-1 ß and 30 additional amino acids in the carboxy- terminal area, which show no homology to SDF-1 ß (see. Fig. 3).
• the theoretical molecular weight of the 119 amino acid long SDF-1 ⁇ peptide is 13.5 Kd.
• the amino acid sequence of the SDF-1 ⁇ from rat shows a strong homology (96.8%) to the corresponding mouse protein (98.9% taking conservative amino acid exchanges into account).
• a comparison of the amino acid sequences of the new SDF-1 isoforms SDF-1 - ⁇ and SDF-1 ⁇ with the known SDF-1 sequences is shown in FIG. 4.
• RNA samples 10 ⁇ g total RNA were fractionated in 1.2% agarose gels containing 15% formaldehyde and then transferred to Nytran NY 13 N membranes (Schleicher and Schuell, Keene, NH) by standard methods.
• the filters were irradiated with UV light and stained with methylene blue (Sambrook et at, 1989), prehybridized in a 0.5 M sodium phosphate solution (pH 7.0) with 7% SDS and with 1-5 x 10 6 cpm / ml of a 32 P-labeled cDNA probe hybridized in the same solution.
• cDNA fragments corresponding to (i) the common 3'-UTR region of SDF-1 ⁇ / ⁇ (nucleotides 743-1368 in SDF-1ß), (ii) the coding region common to all SDF-1 isoforms (nucleotides 49 -239) and (iii) a 1702 nucleotide long section of the SDF-1 ⁇ -specific insert (nucleotides 625-2327 in the SDF-1 ⁇ cDNA) were radioactively labeled by unidirectional PCR (Sezl et al., 1991).
• the filters were washed for at least 15 minutes at 60 ° C in 2 x SSC / 1% SDS and for 15 minutes at 60 ° C in 0.1 x SSC / 1% SDS.
• the filters were either exposed together with an X-ray film (X-Omat AR, Kodak) or directly quantified with a BAS-1050 bioimager (Fuji).
• the Northern blot hybridization experiments shown in FIG. 5A were carried out with total RNA from different tissues of adult rats and a 602 nucleotide long fragment from the common 3'-UTR region of SDF-1 ⁇ / ⁇ , which was labeled with 32 P-dCTP had been radiolabelled.
• the distribution of the SDF-1 ß- and SDF-1 ⁇ mRNA across different tissues from adult rats showed a complementary pattern. While the ß-isoform was found primarily in the liver, kidney, spleen and thymus, SDF-1 ⁇ occurred predominantly in the heart and lung tissue and in the mature tissue of the nervous system (FIG. 5).
• SDF-1 ß transcript occurs primarily in embryonic and neonatal brain tissue and in the sciatic nerve indicates a differential regulation of SDF-1 expression during the development of the nervous system. Neither SDF-1 ⁇ nor SDF-1 ⁇ mRNA could be detected in muscle and testicular tissue (FIG. 5A).
• RNA from brain tissue at various stages of development in rats was analyzed with a 602 nucleotide fragment of the common 3'- UTR range of SDF-1 ß / ⁇ sampled.
• SDF-1 ß-mRNA was predominantly detected in the brain tissue of E17 embryos, but the amount of transcript decreased with increasing age and the transcript could no longer be detected in the brain tissue of adult rats.
• the amount of SDF-1 ⁇ transcript was very low in E17 embryos, but increased steadily and peaked in adult rats (Fig. 5B).
• SDF-1 ß and SDF-1 ⁇ mRNAs seem to show a different expression pattern during development and in the nervous system of the adult rat. While the SDF-1 ß-isoform predominantly in embryonic or perinatal CNS and PNS occurs, SDF-1 ⁇ is the most important variant in the nervous system of adult rats (Fig. 5B, C). In the period between the 4th and the 7th day after birth, in which the differentiation of the glial cells and neuron maturation begins, the SDF-1 ⁇ and SDF-1 ⁇ transcripts occur in almost equal amounts.
• tissue samples were embedded in Tissue Tee II (Miles, Napperville, IL), frozen in methylbutane at -70 ° C and cut into 20 ⁇ m sections. The sections were fixed and then acetylated and 4 hours at 55 ° C according to Angerer et al. (1987) pre-hybridized.
• vtfro transcripts (i) a subclone of the common 3'-UTR region of SDF-1 ß / ⁇ (nucleotides 1758-2199 in SDF-1 ß), (ii) a subclone that contains all SDF-1 mRNA Isoform common 5'-UTR and coding regions (nucleotides 1-596 in the SDF-1 ß-cDNA) and (iii) a subclone of the SDF-1 ⁇ -specific insert (nucleotides 661-1313 in the SDF-1 ⁇ - cDNA) were generated using the DIG-RNA labeling kit from Boehringer Mannheim using digoxigenin-UTP.
• antisense transcripts from (a) the common 3'-UTR area of SDF-1 ß / ⁇ , (b) the common 5'-UTR and coding area of all SDF 1 isoforms and (c) the SDF-1 ⁇ -specific insert is labeled with digoxigenin-UTP.
• the sense transcripts served as negative controls.
• both the SDF-1 isoform common and the SDF-1 ⁇ -specific probe showed strong and extensive hybridization signals in areas with gray brain matter as well as in "pearl-chain" -like sequences of oligodendrocytes in myelinated nerve fibers such as such as the corpus callosum observed (Fig. 6A, B). Further hybridization signals occurred especially in connection with Purkinje and granular neurons in the cerebellum (Fig. 6D, E), in pyramidal and granular neurons in the hippocampus (Fig. 6G, H) as well as in neurons of all major layers of the neocortex (Fig. 7A, B). No hybridization signals were obtained with the corresponding sense transcripts (see Fig. 6C, F, I for the sense probes for SDF-1 ⁇ ).
• SDF-1 ⁇ -specific antisense transcript The signals obtained with the SDF-1 ⁇ -specific antisense transcript were almost identical to the hybridization pattern obtained with the common SDF-1 antisense probe, which indicates that the SDF-1 ⁇ -isoform in neurons and Brain oligodendrocytes are expressed by adult rats. SDF-1 ß transcripts, insofar as they occur in the brain of adult rats, appear to be present in the same areas and cell populations as SDF-1 ⁇ .
• an antisense probe from the common 3'-UTR region of SDF-1 ⁇ / ⁇ generates spindle-shaped hybridization signals which resemble the typical shape of Schwann cells near the axon fibers (Fig. 8A, B).
• the identical localization of the S-100 immunoreactivity and the hybridization signals in cross sections of the sciatic nerve confirmed the expression of SDF-1 ⁇ / ⁇ -mRNA in Schwann cells (FIG. 8D, E).
• a marking pattern FIG.
• M-mhSDF-1 ⁇ -H6 (methionine / matures human SDF 1 ⁇ / His day).
• the sequence of the mature human SDF-1 ⁇ (amino acids 20-119 in SEQ ID NO: 12) is located behind an N-terminal methionine residue. Since there is no leader sequence, cytosolic localization was expected. There are six histidine residues (His tag) at the C terminus.
• hSDF-1 ⁇ -H6 (immature human SDF-1 ⁇ / His tag). This construct comprises amino acids 1-119 of SDF-1 ⁇ (SEQ ID NO: 12), followed by a C-terminal His tag. It therefore contains the natural leader sequence recognized in human cells. Since leader sequences are sometimes also recognized in heterologous host cells, this construct was used to investigate whether H. polymorpha recognizes the authentic SDF-1 ⁇ leader peptide.
• the starting construct was the plasmid SDF-1 ⁇ -PCRII-TOPO, which contained the 439 bps long SDF-1 ⁇ insert (FIG. 14).
• a first step six codons for a C-terminal His tag were added to the hSDF-1 ⁇ sequence by PCR mutagenesis (hSDF-1 ⁇ -H6, FIG. 15).
• the integrative plasmid pFPMT121 (Gellissen, 2000) was used as the basis vector for the later expression of SDF-1 ⁇ constructs in H. polymorpha, in which the foreign gene to be expressed is under the control of the FMD promoter.
• the following expression vectors were constructed on the basis of this plasmid:
• pFPMT-M-mhSDF-1 ⁇ -H6 A DNA fragment of the M-mhSDF-1 ⁇ -H6 ORF was generated by means of PCR, in which the coding sequence of SDF-1 ⁇ is flanked by an EcoRI (before the start codon) and a ßamHI restriction site. HSDF-1 ⁇ -H6 in PCRII-TOPO (Invitrogen, Groningen, NL) served as template DNA. The PCR product was digested with EcoRI / ßamHI and cloned between the corresponding sites of the pFPMT121 plasmid. The map of the resulting plasmid pFPMT-M-mhSDF-1 ⁇ -H6 is shown in Figure 16.
• the first PCR product (PCR IA) comprised the codons of the prepro sequence of the mating factor ⁇ , flanked by an EcoRI interface (before the start codon) and at the other end of bases with homology to the first codons of the mature hSDF-1 ⁇ -Sequence.
• the second PCR product (PCR IB) contained the sequence of the mature hSDF-1 ⁇ , flanked on the front part by bases with homology to the back part of the prepro sequence of the mating factor ⁇ , flanked on the back part by a SamHI interface (behind the stop codon).
• PCR II another PCR reaction
• the Hin primer from PCR IA (the one with the EcoRI interface) and the back primer from PCR IB (the one with the ßamHI interface) were used as primers.
• the resulting PCR product comprised the prepro sequence of the mating factor ⁇ fused to the sequence of the mhSDF-1 ⁇ -H6, flanked by EcoRI (before the start codon) and ßamHI interfaces (behind the stop codon).
• EcoRI / ⁇ amHI the fragment was cloned between the corresponding sites of the pFPMTl 21 plasmid.
• the map of the resulting plasmid pFPMT-MF ⁇ -mhSDF-1 ⁇ -H6 is shown in Fig. 19.
• H. polymorpha cells were prepared for electroporation, 5 ml of YPD medium were used with a single colony of H. polymorpha RB11 (odc, orotidine-5-phosphate-decarboxylase-deficient (uracil auxotrophic) H. po / ymorp ⁇ a strain (Weydemann et al., 1995)) inoculated and shaken at 37 ° C for 16 hours. 100 ml of YPD medium were then inoculated with 3 ml of this preculture in a 2 l Erlen-Meyer flask and incubated to an OD 6 oo of 0.8-1 at 37 ° C. (shaking frequency 140 rpm).
• the cells were harvested by centrifuging the culture in 50 ml Falcon tubes (4000 rpm; 6 ' ) in a Beckmann centrifuge. After the supernatants had been removed, the cells were resuspended in 20 ml of 50 mM potassium phosphate buffer (pH 7.5; preheated to 37 ° C.), mixed with 0.5 ml of 1 M DTT and incubated for 15 minutes at 37 ° C.
• 50 mM potassium phosphate buffer pH 7.5; preheated to 37 ° C.
• the cells were then centrifuged again (3000 rpm; 10 ', Beckmann centrifuge) and washed with 100 ml, then with 50 ml of STM buffer (270 mM sucrose; 10 mM Tris-HCl pH 7.5; 1 mM MgCl 2 ). After centrifuging again, the cells were resuspended in 0.5 ml of STM buffer and used as 60 ⁇ l aliquots either directly for transformations or frozen at -70 ° C. for later use.
• STM buffer 270 mM sucrose
• 10 mM Tris-HCl pH 7.5 1 mM MgCl 2
• Competent cells from H. polymorpha RB11 were transformed with the three expression plasmids generated (see above) as follows: 60 ⁇ l of competent H. polymorpha cells were mixed with 1-2 ⁇ g of the circular plasmid DNA to be introduced and with 2 mm in electroporation cuvettes Gap width transferred. The electroporation was carried out at 2 kV, 25 ⁇ F and 200 ohms. The cells were then transferred to test tubes with 1 ml of YPD medium and shaken for 1 hour at 37 ° C. (angle 45 °; 160 rpm). To of this recovery, 330 ⁇ l of the cells were plated onto YNB agar plates (1% glucose; without uracil). The plates were incubated at 37 ° C until macroscopic uracil-prototrophic colonies became visible (approx. 1 week).
• uracil-prototrophic colonies were converted into stable strains by passage four times and stabilization twice.
• 2 ml of YNB medium (1% glucose) with individual uracil-prototrophic colonies were inoculated from the transformant plates and incubated for 2 days at 37 ° C. (angle 45 °; shaking frequency 160 rpm).
• 150 ⁇ l of the cultures from the last passage were transferred to 2 ml of YPD medium and incubated for 2 days at 37 ° C. (see above). Aliquots of these cultures were then plated onto YNB agar plates (1% glucose; without uracil). A single colony was isolated for each smear and defined as a strain.
• the cell pellets from the induction cultures were each in 500 ⁇ l extraction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% v / v Triton X -100 or PBS buffer) and resuspended with 12.5 ⁇ l PMSF. The samples were then transferred to 1.5 ml Eppendorf tubes. After 500 ⁇ l of glass beads had been added, the cells were disrupted in a Vibrax at 2500 rpm. The supernatants were transferred to fresh Eppendorf tubes and centrifuged for 10 minutes at 10,000 rpm (Eppendorf centrifuge with cooling function). The supernatants from this centrifugation represented the so-called intracellular soluble fraction. These were used for direct protein gel electrophoresis with 1 %. 4 x SAB added and denatured for 5 'at 95 ° C, or frozen for further use without SAB addition at -20 ° C.
• 500 ⁇ l extraction buffer 50 mM Tris pH 7.5, 150 mM
• the denatured samples were separated by protein gel electrophoresis on 4-20% Tricine-SDS gels (Novex) according to the manufacturer's instructions.
• the protein bands were then transferred to nitrocellulose membranes in a semi-dry blot apparatus (Trans-Blot SD; Biorad) according to the manufacturer's instructions.
• a primary His-specific monoclonal antibody from mouse (RGS-His antibody, Qiagen, Hilden, FRG) or an SDF-1-specific polyclonal serum from goat ( SDF-1 (C19); # sc6193; Santa Cruz Biotechnology, USA).
• the Western blots were carried out with the Western Breeze kits mouse or goat (Novex) according to the manufacturer's instructions.
• strains could be identified for each of the three constructs that produced significant amounts of the respective hSDF-1 ⁇ -H6 derivative.
• the most productive strain was selected for further product analyzes. For pFPMT-M-mhSDF-1 ⁇ -H6 this was strain g7-5 / 36; strains g8-28 / 7 and g9c-20/6 were selected for pFPMT-hSDF-1 ⁇ -H6 and pFPMT-MF ⁇ -mhSDF-1 ⁇ -H6, respectively.
• M-mhSDF-1 ⁇ -H6 calculated 12.692 kDa, observed about 16 kDa (Fig. 20, lanes 2 and 3 (A), lanes 3 and 4 (B)); hSDF-1 ⁇ -H6: 14.529 kDa calculated, about 17 kDa observed (Fig. 20, lanes 4 and 5 (A), lanes 5 and 6 (B)); MF ⁇ -mhSDF-1 ⁇ -H6: 21, 468 kDa calculated, about 30 kDa observed (Fig. 20, lanes 6 and 7 (A), lanes 7 and 8 (B)).
• the amino acid sequences of M-mhSDF-1 ⁇ -H6 and hSDF-1 ⁇ -H6 contain no potential N-glycosylation sites. Accordingly, PNGaseF digestion has no influence on the apparent molecular weight of the respective main product band (Fig. 20, lanes 2/3 and 4/5 (A), lanes 3/4 and 5/6 (B)).
• MF ⁇ -mhSDF-1 ⁇ -H6 has three N-glycosylation sites in the area of the MF ⁇ prepro sequence, which are typically N-glycosylated in the ER.
• the lack of reduction in the apparent molecular weight of the 30 kDa product after PNGaseF digestion indicates that this product is the prepro form which was not introduced into the ER (Fig.
• FIG. 21 shows the result of Ca imaging experiments in which the effect of SDF-1 ⁇ and SDF-1 ⁇ on the Ca concentration in astrocytes was compared.
• FIG. 21 C shows the quantitative evaluation of the intracellular calcium increases in SDF-1 ⁇ and the control extract based on the calcium increase caused by SDF-1 ⁇ .
• FIG. 22 shows the result of a Ca imaging experiment in astrocytes for SDF-1 ⁇ (A) without and (B) with antibody against CXCR4,
• FIG. 23 shows the result of the corresponding experiments for SDF-1 ⁇ .
• SDF-1 ⁇ 50 nM; R&D Systems, Wiesbaden, Germany
• the intracellular calcium concentration in cultured astrocytes rises sharply (FIG. 22 A).
• SDF-1 ⁇ is given after 5 minutes of preincubation with the monoclonal antibody 12G5
• cultured astrocytes show an approximately 50% lower intracellular calcium release (FIG. 22 B).
• FIG. 24 shows the result of a corresponding Ca imaging experiment in cortex neurons.
• the application of both 35 ⁇ g and 124 ⁇ g (total protein) of a yeast cell extract with recombinant SDF-1 ⁇ (M-mhSDF-1 ⁇ -H6) leads to a significant increase in the intracellular calcium concentration in cultivated primary cortex neurons (FIG. 24 A).
• the monoclonal Antibody 12G5 antioxidant against CXCR4
• Example 6 Effect of the C-terminal basic peptide from SDF-1 y and the synthetically produced peptide cleavage products derived therefrom on the intracellular Ca concentration in
• FIG. 25 shows the result of a Ca imaging experiment with the C-terminal basic peptide of SDF-1 ⁇ in astrocytes.
• FIG. 26 shows that the application of 1 mg / ml of peptide 2 (KKEKIG; SEQ ID NO: 6) (Fi gur 26 A) or peptide 3 (KKKRQ; SEQ ID NO: 8) (FIG. 26 B) leads to a significant increase in the intracellular calcium concentration in cultured primary astrocytes. In contrast, peptides 4 and 5 did not cause an increase in the Ca concentration in astrocytes.
• TNF ⁇ reduces glutamate induced intracellular Ca 2+ increase in eultured cortical astrocytes. Brain Research 893: 237-243.

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