WO2004003553A1 - Procedes pour identifier de nouveaux inhibiteurs de transcription - Google Patents
Procedes pour identifier de nouveaux inhibiteurs de transcription Download PDFInfo
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- WO2004003553A1 WO2004003553A1 PCT/SE2003/001112 SE0301112W WO2004003553A1 WO 2004003553 A1 WO2004003553 A1 WO 2004003553A1 SE 0301112 W SE0301112 W SE 0301112W WO 2004003553 A1 WO2004003553 A1 WO 2004003553A1
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- hrp65
- actin
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- interaction
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
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- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- the present invention relates to a method for identifying compounds that will inhibit DNA transcription, and to compounds identified by this method. Furthermore, it relates to a compound that is useful in the treatment of hyperproliferative disorders. The compound may also be used as a tool in the field of cell and molecular biology.
- a large number of diseases are caused by an increase in cell proliferation.
- cancer is a major cause of suffering and death around the world and new therapies are continuously sought.
- a drug with antiproliferative effect would desirably be directed against a target or process which is vital for the proliferating cells.
- targets or processes may be envisaged, such as a DNA polymerase for inhibiting replication, or an RNA polymerase for inhibiting transcription.
- a central activity in cells that are undergoing rapid cell proliferation is the transcription of DNA to RNA. This forms the basis of an opportunity to treat diseases caused by an increased cell proliferation.
- the transcription-inhibiting compound Actinomycin D has been used as a cytostatic drug in cancer treatment, but carries with it non desirable side effects. Consequenly, there is a need for other anti-hyperproliferative drugs, to be used either on its own or in combination therapies together with other drugs.
- the mammalian cell membrane is refractory to most proteins and peptides.
- the most widely used methods to introduce antibodies, peptides, or other membrane-impermeable molecules into cells were microinjection and electroporation- both invasive techniques that disrupt the cell membrane.
- Microinjection involves loading or transferring a dissolved substance into a living cell using the microscopic tip of a glass capillary.
- the technique requires technical skill and can only be performed one cell at a time.
- Electroporation uses high-voltage pulses to produce transient pores through which proteins can enter. While this technique can be applied to many cells at once, it is toxic and non-specific-that is, anything can enter or exit the cell once the membrane is disrupted.
- the present invention relates to a compound and its analogues with a inhibitory effect on transcription.
- the compound or analogues thereof according to the invention are useful in the treatment of diseases caused by hyperproliferating cells. Examples of such diseases include various types of cancers, and also non-cancer like diseases such as rheumatoid arthritis or psoriasis.
- Another aspect of the invention is to use the identified inhibitors as tools used in cell and molecular biology in experiments where it is necessary to inhibit transcription, or in protein transduction methods.
- the protein actin is a nuclear component.
- the inventors sought to identify proteins which bind to actin in the cell nucleus. Affinity chromatography experiments were performed using nuclear extracts of
- Hrp65 amino acid sequence shown in SEQ ID NO 4
- PSF protein sequence shown in SEQ ID NO 4
- p54nrb protein sequence shown in SEQ ID NO 4
- PSP1 protein sequence shown in SEQ ID NO 4
- Multiple functions have been attributed to these proteins, from splicing to transcription regulation to retention of RNA in the nucleus and it is not known whether these proteins bind actin.
- actin is able to bind to the isoforms Hrp65-1 and Hrp65-2, but not Hrp65-3. Since the Hrp65 isoforms are identical from amino acid residue 1 to 499, their differential behaviour in the actin-binding assay suggested that the variable C-terminal sequences were involved in actin binding.
- a peptide termed 65-2CTS hereinafter also termed TIP (as in transcription inhibiting peptide), sequence shown in SEQ ID NO 1) containing the last 15 C- terminal amino acid residues of Hrp65-2 was chemically synthesized and conjugated to a carrier, keyhole limpet hemocyanin (KLH).
- Actin-Hrp65 binding assays were then performed in the presence of saturating amounts of either KLH-65-2CTS or KLH alone.
- KLH65-2CTS was able to compete specifically for binding of actin to Hrp65-2.
- Hrp65 as an actin binding protein, together with the further results presented herein, i. e. the finding that disruption of the actin/Hrp65 interaction causes inhibition of transcription, demonstrates that the actin/Hrp65 system would be a useful tool for identifying compounds that may be used as drugs for treating hyperproliferative diseases, such as cancer. Consequently, one aspect of the present invention is an assay which can be used for identifying compounds that are capable of inhibiting, preventing or in other ways disturbing, blocking or abolishing the interaction between an actin molecule and an Hrp65 molecule. As described herein, blocking this interaction has the effect of inhibiting transcription of DNA into RNA.
- the method according to the invention may be adapted to suit a high through-put screening format, but other variants are also envisaged.
- a general assay according to the invention may comprise the following steps;
- the order of dispensing is not crucial for the assay, it is therefore possible to start with the actin molecule, followed by addition of the Hrp65 molecule.
- the test compound may be added after the complex has formed, or it may be added together with one of the protein molecules. It is well within the capacity of the skilled person to determine if a test compound has an effect on the interaction between Hrp65 and actin.
- Such methods may include mass spectroscopy, gel filtration, CD spectroscopy, in which one determines the molecular mass of a protein.
- FRET fluorescence resonance energy transfer
- An assay to identify compounds which will inhibit the interaction may comprise the following steps; (a) conjugating Hrp65 to a solid support, such as a bead, resin, surface, or in a well in a multiwell plate, (b) removing any non-conjugated Hrp65 by washing in a suitable buffer, (c) blocking non-conjugated sites on the solid support by adding another protein (e.g. bovine serum albumin, or a blocking mixture such as milk powder), (d) adding labeled (e.g. radioactively, by e.g. 35 S, or fluorescently, by e.g.
- FITC or fluorescein-5-maleimide actin to the conjugated Hrp65, (e) washing away unbound actin, by washing with a suitable buffer, (f) optionally calculating the amount of formed actin/Hrp65 complexes by measuring the amount of bound labeled , molecule, (g) adding a test compound to the formed complexes, (h) collecting the displaced actin and measuring either the amount of displaced actin or the amount of actin remain in complex with Hrp65 (e.g.
- a compound which prevents more than half of the actin molecules to bind to Hrp65 at a concentration lower than lOO ⁇ M, or preferably lO ⁇ M, or more preferably l ⁇ M, is considered to be an inhibitor of the actin/Hrp65 interaction and may therefore be useful in treating antiproliferative diseases.
- FITC is fluorescein isothiocyanate, and is obtainable from Molecular Probes, or from Sigma. It should be noted that either Hrp65 or actin could be conjugated to the solid support. In the case of conjugating actin to the solid support, labeled Hrp65 is then added in step (d).
- sites on the solid support which has not been conjugated to the actin or Hrp65, respectively, is preferably blocked by adding another protein or blocking mixture, such as bovine serum albumin or milk powder.
- Another way to detect binding between Hrp65 and actin is by immunochemical detection. In this case, one does not need to label the proteins, but the presence of bound protein after step (h) is detected by the use of an antibody against the non-conjugated protein.
- an antibody directed against Hrp65 may be used to detect the presence of Hrp65 after washing off unbound protein.
- Actin may be obtained according to the method disclosed in Zechel, K., (1980).
- Hrp65 can be obtained according to the procedure described herein (Example 5). Alternatively, recombinant Hrp65 can be produced in bacteria with a histidine tag and affinity purified on Ni-agarose beads according to standard procedures. It should be noted that functionally equivalent forms of the two proteins in the assay may be used. Therefore, analogues, such as homologous proteins may be used. The only provision is that the proteins can interact in essentially the same way as the proteins that are used in the work described herein. Test compounds may be any compound, including peptides, polypeptides, organic or inorganic molecules.
- the inventors have used a variant of the above mentioned method, in which both Hrp65 and actin have been radioactively labeled, to be able to detect, by autoradiography, both proteins on a sodium dodecyl sulphate-polyacrylamide (SDS- PAGE) gel.
- SDS- PAGE sodium dodecyl sulphate-polyacrylamide
- a polypeptide in which the amino acids in position 7 and 10 of SEQ ID NO 5 (and 12 and 14 in SEQ ID NO 6) are basic has TIP-activity. It is believed that basic amino acids in said positions are necessary for activity, and consequently, the amino acids in positions 7 and 10, denoted by an X may be any amino acid, but the basic amino acids arginine and lysine are preferred, or most preferably, the amino acids are Q at position 7 and K at position 10.
- one aspect of the invention is a compound comprising the sequence according to SEQ ID NO 5, further comprising the variants mentioned above. Also included in the present invention is a compound comprising the sequence CPYVNQXPQXAXYXNG (SEQ ID NO 6) in which X may be any amino acid, preferably any of the basic amino acids arginine or lysine or it may also be alanine.
- the invention is a compound chosen from the group comprising; a polypeptide according to SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, or SEQ ID NO 6, analogues or oligomers thereof, fragments thereof, or oligomers thereof.
- the compound may be a compound having at least 80% similarity, preferably at least 90% similarity, more preferably at least 95%, further more preferably at least 96%, even more preferably at least 97%, or most preferably at least 98% similarity to the polypeptide shown in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, OR SEQ ID NO 6.
- a compound according to the invention will have at least 80% identity, preferably at least 90% identity, more preferably at least 95%, further more preferably at least 96%, even more preferably at least 97%, or most preferably at least 98%, identity to the polypeptide shown in SEQ ID NO 1 , SEQ ID NO 3, SEQ ID NO 5, OR SEQ ID NO 6.
- the compounds according to the invention are meant for medical use.
- the compounds according to the invention are used for manufacturing a medicament which is to be used in treating a mammal suffering from a disease caused by hyperproliferative cells.
- diseases are exemplified by melanoma, non-small-cell lung cancer, small-cell lung cancer, lung cancer, hepatocarcinoma, retioblastoma, astrocytoma, glioblastoma, leukemia, neuroblastoma, cancer in the gum, tongue, head, neck, breast, pancreas, prostate, kidney, bone, testicle, ovary, mesothelia, cervix, gastrointestinal tract, lymphoma, brain, colon, sarcoma, bladder, rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemagioomas, fibromas, vascular occlusion, retenosis, atherosclerosis, pre-neoplastic les
- the compounds according to the invention will be administered to patients suffering from the above mentioned diseases such that these patients are conferred a therapeutic benefit as a result of the treatment.
- therapeutic benefit used throughout this application refers to anything that promotes or enhances the well-being of the patient with respect to the medical treatment of his hyperproliferative disease.
- a list of nonexhaustive examples of this includes a reduction of the symptoms of the disease, extension of the patient's life by any period of time; decrease or delay in the neoplastic development of the disease; decrease in hyperproliferation, reduction in tumor growth, delay of metastasis, reduction in the proliferation rate of a cancer cell, tumor cell, or any other hyperproliferative cell; and a decrease in pain to the patients that can be attributed to the patient's condition.
- the compounds according to the invention will be used in a pharmaceutical composition together with pharmaceutically acceptable excipitents and additives.
- the compositions may be formulated according to conventional pharmaceutical practice, see, e.g., "Remington: The science and practice of pharmacy” 20 th ed. Mack Publishing, Easton PA, 2000 ISBN 0-912734-04-3 and "Encyclopedia of Pharmaceutical Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988 ISBN 0-8247-2800-9.
- weaker preparations should be used on more sensitive areas of the body such as the genitals, groin, and face. Stronger preparations will usually be needed to control lesions on the scalp, elbow, knees, palms and soles, and parts of the torso and may need to be applied under dressings. These must be used cautiously and with the dermatologist's instruction.
- the compounds according to the invention and their physiologically tolerable salts and derivatives can be administered according to the invention to animals, preferably to mammals, and in particular to humans, as pharmaceuticals for therapy or prophylaxis. They can be administered per se, in mixtures with one another or in the form of pharmaceutical preparations which permit enteral or parenteral administration and which as active constituent contain an efficacious dose of at least one compound according to the invention and/or its physiologically tolerable salts and derivatives in addition to customary pharmaceutically excipients and/or additives.
- the pharmaceuticals can be administered systemically or locally. They can be administered, for example, in the form of pills, tablets, film-coated tablets, sugar- coated tablets, granules, hard and soft gelatin capsules, powders, solutions, syrups, emulsions, suspensions or in other pharmaceutical forms. However, administration can also be carried out vaginally or rectally, for example in the form of suppositories, or parenterally or by implantation, for example in the form of injection solutions or infusion solutions, microcapsules or rods, or topically or percutaneously, for example in the form of ointments, solutions or tinctures, or in another way, for example in the form of nasal sprays or aerosol mixtures or as inhalable dry powder preparations.
- solutions are parenterally administered they can be administered, for example, intravenously, intramuscularly, subcutaneously, intraarticularly, intrasynovially or in another manner, e.g. by inhalation of wet aerosols or dry powder preparations.
- the pharmaceutical preparations according to the invention are prepared in a manner known per se, it being possible to use pharmaceutically inert inorganic and/or organic excipients in addition to the compound(s) according to the invention and/or its/their physiologically tolerable salts and derivatives.
- pharmaceutically inert inorganic and/or organic excipients in addition to the compound(s) according to the invention and/or its/their physiologically tolerable salts and derivatives.
- for the preparation of pills, tablets, sugar-coated tablets and hard gelatin capsules it is possible to use, for example, lactose, cornstarch or derivatives thereof, talc, stearic acid or its salts etc.
- Excipients for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, polyethylene glycols, natural or hardened oils etc.
- Suitable excipients for the preparation of solutions for example injection solutions, or of emulsions or syrups are, for example, water, alcohols, glycerol, diols, polyols, sucrose, invert sugar, glucose, vegetable oils etc.
- Suitable excipients for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.
- the pharmaceutical preparations normally contain approximately 0.5 to 90% by weight of the compounds according to the invention and/or their physiologically tolerable salts and derivatives.
- the pharmaceutical preparations can additionally contain auxiliaries or additives, such as, for example, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatizers, thickeners, diluents, buffer substances, solvents or solubilizers, means for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants. They can also contain two or more compounds according to the invention and/or their physiologically tolerable salts and derivatives.
- auxiliaries or additives such as, for example, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavorings or aromatizers, thickeners, diluents, buffer substances, solvents or solubilizers, means for achieving a depot effect, salts for altering the
- compositions can also contain one or more other therapeutically or prophylactically active substances in addition to at least one compound according to the invention and/or its physiologically tolerable salts and derivatives.
- the pharmaceutical preparations normally contain 0.2 to 500 mg, preferably 1 to 100 mg, of active compound according to the invention and/or its physiologically tolerable salts and derivatives per dose.
- the compounds according to the invention or pharmaceutical preparations containing them are administered as aerosols, for example as nasal aerosols or by wet aerosols or dry powder inhalation, this can be effected, for example, using a spray, an atomizer, a pump atomizer, an inhalation apparatus, a metered inhaler or a dry powder inhaler, respectively.
- Pharmaceutical forms for administration of the compounds according to the invention as an aerosol can be prepared by the process well known to the person skilled in the art.
- solutions or dispersions of the compounds according to the invention in water, water-alcohol mixtures or suitable saline solutions using customary additives for example benzyl alcohol or other suitable preservatives, absorption enhancers for increasing the bioavailability, solubilizers, dispersants and others, and, if appropriate, customary propellants, for example chlorofluorohydrocarbons and/or fluorohydrocarbons are suitable
- customary propellants for example chlorofluorohydrocarbons and/or fluorohydrocarbons
- dry powder preparations of the compounds according to the invention and/or their physiologically tolerable salts may be obtained by freeze drying or preferably spray drying aqueous solutions of the compounds according to the invention and/or their physiologically tolerable salts and of suitable water soluble additives, such as sugars or sugar derivatives and amino acids.
- the dose when using the compounds according to the invention can vary within wide limits, and as customary it is to be tailored to the individual conditions in each individual case, as is known to the physician. It depends, for example, on the nature and severity of the disease to be treated, on the compound employed or whether an acute or chronic disease state is treated or prophylaxis is conducted or on whether further active compounds are administered in addition to the compounds according to the invention.
- a daily dose of approximately 0.01 to 100 mg/kg, preferably 0.1 to 10 mg/kg, in particular 0.3 to 2 mg/kg (in each case per kg of body weight) is appropriate in an adult to achieve effective results.
- the daily dose is in general approximately 0.01 to 50 mg/kg, preferably 0.01 to 10 mg/kg of body weight.
- the daily dose can be divided into a number, for example 2, 3 or 4, of part administrations. If appropriate, depending on individual behavior, it may be necessary to deviate upward or downward from the indicated daily dose.
- the compounds according to the invention and their salts according to the present invention can be used as intermediates for the preparation of other compounds, in particular of other pharmaceutical active compounds which are obtainable from compounds according to the invention, for example, by modification or introduction of radicals or functional groups, for example by esterification, reduction, oxidation or other conversions of functional groups.
- the peptide analogues according to the present invention thus found can on the one hand be used directly as therapeutic agent, but they can also form the basis for related structures, which are also suitable for use as therapeutic agent for treating diseases relating to an increased cell proliferation.
- An effective amount of the pharmaceutical composition is defined as that amount sufficient to detectably and repeatedly to ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. More rigorous definitions may apply, including elimination, eradication or cure of disease.
- the routes of administration will vary, naturally, with the location and nature of the lesion, and include, e.g. intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration and formulation.
- Intratumoral injection, or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate.
- the volume to be administered will be about 4-10 ml (preferably 10 ml), while for tumors of ⁇ 4 cm, a volume of about 1-3 ml will be used (preferably 3 ml).
- Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes.
- the hyperproliferating cells may be contacted by administering multiple injections to the tumor, spaced at approximately 1 -cm intervals.
- the present invention may be used preoperatively, to render an inoperable tumor subject to resection.
- the present invention may be used at the time of surgery, and/or thereafter, to treat residual or metastatic disease.
- a resected tumor bed may be injected or perfused with a formulation comprising the compounds according to the invention.
- the perfusion may be continued post-resection, for example, by leaving a catheter implanted at the site of the surgery. Periodic post-surgical treatment also is envisioned.
- Continuous administration also may be applied where appropriate, for example, where a tumor is excised and the tumor bed is treated to eliminate residual, microscopic disease. Delivery via syringe or catherization is preferred. Such continuous perfusion may take place for a period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about 1-2 weeks or longer following the initiation of treatment. Generally, the dose of the therapeutic composition via continuous perfusion will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the perfusion occurs. It is further contemplated that limb perfusion may be used to administer therapeutic compositions of the present invention, particularly in the treatment of melanomas and sarcomas.
- Treatment regimens may vary as well, and often depend on tumor type, tumor location, disease progression, and health and age of the patient. Obviously, certain types of tumor will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the known efficacy and toxicity (if any) of the therapeutic formulations.
- the tumor being treated may not, at least initially, be resectable.
- Treatments with therapeutic viral constructs may increase the resectability of the tumor due to shrinkage at the margins or by elimination of certain particularly invasive portions. Following treatments, resection may be possible. Additional treatments subsequent to resection will serve to eliminate microscopic residual disease at the tumor site.
- a typical course of treatment, for a primary tumor or a post-excision tumor bed, will involve multiple doses.
- Typical primary tumor treatment involves a 6-dose application over a two-week period.
- the two-week regimen may be repeated one, two, three, four, five, six or more times.
- the need to complete the planned dosings may be re-evaluated.
- the invention relates to the use of the compounds according to the invention as a tool within the fields of cell and molecular biology.
- An experimental approach used in cell biology research is to disturb cellular processes and to analyze the effect(s) of the disturbance on the cell function(s).
- Several commercially available drugs can be used to inhibit transcription. Actinomycin D (also used as a cytostatic drug in cancer treatment), alpha-amanitin, and 5,6-dichloro-l-beta- D-ribofuranosylbenzimidazole (DRB) are the most commonly used.
- TIP constitutes an alternative to the use of conventional drugs, such as the ones mentioned above.
- the compounds should be able to enter the cells and reach their target(s).
- the compounds according to the invention are efficiently taken up by living cells from the medium and transported into the cell nucleus, as shown in Example 15 herein.
- Protein transduction is the process by which peptide or protein motifs cross the cellular plasma membrane.
- the direct application of functional peptides and proteins to cells has been used to probe signal transduction pathways, block transcription factors, and conduct detailed structure/function analyses of integrin and other receptors' cytoplasmic domains, among other research applications (J. Hawiger, Current Opinion in Chemical Biology, 3 :89-94, 1999).
- Protein transduction is not the only protein-delivery method.
- scientists can achieve the same effect using other, more invasive techniques.
- a growing range of protein delivery products and kits are coming onto the market.
- Other limitations of these methods include the efficiency and cell-to-cell variability. Electroporation, for instance, delivers proteins to only 1-2% of a primary cell population and 10- 15% of an established cell line population.
- the cells that do not receive the protein can easily mask any effect produced by those cells that do.
- not all of the cells that take up protein end up with the same concentration of exogenous protein, leading to variable and difficult-to-interpret results.
- PTDs are generally short peptides, about 10-16 residues in length. Structurally dissimilar, their only common feature appears to be the presence of numerous positively charged lysine and arginine residues (S.R. Schwarze, S.F. Dowdy, Trends in Pharmacological Sciences, 21 :45-8, February 2000). PTD-mediated transduction evidently occurs by a mechanism other than classical endocytosis: Cells can take up these peptides at 4°C and in the presence of cellular transport process inhibitors(S.R. Schwarze, S.F. Dowdy, Trends in Pharmacological Sciences, 21 :45-8, February 2000).
- PTD-mediated transduction seems to be independent of protein size.
- PTDs have delivered covalently attached proteins in excess of 700 kDa to cells. They have also transduced liposomes over 200 nm in diameter-about the size of a mitochondrion- directly across the cell membrane by anchoring PTDs to the liposome surface.6
- the covalent attachments that link the PTD to its cargo can be made reversible, for example, by using disulfide or ester linkages that are reduced or cleaved once inside the cell.
- Tat transduction occurs through a poorly defined mechanism that is independent of receptors, transporters, and endocytosis.
- the 101 -residue-long Tat protein crosses the plasma membrane, localizes to the nucleus, and initiates the replication of viral DNA.
- Tat's PTD consists of an arginine- rich stretch of nine amino acids (RKKRRQRRR) embedded within the protein.
- Tat When synthesized as a recombinant fusion protein or covalently cross-linked to other peptides or proteins, Tat is capable-albeit with poor efficiency-of delivering biologically active cargo to a cell. However, if the cargo is instead covalently linked at the N- or C-terminus to a truncated Tat PTD, the transduction efficiency improves dramatically. Transduction then occurs via a rapid process that targets cells in a concentration-dependent fashion at both 37°C and 4°C.
- Tat-coupled proteins have been transduced into cells and tissues in vivo, as well as in culture. It ha been demonstrated that intraperitoneal injection by the Tat peptide into a live mouse can deliver functional protein-in this case, b-galactosidase (120 kDa)-to all murine tissues tested, including the brain (S.R. Schwarze et al., Science, 285: 1569-72, 1999). Only proteins that are covalently linked to the PTD can get in and out of cells, thus ensuring that the process is specific-a particularly important consideration for in vivo applications of this technology.
- Pep-1 is a 21 -residue-long carrier consisting of three domains: a hydrophobic, tryptophan-rich motif that targets the cell membrane and forms hydrophobic interactions with proteins; a hydrophilic, lysine-rich domain derived from the simian virus 40 large T antigen nuclear localization sequence, which improves intracellular delivery and solubility of the peptide vector; and a spacer sequence (M.C. Morris et al., Nature Biotechnology, 19: 1 173-6, December 2001).
- GFP green fluorescent protein
- Pep-1 one molecule of green fluorescent protein (GFP; 30 kDa) interacts with 12-14 molecules of Pep-1 (M.C.
- the compounds according to the present invention may also have a potential application to clinical medicine. While research in this field is still in its infancy, it is possible that protein delivery will have some application to current gene therapy protocols, in cases where the direct delivery of the gene product itself may be more beneficial then the delivery of the gene.
- protein transduction technology has potential applications for vaccine development. For instance, Phogen has licensed its VP22 technology to Palo Alto, Calif.-based Genencor International Inc. for the development of therapeutic vaccines against hepatitis B, hepatitis C, and human papilloma virus.
- the compounds according to the invention may be used as a vehicle for protein transduction.
- test compounds are brought in contact with preformed complexes between actin and Hrp65 is useful for identifying such compounds which will inhibit said interaction. They further show that compounds which inhibit said interaction also inhibit transcription, by the demonstration of inhibition of the formation of transcription-active BR-puffs and by the inhibition of BrUTP incorporation into nascent RNA.
- TIP-1 sequence shown in SEQ ID NO 2
- TIP-2 sequence shown in SEQ ID NO 3
- Figure ID The sequences of TIP, TIP-1 and TIP-2 are shown below, and also in the sequence listing. The sequences are shown in N- to C-terminal direction and differences to TIP in bold.
- An actin/Hrp65-assay performed in vivo is also described. It is useful to perform in vivo cross-linking experiments to establish whether a compound can disturb the interaction between actin and Hrp65 in vivo.
- TIP was also injected into the nucleus of salivary gland cells of C. tentans and the effects of the injection on the expression of the BR genes were analyzed.
- the BR genes display all the typical features of protein-coding genes and their expression can be easily monitored at the cytological level using immunofluorescence microscopy.
- Figure 3 A panel a
- two giant BR puffs, BR1 and BR2 are observed under normal growth conditions in the nuclei of salivary gland cells immuno-stained with antibodies against an abundant non-shuttling SR protein, Hrp45.
- FIG. 1 Actin Binds Directly to the C-Terminus of Hrp65-2.
- a Nuclear actin- associated proteins studied by DNase I affinity chromatography and Western blotting. Nuclear extracts were prepared from C. tentans tissue culture cells and either fractionated by SDS-PAGE (N), or mixed with DNase I-Sepharose beads. The proteins in the DNase I-bound fraction (B) were eluted and separated by SDS-PAGE. Gels were silver stained (lanes 1 and 2) or analyzed by Western blotting using mAbs against three C. tentans proteins: Hrp23 (lanes 3 and 4), Hrp36 (lanes 5 and 6), and Hrp65 (lanes 7 and 8).
- the actin band previously identified by mass spectrometry is marked with a dot next to the silver stained gel.
- b Schematic structure of the Hrp65 isoforms containing an N-terminal T7 tag.
- c Reconstitution experiments using purified S- Hrp65 isoforms immobilized on T7-beads and saturating amounts of 35 S-labelled G- actin. Bound proteins were separated by SDS-PAGE and visualized by autoradiography.
- d Specific binding of actin to Hrp65-2.
- the wild type 65-2CTS peptide, two mutated versions (65- 2mut-l and 65mut-2) and two unrelated peptides (118B and 2D 13) were coupled to Sulpholink beads via their terminal cysteines, and incubated with purified 35 S-labelled G-actin. Bound actin was fractionated by SDS-PAGE and visualized by autoradiography. The bound actin was quantified by phosphoimaging and the results are given under each lane as percentages of input actin.
- FIG. 1 Actin Binds to Hrp65 In Vivo, a, Co-immunoprecipitation after in vivo cross-linking.
- Cultured C. tentans cells were incubated in the absence (lanes 1 to 4) or , the presence of peptides 65-2CTS (lanes 5 and 6) and 1 18B (lanes 7 and 8), and then treated with the cell permeable cross-linking reagent DSP. Nuclear extracts were prepared, and each extract was split into two equal portions, one being treated with urea. Nuclear extracts were also prepared from DSP-untreated cells (lines 1 and 2).
- Peptides 65-2CTS and 1 18B are expontaneously internalized into C. tentans tissue culture cells. Peptides were labeled with fluorescein maleimide and added to the cell medium at a final concentration of 15 ⁇ M. The internalization of the peptides was analyzed by confocal microscopy. Fluorescein-labelled 65-2CTS was detected almost exclusively in the nucleus (cf.
- fluorescein-labelled 1 18B is found in both the nucleus and the cytoplasm (cf panels c, g and k).
- the fluorescein labeled 65- 2CTS (cf. panels b,fmdj) and 1 18B (cf. panels d, h and /) were digested with trypsin before incubation with the cells.
- FIG. 3 Disruption of the Actin-Hrp65-2 Complex Down-Regulates mRNA Transcription
- a Salivary glands were isolated from C. tentans fourth instar larvae, immunostained with mAb 2E4 against Hrp45, and visualized by confocal microscopy.
- a-d show individual salivary gland cells untreated (a), treated with the transcription inhibitor actinomycin D at 4 ⁇ g/ml for 90 min before immunostaining (b), injected with peptide 65-2CTS (TIP) at 30-60 mg/ml before immunostaining (c), or injected with control peptides, either 118B or 65-2mutl, at 30-60 mg/ml before immunostaining (d).
- TIP peptide 65-2CTS
- c control peptides, either 118B or 65-2mutl, at 30-60 mg/ml before immunostaining
- BrUTP was injected into the cytoplasm of salivary gland cells at 100 mg/ml, and the incorporation of BrUTP into nascent RNA was visualized by immunofluorescence after 20 min incubation in hemolymph at 18°C, using a mouse anti-BrUTP antibody and a FITC-coupled secondary antibody.
- the glands were incubated with hemolymph containing actinomycin D at 4 ⁇ g/ml for 60 min before BrUTP injection and immunostaining.
- peptide 65-2CTS TIP
- control peptides 65- 2mut-l or 1 18B
- FIG. 4 Specificity of the Anti-Hrp65-2 Antibody, a, C. tentans nuclear proteins probed with either antibody 282-296 against all the Hrp65 isoforms (lane 1), or the peptide-specific antibody raised against the C-terminal sequence of Hrp65-2 (lane 2). The mobility of molecular mass standards is given in kDa.
- b Polytene chromosomes were manually isolated as described by Bj ⁇ rkroth et al. and immunostained with the Hrp65-2 specific antibody followed by a gold-conjugated secondary antibody and silver enhancement. In some cases, the isolated chromosomes were treated with 100 ⁇ g/ml RNase A for 60 min at room temperature prior to post-fixation and immunostaining.
- Hrp65-2 is Associated with the Nascent BR pre-mRNP Particles.
- A Immuno-electron microscopic localization of Hrp65-2 in the BR transcription unit. Polytene chromosomes were isolated as in Figure 4, incubated with the anti-Hrp65-2 antibody at 0.5 mg/ml, and detected with a secondary antibody conjugated to 12-nm colloidal gold markers.
- the immunolabeled chromosomes were fixed, dehydrated, embedded in Agar resin and sectioned as previously described, a, Distribution of gold markers in the proximal (p), middle (m) and distal (d) portions of the BR transcription unit, b, Schematic representation of the BR transcription unit showing the progressive growth of the pre-mRNPs along the gene and their release into the nucleoplasm upon transcription termination, c-h, Examples of immunolabeling in nascent BR pre-mRNP particles. Schematic interpretations of the images are provided under each micrograph. The bar represents 100 nm.
- FIG. 6 The figure demonstrates the effect of TIP on the incorporation of j2 P-ATP isotope into newly synthesized RNA. It is clear that there is a dramatic reduction of the total RNA produced when the peptide is added (squares on the chart vs triangles for the control).
- Amino acids are denoted by the conventional one-letter abbreviations. Polypeptides are written in N-terminal to C-terminal direction.
- similarity or “similar substitutions” mean that chemically similar amino acids replace each other.
- the basic residues Lys and Arg are considered chemically similar and often replace each other, as do the acidic residues Asp and Glu, the hydroxyl residues Ser and Thr, the aromatic residues Tyr, Phe and Trp, and the non-polar residues Ala, Val, He, Leu and Met. Similarity is measured by dividing the number of similar residues by the total number of residues and multiplying the product by 100 to achieve a percentage.
- identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity.
- BLAST Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410) are available for determining sequence identity.
- Hrp65-like activity means the ability of a compound to interact with actin, in the same way as Hrp65 does. It is well within the capacity of the skilled person to determine, with reference to the present description, whether a compound is capable of interacting with actin or not.
- a compound with Hrp65-like activity is normally a protein, similar or identical to the molecule disclosed in SEQ ID NO 4.
- Hrp65 and Hrp65-2 are used interchangeably, and are used to refer to the amino acid sequence shown in SEQ ID NO 4.
- TIP transcription inhibiting peptide
- analogue in the context of the TIP polypeptide, is meant a polypeptide having TIP-like activity, in which polypeptide one or more amino acids are replaced by a different, natural or artificial, amino acid. Also included are variants of TIP in which deletions, substitutions, additions or repeats of one or more amino acids have been introduced. Furthermore, fragments of the peptide, or oligomers of these fragments are included.
- TIP transcription inhibiting peptide
- TIP-activity As defined herein, the terms “TIP-activity”, “TIP-like activity”, or “transcription inhibiting activity” relate to the activity of TIP which inhibits transcription and consequently cell proliferation. The terms relate to the effect TIP has on transcription. It is well within the capacity of the skilled person to determine, with the help of the description given herein, whether or not a compound has any transcription inhibiting activity.
- a compound will be considered to be an inhibitor of transcription if it blocks transcription at least by 30%, preferably at least by 40%, more preferably at least by 50%o or most preferably at least by 60%, compared to control experiments.
- Another definition of an inhibitor may be one which, at a concentration of 10 ⁇ M, or preferably 1 ⁇ M, will disrupt 50% of the complexes between actin and Hrp65. Yet another definition of an inhibitor will be one which at a concentration of 10 ⁇ M, or preferably 1 ⁇ M, will decrease the incorporation of BrUTP into RNA by 50%.
- Hrp65 refers to the polypeptide according to SEQ ID NO 4, and to functional equivalents thereof. Being a functional equivalent of Hrp65 implies that a molecule has essentially the same actin-interacting property as Hrp65. It is well within the capacity of the skilled person, with reference to the description disclosed herein, to establish whether a molecule interacts with actin or not. Consequently, variants are included in the scope of the invention. Such variants include polypeptides according to SEQ ID NO 4, in which additions, deletions, or substitutions have been made, while still retaining the Hrp65-like activity.
- the treatments may include various "unit doses.”
- Unit dose is defined as containing a predetermined-quantity of the therapeutic composition.
- the quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts.
- a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
- Unit dose of the present invention may conveniently be described in terms of mg/kg body weight.
- TIP (SEQ ID NO 1) corresponding to the C-terminal sequence of Hrp65-2, amino acids 503-517, plus an N-terminal cystein, was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize rabbits according to standard procedures.
- KLH keyhole limpet hemocyanin
- the anti-Hrp65-2 antibody was purified by peptide-affinity chromatography using the UltraLink system (Pierce). The activity of the affinity purified antibody was confirmed by ELISA assay. For microinjection assays, the purified antibody was further concentrated using a Nanosep centrifugal device (Pall Gelman Sciences).
- All peptides (65-2 CTS, 65-2 CTS mut-1, 65-2 CTS mut-2, 1 18B and 2D13) were chemically synthesized by solid phase method as described (Atherton & Sheppard, 1989) and purified by reverse phase HPLC to more than 80% homogeneity. Peptides may also be purchased from Innovagen, Lund, Sweden. Mass spectral analysis was performed to confirm their expected molecular weights.
- a Bgl II-Sal I restriction fragment containing the full ORF of hrp65-2 was excised from pEGFP-C3-hrp65-2 (Miralles and Visa, 2001) and cloned into the Bam HI - Sal I sites of pET21b (Novagen) to generate pET21b-hrp65-2.
- Fluorescent labeling of peptides and proteins Aliquots of peptides or proteins were reacted with fluorescein-5-maleimide (obtainable from Roche) via a cysteine amino acid residue placed at the N-terminal end of the TIP and 118B peptides, or at cysteine residues on either Hrp65 or actin. 1-3 mg of peptides TIP and/or 118B or proteins dissolved in PBS were incubated with 2 eq of fluorescein- 5-maleimide dissolved in dimethylformamide (DMF) per SH group of the peptide. The labeling reaction was carried out for 2 hours at room temperature with continuous agitation and in the dark. Fluorescent peptides or proteins were purified on a G-50 spin column and stored at -20°C in the dark until further use.
- fluorescein-5-maleimide obtainable from Roche
- S-labelled native actin from non-muscle cells was prepared by DNase I affinity chromatography essentially as previously described (Zechel, K., (1980)).
- the plasmid containing the full-length ORF of hrp65-2, (pET21b-hrp65-2, encoding the amino acid sequence according to SEQ ID NO 4) was used as template for TNT- coupled in vitro transcription/translation essentially following the manufacturer's instruction manual (TNT Coupled Reticulocyte Lysate Systems, Promega).
- the proteins were labeled by incorporation of S-methionine, electrophoresed in 10% SDS/polyacrylamide gels and detected by autoradiography.
- C. tentans was reared as described by Case and Daneholt (1978), and salivary glands were isolated from fourth instar larvae.
- C. tentans tissue culture cells were grown in suspension at 24°C as devised by Wyss (1982).
- Tissue culture cells were washed with PBS and re-suspended in PBS containing 0.2% NP40 and 0.1 mM PMSF. The cells were homogenized and spun at 2000 g for 5 minutes at 4 ° C. The supernatant constituted the cytosolic extract. The pellet was washed twice in PBS and resuspended in PBS containing 0.1 mg/ml tRNA, sonicated, and clarified. The supernatant was retained as the nuclear extract.
- Microinjections were performed on isolated salivary glands.
- the peptides were injected at a concentration of 30mg/ml or 60mg/ml in PBS. After injection, the glands were incubated at 18°C for 90 min in hemolymph and fixed. Immunofluorescent detection of hrp45 was performed as described herein.
- Proteins were fractionated on 10% polyacrylamide gels, containing 0.1% SDS (SDS- PAGE), transferred electrophoretically to PVDF membranes (Immobilon-P; Millipore) in a semi-dry transfer cell (Trans-Blot; Biorad) and visualized with 0.2% (w/v) Ponceau-S (Biorad) in 10% (v/v) acetic acid. After blocking with 10% (w/v) non-fat dry milk in PBS-0.1% Tween-20, the blots were incubated with monoclonal antisera against hrp36 and hrp65 (1 :200 dilution) in 5% (w/v) non-fat dry milk in PBS-0.1% Tween-20.
- hrp36 and hrp65 were detected using a goat anti-mouse IgG (DAKO) coupled to horse-radish peroxidase and immunodetection was performed using the enhanced chemiluminescent method as described in the manufacturer's instruction manual (ECL-Plus, Amersham).
- DAKO goat anti-mouse IgG
- DNase I (Sigma) was coupled to CNBr-activated Sepharose (Pharmacia) according to the manufacturers instructions.
- 300 ⁇ l DNase I beads pre-equilibrated in lxPBS were incubated in a single step procedure nuclear and cytosolic extracts, prepared from 500 ml of C. tentans tissue culture cells. Following incubation for 30-40 minutes at 4°C, the beads were repeatedly washed to remove non-specifically bound material as previously described. The beads were resuspended in Laemmli buffer, heat denatured to elute the bound proteins which were then resolved by 10% SDS-PAGE. The proteins were detected by silver staining. The results are shown in figure 1 A.
- T7 tag antibody agarose co-immunoprecipitations T7-tagged 35 S-labelled hrp65-l, hrp65-2 were expressed by coupled in vitro transcription/translation and immobilised onto T7-tag antibody agarose following the instruction manual. Hrp65 agarose beads were then incubated with saturating amounts of DNasel affinity-purified 35 S-labelled native actin for 30-40 minutes at 4°C with continuous mixing. In parallel, competition experiments were also performed using Hrp65 beads.
- Hrp65-1 and Hrp65-2 beads were pre-saturated with TIP-peptide conjugated with keyhole limpet hemocyanin (KLH), as well as KLH alone for 20 minutes at 4 ° C with continuous mixing and subsequently incubated with saturating amounts of S-labelled actin as mentioned above. Beads were then spun down and washed with phosphate buffer saline containing 4% glycerol, 1%» NP40, 0.1% deoxycholate, 1 mM DTT as already described. Bound proteins were analyzed as described above and detected by autoradiography of the fixed and dried gel.
- lyophilized peptide typically about 3.0 mg were dissolved in 50 mM Tris, 5 mM EDTA, pH 8.5 and incubated with 1 ml of Sulpholink resin (50% slurry) pre-equilibrated in 50 mM Tris, 5 mM EDTA, pH 8.5 according to the manufacturer's instructions.
- Sulpholink resin 50% slurry
- Example 10 Assay for identifying compounds capable of inhibiting the interaction between Hrp65 and actin.
- Such an assay may comprise the following steps; (a) conjugating Hrp65 to a solid support, such as a bead, surface, or in a well in a multiwell plate, (b) washing away non-conjugated Hrp65, (c) blocking non-conjugated sites on the solid support by adding another protein (e.g. bovine serum albumin, or a blocking mixture such as milk powder), (d) adding labeled (e.g. radioactively, by e.g. 35 S, or fluorescently, by e.g. FITC) actin either in the presence or absence of a test compound, (e) washing away unbound actin, (f) measuring either the amount of actin still bound (e.g.
- a solid support such as a bead, surface, or in a well in a multiwell plate
- another protein e.g. bovine serum albumin, or a blocking mixture such as milk powder
- labeled e.g. radioactively, by e.g.
- FITC is fluorescein isothiocyanate, and is commercially available from Molecular Probes, or from Sigma. It is also possible to use an analog of FITC, fluorescein-5-maleimide, which is commercially available from Pierce.
- Salivary glands were isolated from fourth instar C. tentans in ice-cold TKM buffer (100 mM KC1, 1 mM MgCl 2 and 10 mM triethanolamine-HCl, pH 7.0). All isolation steps were performed at a temperature close to 0°C if not indicated otherwise. The glands were incubated for 60 sec in TKM buffer, containing 2% Nonidet P40 (NP-40), and transferred twice into fresh 0.025% NP-40 in TKM. The polytene chromosomes were released from the glands by a pipetting procedure described by Bjorkroth et al. (1988). Individual chromosomes were collected and transferred to a microscopic slide.
- TKM buffer 100 mM KC1, 1 mM MgCl 2 and 10 mM triethanolamine-HCl, pH 7.0. All isolation steps were performed at a temperature close to 0°C if not indicated otherwise. The glands were incubated for 60 sec in TKM buffer, containing 2% Nonidet
- chromosomes After attaching to the surface they were post-fixed in 4% paraformaldehyde in TKM for 30 min at room temperature and washed three times with fresh TKM. Prior to fixation, some of the chromosomes were treated with RNase A (100 ⁇ g/ml) for 60 min at room with subsequent postfixation and washes as described above. For immunocytological analysis the isolated chromosomes were blocked with 2% bovine serum albumin (BSA) in TKM for 30 min at room temperature in a humid chamber. The primary antibody, anti-hrp65-2, was applied diluted 1 : 100 in TKM containing 0.5% BSA, and incubated for 60 min. As negative control a non-related antibody against synaptonemal complex protein 3 (SCP3) was used.
- SCP3 synaptonemal complex protein 3
- Polytene chromosomes were isolated and immunostained as described above, but with some differences.
- the chromosomes were transferred to a siliconized slide and the colloidal gold on the secondary antibody had a diameter of 12 nm.
- the chromosomes were fixed with 2% glutaraldehyde in TKM for 60 min. They were washed with TKM for three times, 5 min each, dehydrated in three steps with ethanol (90, 95, 100%) and with subsequent embedding in an Agar 100 resin.
- the specimens were sectioned (60-75 nm) with a Leica Ultracut ultramicrotome (Leica, Wien, Austria) and the sections were stained with saturated uranyl acetate and subsequently with lead citrate.
- the individual gold particles along the BR genes were studied in a Philips CM 120 electron microscope at 60 kV. The results of this experiment are shown in Figure 5.
- FITC-conjugated anti mouse secondary antibody diluted 1 : 100 in PBS containing 0.5% nonfat dry milk and 0.5% BSA was used to detect the monoclonal antibody against hrp45; a FITC-conjugated anti rabbit antibody (1 :50) was used to detect the polyclonal antibody against hrp65-2; a FITC-conjugated anti goat antibody (1 :50) was used to reveal the antibody against the large subunit of RNA polymerase II.
- DAPI was added at the end of the incubation to stain DNA; the samples were then washed three times in PBS and mounted in Mowiol.
- C. tentans tissue culture cells were individually incubated for 15 minutes at room temperature with fluorescein-labeled 65-2 CTS and 118B at a final concentration of 5-10 ⁇ M.
- C. tentans cells were also incubated with fluorescein-labeled 65-2 CTS and 118B peptides pre-digested with trypsin for lh at 37°C. The samples were subsequently washed and spun down on poly-lysine coated glass plates for 3 minutes at 800 rpm using a Cytospin apparatus.
- BrUTP (5-Bromouridine 5'-Triphosphate, Sigma) was injected at a concentration of 100 mg/ml, 60 min after peptide injections (either TIP or control peptides). Incorporation of BrUTP into nascent RNA was visualized by immunofluorescence after a 90 minute incubation in hemolymph at 18°C, using a mouse anti BrUTP antibody (Roche) 1 : 10 in phosphate buffered saline (PBS) containing 0.5%) nonfat dry milk and 0.5% BSA) and revealed by a FITC-coupled goat anti-mouse antibody (1 : 100 in PBS containing 0.5% nonfat dry milk and 0.5% BSA).
- PBS phosphate buffered saline
- Salivary glands were isolated from C. tentans fourth instar larvae, immunostained with monoclonal antibody 2E4 against Hrp45, and visualized by confocal microscopy. The results are shown in figure 3, panels a-d. Individual salivary gland cells were either untreated, treated with the transcription inhibitor actinomycin D at 4 ⁇ g/ml for 90 min before immunostaining, injected with peptide 65-2CTS at 30-60 mg/ml before immunostaining, or injected with control peptides, either 1 18B or TIP-1, at 30-60 mg/ml before immunostaining. After peptide injections, the glands were incubated in hemolymph at 18°C for 90 min before fixation and immunostaining. For each treatment, three examples are presented: one showing a full nucleus and two additional ones showing only the BR puffs. The bar in panel a represents 10 ⁇ m and all photographs are at the same magnification.
- cell lysis was performed in lysis buffer containing 10 mM Tris pH 7.5, 1 mM EDTA, 0.5% SDS, 0.15 M LiCl, and followed by incubation at 65° C for 5 minutes. Extracts were then left on ice for 5 minutes and then incubated with 20 microliters of pre- equilibrated oligo(dT)-cellulose for 30 minutes at 4° C under continuous agitation. Beads were subsequently washed with a 10-fold excess of lysis buffer (same as above) and the total poly(A)+ RNA bound to the beads was quantified by standard scintillation methods. As control, the same experiment was performed in parallel, but without peptide. In both cases, the values obtained in cpm (counts per minute) from isotope measurements were then plotted against time. The results are shown in figure 6.
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JP2009502839A (ja) * | 2005-07-29 | 2009-01-29 | エッカルト ゲゼルシャフト ミット ベシュレンクテル ハフツング | 金属様光沢外観を有する化粧用コーティングを得るための化粧料組成物、人工フィンガーネイル、およびpvdアルミニウム顔料の使用 |
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Non-Patent Citations (6)
Title |
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DATABASE SWISSPROT [online] 1 December 2001 (2001-12-01), XP002966567, Database accession no. (Q95ZH0) * |
DATABASE SWISSPROT [online] 1 May 2000 (2000-05-01), XP002962700, Database accession no. (Q9U1N0) * |
MIRALLES FRANCESC ET AL.: "Electron tomography reveals posttranscriptional binding of Pre-mRNPs to specific fibers in the nucleoplasm", THE JOURNAL OF CELL BIOLOGY, vol. 148, no. 2, January 2000 (2000-01-01), pages 271 - 282, XP002950418 * |
MIRALLES FRANCESC ET AL.: "Molecular characterization of CT-hrp65: identification of two novel isoforms originated by alternative splicing", EXPERIMENTAL CELL RESEARCH, vol. 264, 2001, pages 284 - 295, XP002962699 * |
PERCIPALLE PIERGIORGIO ET AL.: "Actin bound to the heterogeneous nuclear ribonucleoprotein hrp36 is associated with Balbiani Ring mRNA from the gene to polysomes", THE JOURNAL OF CELL BIOLOGY, vol. 153, no. 1, April 2001 (2001-04-01), pages 229 - 235, XP002962674 * |
SCHWARZE STEVEN R. ET AL.: "In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA", TIPS, vol. 21, 2000, pages 45 - 48, XP004189118 * |
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JP2009502839A (ja) * | 2005-07-29 | 2009-01-29 | エッカルト ゲゼルシャフト ミット ベシュレンクテル ハフツング | 金属様光沢外観を有する化粧用コーティングを得るための化粧料組成物、人工フィンガーネイル、およびpvdアルミニウム顔料の使用 |
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