Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/10—Anti-acne agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/91—Transferases (2.)
  • G01N2333/91091—Glycosyltransferases (2.4)
  • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
  • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders

Definitions

• the invention relates to the identification and use of modulating compounds of UDP-glucose ceramide glucosyltransferase for the treatment of acne, as well as cutaneous disorders associated with hyperkeratinization. It also relates to methods of diagnosis or prognosis in vitro of these pathologies.
• Acne is generally due to the involvement of three factors: - an excessive production of sebum (hyperseborrhoea), under the effect of hormones and puberty, a thickening of the skin (hyperkeratinization) whose pores and more particularly the sebaceous glands become clogged causing the formation of blackheads and comedones, and - the development of bacteria, causing inflammation and the appearance of red or white pimples often painful.
• Cornification of keratinocytes is a complex process that involves the degradation of a large number of intracellular components. This process is the final step in epidermal differentiation and is associated with the formation of organized lamellar bilayers enriched with ceramides, cholesterol and fatty acids.
• the formation of ceramides is a key point leading to the formation of a normal stratum corneum and regulating skin barrier function and desquamation (Holleran WM et al., J. Lipid Res., 1994, 35, 905- 912). Reduction of stratum corneum ceramide and barrier function is observed in acne patients (Yamamoto A et al., Arch Dermatol Res., 1995, 187, 214-218).
• Topical application of retinoids or oral administration of isotretinoin has been shown to increase ceramide levels in acne patients.
• the increase in ceramide is correlated with a decrease in comedones after treatment with topically applied retinoids (Melnic B et al., Arch Dermatol Res., 1988, 280, 97-102, Thielnitz A, Br. Dermatol., 2001, 1995, 95, 2903-2909).
• Retinoids are generally highly irritating and stripping compounds, causing unattractive facial redness.
• UDP-glucose ceramide glucosyltransferase UGCG
• UGCG UDP-glucose ceramide glucosyltransferase
• acne we mean all forms of acne, namely in particular vulgar acne, comedonal, polymorphic, nodulocystic acne, conglobata, or secondary acne such as solar acne, drug or professional.
• the Applicant also proposes in vitro diagnostic methods or in vitro prognosis, based on the detection of UGCG expression or activity.
• the UGCG enzyme refers to UDP-glucose ceramide glucosyltransferase. This enzyme is involved in the process of keratinization. This process is the last stage of epidermal differentiation, and is associated with the formation of a highly organized lamellar double layer enriched with ceramides, cholesterols and free fatty acids. These lipids are derived from the epidermal lamellar body, secretory organelles containing phospholipids, glucosylceramides and also hydrolytic enzymes. UDP-glucose ceramide glucosyltransferase is the enzyme responsible for the formation of ceramides from the glucosylceramide cell pool.
• Ceramide production has been shown to be a critical step for the formation of normal stratum corneum, and thereby regulates the permeable barrier and desquamation of the skin (Hollerman WM et al., J. Lipid Res 1994, 355: 905). -912).
• a defect in UDP-glucose ceramide glucosyltransferase causes skin abnormalities described in patients with Gaucher disease.
• a new therapeutic protocol has been proposed for the management of Gaucher disease. This approach aims to reduce the biosynthesis of glucosylceramide using glucosylceramide synthase inhibitors.
• One of these inhibitors, N-butyldeoxynojirimycin (Miglustat) has recently been approved by the FDA for the treatment of Gaucher disease.
• glycosylceramides and ceramides appear to regulate cell proliferation and differentiation.
• changes in the level of glucosylceramides stimulated keratinocyte proliferation (Uchida Y et al., J Invest Dermatol, 1994, 102: 594a, Marsh NL et al, J Clin Invest, 1995, 95, -2903-2909).
• UGCG gene or "UGCG nucleic acid” means the gene or nucleic acid sequence that encodes UDP-glucose ceramide glucosyltransferase. If the targeted target is preferably the human gene or its expression product, the invention may also use cells expressing a heterologous UDP-glucose ceramide glucosyltransferase, by genomic integration or transient expression of an exogenous nucleic acid coding for the enzyme.
• a human cDNA sequence of UGCG is reproduced in the appendix (SEQ ID No.1). It is the NM003358.1 sequence whose coding portion is from acid 291 to 1475.
• Another subject of the invention relates to an in vitro method for diagnosing or monitoring the progression of acne lesions or a skin disorder associated with hyperkeratinization in a subject, comprising comparing the expression or the activity of the UDP glucose ceramide glucosyltransferase (UGCG) protein, the expression of its gene or the activity of at least one of its promoters, in a biological sample of a subject relative to a biological sample of a subject control.
• UGCG UDP glucose ceramide glucosyltransferase
• the expression of the UGCG protein can be determined by assaying this protein by radioimmunoassay, for example by ELISA assay. Another method, especially for measuring the expression of the UGCG gene, is to measure the amount of corresponding mRNA by any method as described above. An assay of the activity of the UGCG protein can also be envisaged.
• control is a "healthy” subject.
• control subject refers to the same subject at a different time, which preferably corresponds to the beginning of the treatment (To ).
• This measurement of the expression difference or the activity of the UGCG protein, the expression of its gene or the activity of at least one of its promoters makes it possible in particular to monitor the efficacy of a treatment. , in particular treatment with a UGCG modulator, as envisaged above or by another treatment against acne or a skin disorder associated with hyperkeratinization.
• Another aspect of the present invention relates to an in vitro method for determining susceptibility of a subject to develop acne lesions or skin disorder associated with hyperkeratinization, including comparing the expression or activity of the subject.
• the expression of the UGCG protein can be determined by an assay of this protein by radioimmunoassay, for example by ELISA assay.
• Another method, especially for measuring the expression of the UGCG gene is to measure the amount of corresponding mRNA by any method as described above.
• An assay of the activity of the UGCG can also be envisaged.
• the tested subject is here an asymptomatic subject, presenting no skin disorder linked to hyperkeratinization or acne.
• the subject "control” in this method means a "healthy" reference subject or population. The detection of this susceptibility allows the establishment of a preventive treatment and / or increased monitoring of signs related to acne or skin disorder associated with hyperkeratinization.
• the biological sample tested may be any sample of biological fluid or a sample of a biopsy.
• the sample may be a preparation of skin cells, obtained for example by desquamation or biopsy. It can also be sebum.
• Another subject of the invention is an in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, or skin disorders associated with hyperkeratinization, comprising the determination of the capacity of a compound to modulating the expression or the activity of the UDP-glucose ceramide glucosyltransferase or the expression of its gene or the activity of at least one of its promoters, said modulation indicating the utility of the compound for the preventive or curative treatment acne or skin disorders associated with hyperkeratinization.
• the method therefore makes it possible to select the compounds capable of modulating the expression or the activity of the UDP-glucose ceramide glucosyltransferase, or the expression of its gene, or the activity of at least one of its promoters.
• the invention relates to an in vitro method for screening candidate compounds for the preventive and / or curative treatment of acne, or skin disorders associated with hyperkeratinization, comprising the following steps: a) Preparation of at least two biological samples or reaction mixtures; b) bringing one of the samples or reaction mixtures into contact with one or more of the compounds to be tested; c) measuring the expression or the activity of the UDP-glucose ceramide glucosyltransferase protein, the expression of its gene or the activity of at least one of its promoters, in the biological samples or reaction mixtures; d) Selection of compounds for which a modulation of the expression or activity of UDP-glucose ceramide glucosyltransferase, or a modulation of the expression of its gene or a modulation of the activity of at least one of its promoters, is measured in the sample or mixture treated in b) relative to the untreated sample or mixture.
• step d) preferably inhibit the expression or the activity of the UGCG protein, the expression of its gene or the activity of at least one of its promoters.
• the difference in expression obtained with the test compound compared to a control carried out in the absence of the compound is significant from 25% or more.
• expression of a protein means the amount of that protein
• protein activity is meant its biological activity
• promoter activity is meant the ability of this promoter to trigger the transcription of the coded DNA sequence downstream of this promoter (and thus indirectly the synthesis of the corresponding protein).
• the compounds tested can be of any type. They can be of natural origin or have been produced by chemical synthesis. It can be a library of structurally defined chemical compounds, compounds or uncharacterized substances, or a mixture of compounds. Various techniques can be implemented to test these compounds and to identify the compounds of therapeutic interest, modulators of the expression or the activity of the UDP-glucose ceramide glucosyltransferase.
• the biological samples are cells transfected with a reporter gene operably linked to all or part of the promoter of the UGCG gene, and step c) described above consists in measuring the level of expression.
• the reporter gene may in particular code for an enzyme which, in the presence of a given substrate, leads to the formation of colored products, such as CAT (chloramphenicol acetyltransferase), GAL (beta galactosidase), or GUS (beta glucuronidase). It may also be the gene for luciferase or GFP (Green Fluorescent Protein).
• the assay of the protein encoded by the reporter gene, or its activity is carried out conventionally, by colorimetric, fluorometric or chemiluminescent techniques, among others.
• the biological samples are cells expressing the UGCG gene coding for the UDP-glucose ceramide glucosyltransferase, and step c) above consists in measuring the expression of said gene.
• the cell used here can be of any type. It can be a cell expressing the gene
• UGCG endogenously such as a liver cell, an ovarian cell, or more preferably a keratinocyte or a sebocyte. It is also possible to use organs of human or animal origin, such as, for example, the preputial gland, clitoral gland or the sebaceous gland of the skin. It may also be a cell transformed with a heterologous nucleic acid, encoding a UDP-glucose ceramide glucosyltransferase, preferably human, or mammalian.
• a wide variety of host cell systems can be used, such as, for example, Cos-7, CHO, BHK, 3T3, HEK293 cells.
• the nucleic acid can be stably or transiently transfected by any method known to those skilled in the art, for example by calcium phosphate, DEAE-dextran, liposome, virus, electroporation, or microinjection.
• the expression of the UGCG gene or the reporter gene can be determined by evaluating the transcription rate of said gene, or its translation rate.
• transcription rate of a gene is meant the amount of mRNA produced.
• translation rate of a gene is meant the amount of protein produced.
• RNAs of a gene of interest are the most common (Northern Blot, RT-PCR, Rnase protection). It may be advantageous to use detection markers, such as fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin).
• detection markers such as fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin).
• the expression of the gene can be measured by real-time PCR or by RNase protection.
• RNase protection is meant the detection of a known mRNA from the poly (A) RNAs of a tissue that can be done by means of specific hybridization with a labeled probe.
• the probe is a complementary RNA labeled (radioactive) messenger to look for. It can be constructed from a known mRNA whose cDNA, after RT-PCR, has been cloned into a phage. RNA-poly (A) of the tissue where the sequence is to be searched is incubated with this probe under slow hybridization conditions in a liquid medium. RNA: RNA hybrids are formed between the desired mRNA and the antisense probe. The hybrid medium is then incubated with a mixture of ribonucleases specific for single-stranded RNA, so that only the hybrids formed with the probe can resist this digestion. The digestion product is then deproteinized and repurified, before being analyzed by electrophoresis. The labeled hybrid RNAs are detected by autoradiography.
• the translation rate of the gene is evaluated for example by immunological assay of the product of said gene.
• the antibodies used for this purpose may be of polyclonal or monoclonal type. Their production is based on conventional techniques.
• a polyclonal anti-UDP-glucose ceramide glucosyltransferase antibody may, inter alia, be obtained by immunizing an animal such as a rabbit or a mouse, using the entire enzyme. The antiserum is removed and then exhausted according to methods known to those skilled in the art.
• a monoclonal antibody can, inter alia, be obtained by the conventional method of Kohler and Milstein (Nature (London), 256: 495-497 (1975)). Other methods of preparing monoclonal antibodies are also known.
• monoclonal antibodies can be produced by expression of a cloned nucleic acid from a hybridoma.
• Antibodies can also be produced by the phage display technique, by introducing antibody cDNAs into vectors, which are typically filamentous phages that have V gene libraries on the surface of the phage. (for example fUSE5 for E.coli).
• the immunoassay can be carried out in solid phase or in homogeneous phase; in a time or in two stages; sandwich method or competitive method, by way of non-limiting examples.
• the capture antibody is immobilized on a solid phase.
• solid phase it is possible to use microplates, in particular polystyrene microplates, or particles or solid beads, paramagnetic beads.
• ELISA assays radioimmunoassays, or any other detection technique can be used to reveal the presence of the antigen-antibody complexes formed.
• the characterization of antigen / antibody complexes, and more generally isolated or purified but also recombinant proteins (obtained in vitro and in vivo) can be performed by mass spectrometry analysis. This identification is made possible thanks to the analysis (determination of the mass) of the peptides generated by the enzymatic hydrolysis of the proteins (trypsin in general). In general, the proteins are isolated according to methods known to those skilled in the art, prior to enzymatic digestion.
• Peptide analysis in the form of a hydrolyzate is carried out by separation of the peptides by HPLC (nano-HPLC) based on their physicochemical properties (reverse phase).
• HPLC nano-HPLC
• the determination of the mass of the peptides thus separated is carried out by ionization of the peptides and either by direct coupling to the mass spectrometer (electrospray mode ESI), or after deposition and crystallization in the presence of a matrix known to those skilled in the art (MALDI mode analysis).
• the proteins are then identified through the use of appropriate software (eg Mascot).
• step a) described above consists in preparing reaction mixtures each comprising a UDP-glucose ceramide glucosyltransferase enzyme and a substrate of the enzyme, and step c) described above consists of to measure enzymatic activity.
• the enzyme can be produced according to usual techniques using Cos-7 cells,
• CHO BHK, 3T3, HEK293. It can also be produced using microorganisms such as bacteria (for example E. coli or B. subtilis), yeasts (for example
• Saccharomyces, Pichia Saccharomyces, Pichia
• insect cells such as Sf9 or Sf21.
• the determination of the enzyme activity preferably comprises the determination of the transferase activity, by extraction of the lipids produced and chromatographic analysis. Assays for the enzymatic activity of UGCG are described in the literature (see, for example, Futerman et al., 1991, Biochem J, 280, 295-302). Thus, the activity of UDP-glucose ceramide glucosyltransferase can be evaluated as follows: Liver fractions are incubated with a BSA complex (bovine serum albumin) - [ 14 C] hexanoyl ceramide in the presence of UDP-glucose, then the amount of [ 14 C] hexanoyl glucose-ceramides produced is analyzed.
• BSA complex bovine serum albumin
• the lipids are separated by thin layer chromatography (TLC) and recovered from the plate by friction.
• TLC thin layer chromatography
• the radioactivity is determined by measuring the scintillation related to the incubation of lipids in scintillant. Measurement of background is made by incubating [ 14 C] hexanoyl-ceramides in 25mM KCL / 50mMTris pH 7.4 at 37 ° C in the absence of liver extract.
• the subject of the invention is also the use of a modulator of the human enzyme UDP-glucose ceramide glucosyltransferase obtainable by one of the above methods, for the preparation of a medicinal product intended for preventive treatment. and / or curative acne or skin disorders associated with hyperkeratinization.
• a method for the preventive and / or curative treatment of acne, or skin disorders associated with hyperkeratinization which method comprises the administration of a therapeutically effective amount of a modulator of the human enzyme UDP, is described.
• a modulator of the human enzyme UDP glucose ceramide glucosyltransferase
• the invention finally relates to the cosmetic use of a modulator of the human enzyme UDP-glucose ceramide glucosyltransferase for the aesthetic treatment of desquamation problems.
• the modulator is an inhibitor of the enzyme.
• inhibitor refers to a compound or a chemical substance that substantially eliminates or reduces the enzymatic activity of the UDP-glucose ceramide glucosyltransferase.
• substantially means a reduction of at least 25%, preferably at least 35%, more preferably at least 50%, and more preferably at least 70% or 90%. More particularly it may be a compound that interacts with, and blocks, the catalytic site of the enzyme, as competitive inhibitory type compounds.
• a preferred inhibitor interacts with the enzyme in solution at inhibitor concentrations of less than 1 ⁇ M, preferably less than 0.1 ⁇ M, more preferably less than 0.01 ⁇ M.
• the modulator compound may be an inhibitory anti-UDP-glucose ceramide glucosyltransferase antibody, preferably a monoclonal antibody.
• a such inhibitory antibody is administered in an amount sufficient to obtain a plasma concentration of about 0.01 ⁇ g per ml to about 100 ⁇ g / ml, preferably about 1 ⁇ g per ml to about 5 ⁇ g / ml.
• the modulator compound may also be a polypeptide, an antisense DNA or RNA polynucleotide, an si-RNA, or a PNA ("Peptide nucleic acid", a polypeptide chain substituted with purine and pyrimidine bases, whose spatial structure mimes that of DNA and allows hybridization to it).
• PNA Peptide nucleic acid
• UDP-glucose ceramide glucosyltransferase inhibitor compounds for the preventive and / or curative treatment of acne, or cutaneous disorders associated with hyperkeratinization.
• examples of inhibitors of UDP-glucose ceramide glucosyltransferase include the following compounds:
• PDMP 1-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
• D-PDMP D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol
• modulator compounds identified by the screening method described above are also useful.
• the modulating compounds are formulated within a pharmaceutical composition, in association with a pharmaceutically acceptable vehicle.
• These compositions may be administered, for example, orally, enterally, parenterally, or topically.
• the pharmaceutical composition is applied topically.
• the pharmaceutical composition can be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres or nanospheres or lipid vesicles or polymers for controlled release.
• the pharmaceutical composition may be in the form of solutions or suspensions for infusion or for injection.
• the pharmaceutical composition is more particularly intended for the treatment of skin and mucous membranes and may be in the form of ointments, creams, milks, ointments, powders, soaked swabs, solutions, gels , sprays, lotions or suspensions. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric patches or hydrogels allowing controlled release.
• This composition for topical application may be in anhydrous form, in aqueous form or in the form of an emulsion.
• the pharmaceutical composition is in the form of a gel, a cream or a lotion.
• the composition may comprise a modulator content of UGCG ranging from 0.001 to 10% by weight, especially from 0.01 to 5% by weight relative to the total weight of the composition.
• the pharmaceutical composition may further contain inert additives or combinations of these additives, such as:
• preserving agents such as esters of parahydroxybenzoic acid; stabilizing agents;
• osmotic pressure modifying agents emulsifying agents
• antioxidants such as alpha-tocopherol, butylhydroxyanisole or butylhydroxytoluene, superoxide dismutase, ubiquinol or certain metal chelators.
• Figures 1A and 1B are graphs that show the extent of UGCG gene expression in male gonadectomized and vehicle-treated mice, DHT, DHEA, or the combination of DHEA-Flutamide for a period of 7 days. times a day (long-term treatment).
• the results obtained by the Affymetrix technique (FIG. 1A) were confirmed by the RT-PCR technique in real time (FIG. 1B).
• GDX gonadectomized and vehicle-treated mice
• DHT gonadectomized mice treated with Dihydrotestosterone (androgen receptor agonist)
• DHEA gonadectomized mice treated with dihydroepiandrosterone (precursor of steroid hormones, in the preputial glands metabolized to active androgen)
• DHEA-FIu gonadectomized mice treated with a combination of dihydroepiandrosterone and flutamide (androgen receptor antagonist, which blocks the effects DHT and DHEA agonists).
• Level of expression level of expression of mRNA
• FIG. 2 is a graph reporting a kinetic study from 15 minutes to 96 hours.
• Expression level Square mRNA expression level: expression in gonadectomized mice following treatment with DHT at zero time.
• Rhombus expression in gonadectomized mice without DHT treatment.
• RNA samples were prepared from the sebaceous glands and from the epidermis.
• RNA expression was analyzed on an Affymetrix station (microfluidic module, hybridization oven, scanner, computer) following the protocols provided by the company.
• Affymetrix station microfluidic module, hybridization oven, scanner, computer
• the total RNA isolated from the tissues is transcribed into cDNA.
• biotin-labeled cRNA is synthesized using T7 polymerase and a precursor NTP conjugated to biotin.
• the cRNAs are then fragmented into small fragments. All molecular biology steps are controlled using Agilent's "lab on a chip" system to confirm the good efficiencies of the enzymatic reactions.
• the Affymetrix chip is hybridized with the biotinylated cRNA, rinsed and then fluorescently labeled using a streptavidin-conjugated fluorophore. After washes, the chip is scanned and the results are calculated using the MAS5 software provided by Affymetrix. An expression value is obtained for each gene as well as an indication of the significance of the value obtained. The calculation of the significance of the expression is based on the analysis of the signals that are obtained following the hybridization of the cRNA of a given gene with a perfectly matched oligonucleotide ("perfect match") versus an oligonucleotide that contains a mutation ("single mismatch") in the central region of the oligonucleotide (see Table 1).
• Table 1 Measurement of the expression of UDP-glucose ceramide glycosyltransferase in the epidermis and in the human sebaceous gland via the use of the Affymetrix chip technology.
• mice show differentiation of the sebocyte type and are used as an experimental model of the sebaceous gland. They are of sufficient size to allow isolation of RNA without the use of microdissection technologies.
• UGCG expression in the mouse preputial glands was performed under conditions of steroid hormone deficiency (especially in androgenic hormones) following gonadectomy.
• the gonadectomized animals were then treated with physiological amounts of Dihydrotestosterone (DHT) or Dihydroepiandrosterone (DHEA) to restore a physiological level of the androgenic hormones, or as a control experiment with a combination of DHEA-Flutamide in which Flutamide, a Androgen receptor antagonist blocks the effect of DHEA.
• DHT Dihydrotestosterone
• DHEA Dihydroepiandrosterone
• the comparison of the gene expression under these experimental conditions makes it possible to unambiguously identify the modulation or not of the gene expression of a gene in question by the androgenic hormones.
• Gene expression was analyzed using the Affymetrix technology described above ( Figure 1A) and the results were then confirmed by the real-time PCR technique ( Figure 1B).
• RNA isolated from the tissues is transcribed (RT) into cDNA and this is amplified by PCR (Polymerase Chain Reaction).
• PCR Polymerase Chain Reaction
• the progress of the PCR is monitored in real time using fluorescent TaqMan probes, allowing precise quantification of the amount of mRNA of a given gene present in the biological sample at the start.
• Figure 2 represents the relative level of expression of mRNA as a function of time.
• Gonadectomy which causes a deficiency of steroid hormones induces a slight induction of UGCG expression in the mouse preputial gland.
• the mRNA of UGCG in the mouse preputial gland is decreased by a medium-term treatment with DHT (visible effect at 96 hours).

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