Dermatologically effective yeast extract

Abstract

The invention relates to a method for producing a dermatologically active yeast extract, comprising the following steps: providing a preculture of the yeast cells, culturing the cells for at least fifteen minutes at a pH of 1.8-4, harvesting the cells and lysing the cells, and a yeast extract produced thereby and products comprising said yeast extract.

Claims

1. A method of producing a dermatologically active yeast extract, said method comprising: providing a preculture of yeast cells selected from the group consisting of Yarrowia, Saccharomyces, and Pichia yeast cells; subjecting the preculture of yeast cells to an initial culturing step, wherein said initial culturing step is performed for at least 1 hour at a temperature of 34-39 C. and at a pH>5; culturing the yeast cells for at least fifteen minutes and at a pH of 1.8-4; harvesting the yeast cells; and lysing the yeast cells, wherein said lysis provides said yeast extract.

2. The method according to claim 1, wherein said lysing is carried out using a water-based lysis agent.

3. The method according to claim 1, wherein said culturing step is carried out at a temperature of 34-39 C.

4. The method according to claim 1, wherein said initial culturing step lasts 3-5 hours.

5. The method according to claim 1, wherein said culturing step lasts 45-75 minutes.

6. The method according to claim 1, wherein said initial culturing step and said culturing step are carried out at a temperature of 36-38 C.

7. The method according to claim 1, wherein said culturing step is carried out at a pH of 1.9-2.2.

8. The method according to claim 1, wherein said initial culturing step is carried out for 3-5 hours at a temperature of 36-38 C., said culturing step is carried out at a pH of 1.9-2.2, at a temperature of 36-38 C. and for 45-75 minutes and the yeast cells are yeast cells of the genus Yarrowia.

Description

(1) The present invention is further illustrated with the following figures and nonlimiting examples, from which further features, embodiments, aspects and advantages of the present invention can be seen.

(2) FIG. 1 shows the growth of yeast cells of the genera Yarrowia, Pichia and Saccharomyces over time at pH 2.

(3) FIG. 2 shows two representative sets with chip data, as obtained using the protocol described. The data there were obtained by analysis of the markers of human fibroblasts, which were treated with extract from Yarrowia cells, which were cultured normally at pH 5.4 (FIG. 2a) or were exposed to stress at pH 2 (FIG. 2b).

EXAMPLE 1

Growth of Various Yeast Strains Under Stress by Exposure to Medium with Low pH

(4) In order to investigate whether it is possible to stress yeast strains by exposure to medium with low pH, and to investigate which yeast strains possibly display suitable or advantageous growth properties at low pH, the growth of one representative from each of three genera, namely Saccharomyces, Pichia and Yarrowia, was investigated at pH=2.

(5) A preculture of the respective yeast strain to be investigated is cultured overnight in standard medium (per litre: 33 g glucose monohydrate, 0.88 g magnesium sulphate7H.sub.2O, 0.2 g calcium chloride2H.sub.2O, 4.83 ammonium chloride, 0.06 g sodium chloride, 1 g potassium dihydrogen phosphate, 0.059 g myo-inositol, 20 g MOPS, 0.3 mL trace element solution (per kg: 100 g 8M H.sub.2SO.sub.4, 50 g of citric acid monohydrate, 48 g FeSO.sub.47H.sub.2O, 16.7 g ZnSO.sub.47H.sub.2O, 2.5 g CuSO.sub.45H.sub.2O, 1.88 g MnSO.sub.4H.sub.2O, 2 g H.sub.3BO.sub.3, 2 g NaMoO.sub.42H.sub.2O, 0.5 g KI) and 0.3 mL vitamin solution (per litre: 5 g nicotinic acid, 5 g calcium-D-pantothenic acid, 5 g thiamine, 3.33 p-aminobenzoate, 0.5 g pyridoxine, 0.0167 biotin), the pH of which was adjusted to 2 with sulphuric acid. The optical density is monitored continuously by spectrophotometry.

(6) The results of the test are shown in FIG. 1. It can be seen that all the strains used show detectable growth in these conditions, but the representative of the genus Yarrowia at pH=2 under the same conditions shows faster growth than the comparative genera, and also reaches a higher optical density.

EXAMPLE 2

Comparative Characterization of the Action of Yeast Cells that were Stressed Either by Increased Temperature, Treatment with Hydrogen Peroxide or Exposure to Medium with Low pH, Using Chip Arrays, on Human Fibroblasts

(7) It was investigated whether stress by exposure to medium with low pH exerts the same effect on yeast cells as stress through increased temperature or treatment with hydrogen peroxide and whether differently stressed yeast cells or their extracts differ. Saccharomyces cerevisiae was selected as the model organism.

(8) Production of the Yeast Extract

(9) The preculture is set up as described above and adjusted to a pH of 5.4. The medium is inoculated in the shaking flask without baffles with addition of antifoaming agent. The temperature is 30 C. and the speed is 180 rpm (amplitude: 2.5 cm). The optical density is determined beforehand, it is measured continuously, and at OD>35 the respective stress situation is created, i.e. by increasing the temperature to 37 C., by adding sulphuric acid up to the desired pH or by adding hydrogen peroxide.

(10) Then the cells are harvested by centrifugation and frozen or lysed by aqueous lysis. For this, the extract is suspended and treated by means of a high-pressure homogenizer. The suspension is filtered off or centrifuged and the aqueous solution is taken as the end product.

(11) Test Principle and Execution of the Experiments with Chip Arrays:

(12) In these tests, the effect of yeast extract on the behaviour of human dermal fibroblasts is investigated, which are a recognized model for human skin. For this purpose, after the extract treatment the cells are lysed, their RNA is isolated, and the DNA obtained by reverse transcription is investigated by means of a chip for regulation of the expression of dermatologically relevant markers. Markers whose upregulation or downregulation indicates a positive effect on the morphology and physiology of the skin, especially markers of the Wnt-pathway and of the ECM, are used exclusively. The Wnt genes code for a large family of secreted proteins. To date, 19 Wnt proteins have been identified in humans. Both the various subgroups of the Wnts in individual species, and Wnts in comparison between different species sometimes show very large homologies. In humans, for example, the individual Wnt proteins show agreement from 27 to 83% (Miller, 2001). The central molecule of the canonical Wnt signal cascade is -catenin (Cat), whose stability is regulated by the Wnt proteins. Investigations of embryonic and postnatal skin have shown that Wnt proteins play an essential role in hair follicle development. In particular, the proteins Wnt10a, Wnt10b and Wnt5a are specifically expressed in early stages of hair follicle development (Reddy et al., 2001).

(13) Most of the known communication mechanisms are found in the skin. By the Wnt-pathway, basal cells keep the stem cell character preferentially. As cells age, there is accumulation of cellular and molecular damage, causing reduced regenerative capacity of tissues and organs. This reduced regenerative capacity is partly influenced by the change in self-renewal capacity and differentiation potential of tissue-specific adult stem cells from the fat tissue. It is known that the WNT signal pathway, a known signal pathway that regulates the self-renewal potential and the differentiation potential of many stem cells, therefore has an effect on the growth of stem cells. In addition, with Wnt and -catenin expression, epidermal proliferation, differentiation and migration is increased and therefore wound healing is accelerated.

(14) The physical properties of the connective tissue and the associated skin-firming are determined inter alia by the extracellular matrix, so that the cells are influenced in their differentiation, migration and proliferation. The matrix is formed by the cells located within it (fibroblasts, myofibroblasts, lipoblasts, osteoblasts and chondroblasts). The extracellular matrix is made up of macromolecules, which consist of glycosaminoglycans and fibrous proteins. Interaction of the lymphatic system, of the extracellular matrix and excessive fat-accumulation are involved in skin-firming. The total body fat is determined by the equilibrium between de-novo differentiation, growth and apoptosis (autolysis) of the adipocytes. Adipocyte differentiation is a complex process, which begins at birth and continues throughout life.

(15) A complete list of the markers used is shown in Table 1.

(16) TABLE-US-00001 TABLE 1 List of the markers taken into account for the chip experiments: Gene ID Gene symbol Description of the gene Chromosome 9 NAT1 N-acetyltransferase 1 (arylamine N- 8p23.1-p21.3 acetyltransferase) 10 NAT2 N-acetyltransferase 2 (arylamine N- 8p22 acetyltransferase) 30 ACAA1 acetyl-Coenzyme A acyltransferase 1 3p23-p22 33 ACADL acyl-Coenzyme A dehydrogenase, long chain 2q34-q35 34 ACADM acyl-Coenzyme A dehydrogenase, C-4 to C-12 1p31 straight chain 51 ACOX1 acyl-Coenzyme A oxidase 1, palmitoyl 17q24-q25 90 ACVR1 activin A receptor, type I 2q23-q24 91 ACVR1B activin A receptor, type IB 12q13 92 ACVR2A activin A receptor, type IIA 2q22.3 93 ACVR2B activin A receptor, type IIB 3p22 94 ACVRL1 activin A receptor type II-like 1 12q11-q14 154 ADRB2 adrenergic, beta-2-, receptor, surface 5q31-q32 176 ACAN aggrecan 15q26.1 268 AMH anti-Mullerian hormone 19p13.3 269 AMHR2 anti-Mullerian hormone receptor, type II 12q13 324 APC adenomatous polyposis coli 5q21-q22 325 APCS amyloid P component, serum 1q21-q23 335 APOA1 apolipoprotein A-I 11q23-q24 336 APOA2 apolipoprotein A-II 1q21-q23 345 APOC3 apolipoprotein C-III 11q23.1-q23.2 364 AQP7 aquaporin 7 9p13 387 RHOA ras homolog gene family, member A 3p21.3 595 CCND1 cyclin D1 11q13 633 BGN biglycan Xq28 650 BMP2 bone morphogenetic protein 2 20p12 652 BMP4 bone morphogenetic protein 4 14q22-q23 653 BMP5 bone morphogenetic protein 5 6p12.1 654 BMP6 bone morphogenetic protein 6 6p24-p23 655 BMP7 bone morphogenetic protein 7 20q13 656 BMP8B bone morphogenetic protein 8b 1p35-p32 657 BMPR1A bone morphogenetic protein receptor, type IA 10q22.3 658 BMPR1B bone morphogenetic protein receptor, type IB 4q22-q24 659 BMPR2 bone morphogenetic protein receptor, type II 2q33-q34 (serine/threonine kinase) 815 CAMK2A calcium/calmodulin-dependent protein kinase II 5q33.1 alpha 816 CAMK2B calcium/calmodulin-dependent protein kinase II 22q12 beta 817 CAMK2D calcium/calmodulin-dependent protein kinase II 4q26 delta 818 CAMK2G calcium/calmodulin-dependent protein kinase II 10q22 gamma 894 CCND2 cyclin D2 12p13 896 CCND3 cyclin D3 6p21 948 CD36 CD36 molecule (thrombospondin receptor) 7q11.2 960 CD44 CD44 molecule (Indian blood group) 11p13 961 CD47 CD47 molecule 3q13.1-q13.2 1030 CDKN2B cyclin-dependent kinase inhibitor 2B (p15, 9p21 inhibits CDK4) 1101 CHAD chondroadherin 17q21.33 1277 COL1A1 collagen, type I, alpha 1 17q21.33 1278 COL1A2 collagen, type I, alpha 2 7q22.1 1280 COL2A1 collagen, type II, alpha 1 12q13.11 1281 COL3A1 collagen, type III, alpha 1 2q31 1282 COL4A1 collagen, type IV, alpha 1 13q34 1284 COL4A2 collagen, type IV, alpha 2 13q34 1286 COL4A4 collagen, type IV, alpha 4 2q35-q37 1288 COL4A6 collagen, type IV, alpha 6 Xq22 1289 COL5A1 collagen, type V, alpha 1 9q34.2-q34.3 1290 COL5A2 collagen, type V, alpha 2 2q14-q32 1291 COL6A1 collagen, type VI, alpha 1 21q22.3 1292 COL6A2 collagen, type VI, alpha 2 21q22.3 1293 COL6A3 collagen, type VI, alpha 3 2q37 1301 COL11A1 collagen, type XI, alpha 1 1p21 1311 COMP cartilage oligomeric matrix protein 19p13.1 1374 CPT1A carnitine palmitoyltransferase 1A (liver) 11q13.1-q13.2 1375 CPT1B carnitine palmitoyltransferase 1B (muscle) 22q13.33 1376 CPT2 carnitine palmitoyltransferase II 1p32 1387 CREBBP CREB binding protein 16p13.3 1452 CSNK1A1 casein kinase 1, alpha 1 5q32 1454 CSNK1E casein kinase 1, epsilon 22q13.1 1457 CSNK2A1 casein kinase 2, alpha 1 polypeptide 20p13 1459 CSNK2A2 casein kinase 2, alpha prime polypeptide 16q21 1460 CSNK2B casein kinase 2, beta polypeptide 6p21-p12 1462 VCAN versican 5q14.3 1474 CST6 cystatin E/M 11q13 1487 CTBP1 C-terminal binding protein 1 4p16 1488 CTBP2 C-terminal binding protein 2 10q26.13 1499 CTNNB1 catenin (cadherin-associated protein), beta 1, 3p21 88 kDa 1509 CTSD cathepsin D 11p15.5 1512 CTSH cathepsin H 15q24-q25 1520 CTSS cathepsin S 1q21 1522 CTSZ cathepsin Z 20q13 1544 CYP1A2 cytochrome P450, family 1, subfamily A, 15q24.1 polypeptide 2 1548 CYP2A6 cytochrome P450, family 2, subfamily A, 19q13.2 polypeptide 6 1549 CYP2A7 cytochrome P450, family 2, subfamily A, 19q13.2 polypeptide 7 1553 CYP2A13 cytochrome P450, family 2, subfamily A, 19q13.2 polypeptide 13 1579 CYP4A11 cytochrome P450, family 4, subfamily A, 1p33 polypeptide 11 1581 CYP7A1 cytochrome P450, family 7, subfamily A, 8q11-q12 polypeptide 1 1582 CYP8B1 cytochrome P450, family 8, subfamily B, 3p22-p21.3 polypeptide 1 1593 CYP27A1 cytochrome P450, family 27, subfamily A, 2q33-qter polypeptide 1 1605 DAG1 dystroglycan 1 (dystrophin-associated 3p21 glycoprotein 1) 1622 DBI diazepam binding inhibitor (GABA receptor 2q12-q21 modulator, acyl-Coenzyme A binding protein) 1634 DCN decorin 12q21.33 1634 DCN decorin 12q21.33 1805 DPT dermatopontin 1q12-q23 1855 DVL1 dishevelled, dsh homolog 1 (Drosophila) 1p36 1856 DVL2 dishevelled, dsh homolog 2 (Drosophila) 17p13.2 1857 DVL3 dishevelled, dsh homolog 3 (Drosophila) 3q27 1874 E2F4 E2F transcription factor 4, p107/p130-binding 16q21-q22 1875 E2F5 E2F transcription factor 5, p130-binding 8q21.2 1962 EHHADH enoyl-Coenzyme A, hydratase/3-hydroxyacyl 3q26.3-q28 Coenzyme A dehydrogenase 2006 ELN elastin 7q11.23 2033 EP300 E1A binding protein p300 22q13.2 2033 EP300 E1A binding protein p300 22q13.2 2131 EXT1 exostoses (multiple) 1 8q24.11-q24.13 2132 EXT2 exostoses (multiple) 2 11p12-p11 2134 EXTL1 exostoses (multiple)-like 1 1p36.1 2135 EXTL2 exostoses (multiple)-like 2 1p21 2137 EXTL3 exostoses (multiple)-like 3 8p21 2167 FABP4 fatty acid binding protein 4, adipocyte 8q21 2168 FABP1 fatty acid binding protein 1, liver 2p11 2169 FABP2 fatty acid binding protein 2, intestinal 4q28-q31 2170 FABP3 fatty acid binding protein 3, muscle and heart 1p33-p32 (mammary-derived growth inhibitor) 2171 FABP5 fatty acid binding protein 5 (psoriasisassociated) 8q21.13 2172 FABP6 fatty acid binding protein 6, ileal 5q33.3-q34 2173 FABP7 fatty acid binding protein 7, brain 6q22-q23 2180 ACSL1 acyl-CoA synthetase long-chain family member 1 4q34-q35 2181 ACSL3 acyl-CoA synthetase long-chain family member 3 2q34-q35 2182 ACSL4 acyl-CoA synthetase long-chain family member 4 Xq22.3-q23 2331 FMOD fibromodulin 1q32 2335 FN1 fibronectin 1 2q34 2530 FUT8 fucosyltransferase 8 (alpha (1,6) 14q24.3 fucosyltransferase) 2535 FZD2 frizzled homolog 2 (Drosophila) 17q21.1 2683 B4GALT1 UDP-Gal:betaGlcNAc beta 1,4- 9p13 galactosyltransferase, polypeptide 1 2710 GK glycerol kinase Xp21.3 2712 GK2 glycerol kinase 2 4q13 2713 GK3P glycerol kinase 3 pseudogene 4q32.1 2811 GP1BA glycoprotein Ib (platelet), alpha polypeptide 17pter-p12 2812 GP1BB glycoprotein Ib (platelet), beta polypeptide 22q11.21q11.23 2814 GP5 glycoprotein V (platelet) 3q29 2815 GP9 glycoprotein IX (platelet) 3q21.3 2817 GPC1 glypican 1 2q35-q37 2932 GSK3B glycogen synthase kinase 3 beta 3q13.3 3026 HABP2 hyaluronan binding protein 2 10q25.3 3036 HAS1 hyaluronan synthase 1 19q13.4 3037 HAS2 hyaluronan synthase 2 8q24.12 3038 HAS3 hyaluronan synthase 3 16q22.1 3158 HMGCS2 3-hydroxy-3-methylglutaryl-Coenzyme A 1p13-p12 synthase 2 (mitochondrial) 3161 HMMR hyaluronan-mediated motility receptor 5q33.2-qter (RHAMM) 3339 HSPG2 heparan sulfate proteoglycan 2 1p36.1-p34 3339 HSPG2 heparan sulfate proteoglycan 2 1p36.1-p34 3340 NDST1 N-deacetylase/N-sulfotransferase (heparan 5q33.1 glucosaminyl) 1 3371 TNC tenascin C 9q33 3381 IBSP integrin-binding sialoprotein 4q21-q25 3383 ICAM1 intercellular adhesion molecule 1 19p13.3-p13.2 3397 ID1 inhibitor of DNA binding 1, dominant negative 20q11 helix-loop-helix protein 3398 ID2 inhibitor of DNA binding 2, dominant negative 2p25 helix-loop-helix protein 3399 ID3 inhibitor of DNA binding 3, dominant negative 1p36.13-p36.12 helix-loop-helix protein 3400 ID4 inhibitor of DNA binding 4, dominant negative 6p22-p21 helix-loop-helix protein 3458 IFNG interferon, gamma 12q14 3569 IL6 interleukin 6 (interferon, beta 2) 7p21 3611 ILK integrin-linked kinase 11p15.5-p15.4 3624 INHBA inhibin, beta A 7p15-p13 3625 INHBB inhibin, beta B 2cen-q13 3626 INHBC inhibin, beta C 12q13.1 3655 ITGA6 integrin, alpha 6 2q31.1 3672 ITGA1 integrin, alpha 1 5q11.2 3673 ITGA2 integrin, alpha 2 (CD49B, alpha 2 subunit of 5q23-q31 VLA-2 receptor) 3674 ITGA2B integrin, alpha 2b (platelet glycoprotein IIb of 17q21.32 IIb/IIIa complex, antigen CD41) 3675 ITGA3 integrin, alpha 3 (antigen CD49C, alpha 3 subunit 17q21.33 of VLA-3 receptor) 3676 ITGA4 integrin, alpha 4 (antigen CD49D, alpha 4 subunit 2q31.3 of VLA-4 receptor) 3678 ITGA5 integrin, alpha 5 (fibronectin receptor, alpha 12q11-q13 polypeptide) 3679 ITGA7 integrin, alpha 7 12q13 3680 ITGA9 integrin, alpha 9 3p21.3 3685 ITGAV integrin, alpha V (vitronectin receptor, alpha 2q31-q32 polypeptide, antigen CD51) 3688 ITGB1 integrin, beta 1 (fibronectin receptor, beta 10p11.2 polypeptide, antigen CD29 includes MDF2, MSK12) 3690 ITGB3 integrin, beta 3 (platelet glycoprotein IIIa, antigen 17q21.32 CD61) 3691 ITGB4 integrin, beta 4 17q25 3693 ITGB5 integrin, beta 5 3q21.2 3694 ITGB6 integrin, beta 6 2q24.2 3695 ITGB7 integrin, beta 7 12q13.13 3696 ITGB8 integrin, beta 8 7p15.3 3725 JUN jun oncogene 1p32-p31 3908 LAMA2 laminin, alpha 2 6q22-q23 3909 LAMA3 laminin, alpha 3 18q11.2 3910 LAMA4 laminin, alpha 4 6q21 3911 LAMA5 laminin, alpha 5 20q13.2-q13.3 3912 LAMB1 laminin, beta 1 7q22 3913 LAMB2 laminin, beta 2 (laminin S) 3p21 3914 LAMB3 laminin, beta 3 1q32 3915 LAMC1 laminin, gamma 1 (formerly LAMB2) 1q31 3918 LAMC2 laminin, gamma 2 1q25-q31 4023 LPL lipoprotein lipase 8p22 4040 LRP6 low density lipoprotein receptor-related protein 6 12p11-p13 4041 LRP5 low density lipoprotein receptor-related protein 5 11q13.4 4052 LTBP1 latent transforming growth factor beta binding 2p22-p21 protein 1 4053 LTBP2 latent transforming growth factor beta binding 14q24 protein 2 4054 LTBP3 latent transforming growth factor beta binding 11q12 protein 3 4060 LUM lumican 12q21.3-q22 4086 SMAD1 SMAD family member 1 4q31 4087 SMAD2 SMAD family member 2 18q21.1 4088 SMAD3 SMAD family member 3 15q22.33 4089 SMAD4 SMAD family member 4 18q21.1 4089 SMAD4 SMAD family member 4 18q21.1 4090 SMAD5 SMAD family member 5 5q31 4091 SMAD6 SMAD family member 6 15q21-q22 4092 SMAD7 SMAD family member 7 18q21.1 4093 SMAD9 SMAD family member 9 13q12-q14 4146 MATN1 matrilin 1, cartilage matrix protein 1p35 4147 MATN2 matrilin 2 8q22 4148 MATN3 matrilin 3 2p24-p23 4166 CHST6 carbohydrate (N-acetylglucosamine 6-O) 16q22 sulfotransferase 6 4199 ME1 malic enzyme 1, NADP(+)-dependent, cytosolic 6q12 4237 MFAP2 microfibrillar-associated protein 2 1p36.1-p35 4312 MMP1 matrix metallopeptidase 1 (interstitial 11q22.3 collagenase) 4313 MMP2 matrix metallopeptidase 2 (gelatinase A, 72 kDa 16q13-q21 gelatinase, 72 kDa type IV collagenase) 4314 MMP3 matrix metallopeptidase 3 (stromelysin 1, 11q22.3 progelatinase) 4316 MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 11q21-q22 4316 MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 11q21-q22 4319 MMP10 matrix metallopeptidase 10 (stromelysin 2) 11q22.3 4320 MMP11 matrix metallopeptidase 11 (stromelysin 3) 22q11.2 4321 MMP12 matrix metallopeptidase 12 (macrophage elastase) 11q22.3 4322 MMP13 matrix metallopeptidase 13 (collagenase 3) 11q22.3 4323 MMP14 matrix metallopeptidase 14 (membraneinserted) 14q11-q12 4324 MMP15 matrix metallopeptidase 15 (membraneinserted) 16q13-q21 4609 MYC v-myc myelocytomatosis viral oncogene homolog 8q24.21 (avian) 4772 NFATC1 nuclear factor of activated T-cells, cytoplasmic, 18q23 calcineurin-dependent 1 4773 NFATC2 nuclear factor of activated T-cells, cytoplasmic, 20q13.2-q13.3 calcineurin-dependent 2 4775 NFATC3 nuclear factor of activated T-cells, cytoplasmic, 16q22.2 calcineurin-dependent 3 4776 NFATC4 nuclear factor of activated T-cells, cytoplasmic, 14q11.2 calcineurin-dependent 4 4838 NODAL nodal homolog (mouse) 10q22.1 4973 OLR1 oxidized low density lipoprotein (lectin-like) 12p13.2-p12.3 receptor 1 4982 TNFRSF11B tumor necrosis factor receptor superfamily, 8q24 member 11b 5054 SERPINE1 serpin peptidase inhibitor, clade E (nexin, 7q21.3-q22 plasminogen activator inhibitor type 1), member 1 5055 SERPINB2 serpin peptidase inhibitor, clade B (ovalbumin), 18q21.3 member 2 5105 PCK1 phosphoenolpyruvate carboxykinase 1 (soluble) 20q13.31 5106 PCK2 phosphoenolpyruvate carboxykinase 2 14q12 (mitochondrial) 5170 PDPK1 3-phosphoinositide dependent protein kinase-1 16p13.3 5308 PITX2 paired-like homeodomain 2 4q25-q27 5327 PLAT plasminogen activator, tissue 8p12 5330 PLCB2 phospholipase C, beta 2 15q15 5331 PLCB3 phospholipase C, beta 3 11q13 (phosphatidylinositolspecific) 5332 PLCB4 phospholipase C, beta 4 20p12 5346 PLIN perilipin 15q26 5360 PLTP phospholipid transfer protein 20q12-q13.1 5465 PPARA peroxisome proliferator-activated receptor alpha 22q12-q13.1 5467 PPARD peroxisome proliferator-activated receptor delta 6p21.2-p21.1 5468 PPARG peroxisome proliferator-activated receptor gamma 3p25 5515 PPP2CA protein phosphatase 2 (formerly 2A), catalytic 5q31.1 subunit, alpha isoform 5516 PPP2CB protein phosphatase 2 (formerly 2A), catalytic 8p12 subunit, beta isoform 5518 PPP2R1A protein phosphatase 2 (formerly 2A), regulatory 19q13.33 subunit A, alpha isoform 5519 PPP2R1B protein phosphatase 2 (formerly 2A), regulatory 11q23.2 subunit A, beta isoform 5525 PPP2R5A protein phosphatase 2, regulatory subunit B, 1q32.2-q32.3 alpha isoform 5526 PPP2R5B protein phosphatase 2, regulatory subunit B, beta 11q12-q13 isoform 5527 PPP2R5C protein phosphatase 2, regulatory subunit B, 14q32 gamma isoform 5528 PPP2R5D protein phosphatase 2, regulatory subunit B, delta 6p21.1 isoform 5529 PPP2R5E protein phosphatase 2, regulatory subunit B, 14q23.1 epsilon isoform 5530 PPP3CA protein phosphatase 3 (formerly 2B), catalytic 4q21-q24 subunit, alpha isoform 5532 PPP3CB protein phosphatase 3 (formerly 2B), catalytic 10q21-q22 subunit, beta isoform 5533 PPP3CC protein phosphatase 3 (formerly 2B), catalytic 8p21.3 subunit, gamma isoform 5534 PPP3R1 protein phosphatase 3 (formerly 2B), regulatory 2p15 subunit B, alpha isoform 5535 PPP3R2 protein phosphatase 3 (formerly 2B), regulatory 9q31.1 subunit B, beta isoform 5566 PRKACA protein kinase, cAMP-dependent, catalytic, alpha 19p13.1 5567 PRKACB protein kinase, cAMP-dependent, catalytic, beta 1p36.1 5568 PRKACG protein kinase, cAMP-dependent, catalytic, 9q13 gamma 5578 PRKCA protein kinase C, alpha 17q22-q23.2 5579 PRKCB protein kinase C, beta 16p11.2 5582 PRKCG protein kinase C, gamma 19q13.4 5594 MAPK1 mitogen-activated protein kinase 1 22q11.2 5595 MAPK3 mitogen-activated protein kinase 3 16p11.2 5599 MAPK8 mitogen-activated protein kinase 8 10q11.22 5601 MAPK9 mitogen-activated protein kinase 9 5q35 5602 MAPK10 mitogen-activated protein kinase 10 4q22.1-q23 5613 PRKX protein kinase, X-linked Xp22.3 5616 PRKY protein kinase, Y-linked Yp11.2 5649 RELN reelin 7q22 5663 PSEN1 presenilin 1 14q24.3 5879 RAC1 ras-related C3 botulinum toxin substrate 1 (rho 7p22 family, small GTP binding protein Rac1) 5880 RAC2 ras-related C3 botulinum toxin substrate 2 (rho 22q13.1 family, small GTP binding protein Rac2) 5881 RAC3 ras-related C3 botulinum toxin substrate 3 (rho 17q25.3 family, small GTP binding protein Rac3) 5933 RBL1 retinoblastoma-like 1 (p107) 20q11.2 5934 RBL2 retinoblastoma-like 2 (p130) 16q12.2 5950 RBP4 retinol binding protein 4, plasma 10q23-q24 6093 ROCK1 Rho-associated, coiled-coil containing protein 18q11.1 kinase 1 6198 RPS6KB1 ribosomal protein S6 kinase, 70 kDa, polypeptide 1 17q23.1 6199 RPS6KB2 ribosomal protein S6 kinase, 70 kDa, polypeptide 2 11q13.1 6256 RXRA retinoid X receptor, alpha 9q34.3 6257 RXRB retinoid X receptor, beta 6p21.3 6258 RXRG retinoid X receptor, gamma 1q22-q23 6319 SCD stearoyl-CoA desaturase (delta-9-desaturase) 10q24.31 6342 SCP2 sterol carrier protein 2 1p32 6382 SDC1 syndecan 1 2p24.1 6383 SDC2 syndecan 2 8q22-q23 6385 SDC4 syndecan 4 20q12 6422 SFRP1 secreted frizzled-related protein 1 8p12-p11.1 6423 SFRP2 secreted frizzled-related protein 2 4q31.3 6424 SFRP4 secreted frizzled-related protein 4 7p14.1 6425 SFRP5 secreted frizzled-related protein 5 10q24.1 6477 SIAH1 seven in absentia homolog 1 (Drosophila) 16q12 6482 ST3GAL1 ST3 beta-galactoside alpha-2,3sialyltransferase 1 8q24.22 6483 ST3GAL2 ST3 beta-galactoside alpha-2,3sialyltransferase 2 16q22.1 6487 ST3GAL3 ST3 beta-galactoside alpha-2,3sialyltransferase 3 1p34.1 6500 SKP1 S-phase kinase-associated protein 1 5q31 6667 SP1 Sp1 transcription factor 12q13.1 6696 SPP1 secreted phosphoprotein 1 4q21-q25 6885 MAP3K7 mitogen-activated protein kinase kinase kinase 7 6q16.1-q16.3 6907 TBL1X transducin (beta)-like 1X-linked Xp22.3 6932 TCF7 transcription factor 7 (T-cell specific, HMG-box) 5q31.1 6934 TCF7L2 transcription factor 7-like 2 (T-cell specific, 10q25.3 HMG-box) 7027 TFDP1 transcription factor Dp-1 13q34 7040 TGFB1 transforming growth factor, beta 1 19q13.2 7042 TGFB2 transforming growth factor, beta 2 1q41 7043 TGFB3 transforming growth factor, beta 3 14q24 7044 LEFTY2 left-right determination factor 2 1q42.1 7046 TGFBR1 transforming growth factor, beta receptor 1 9q22 7048 TGFBR2 transforming growth factor, beta receptor II 3p22 (70/80 kDa) 7057 THBS1 thrombospondin 1 15q15 7058 THBS2 thrombospondin 2 6q27 7059 THBS3 thrombospondin 3 1q21 7060 THBS4 thrombospondin 4 5q13 7076 TIMP1 TIMP metallopeptidase inhibitor 1 Xp11.3-p11.23 7077 TIMP2 TIMP metallopeptidase inhibitor 2 17q25 7124 TNF tumor necrosis factor (TNF superfamily, member 6p21.3 2) 7143 TNR tenascin R (restrictin, janusin) 1q24 7146 TNXA tenascin XA pseudogene 6p21.3 7148 TNXB tenascin XB 6p21.3 7157 TP53 tumor protein p53 17p13.1 7316 UBC ubiquitin C 12q24.3 7350 UCP1 uncoupling protein 1 (mitochondrial, proton 4q28-q31 carrier) 7448 VTN vitronectin 17q11 7450 VWF von Willebrand factor 12p13.3 7471 WNT1 wingless-type MMTV integration site family, 12q13 member 1 7472 WNT2 wingless-type MMTV integration site family 7q31.2 member 2 7473 WNT3 wingless-type MMTV integration site family, 17q21 member 3 7474 WNT5A wingless-type MMTV integration site family, 3p21-p14 member 5A 7475 WNT6 wingless-type MMTV integration site family, 2q35 member 6 7476 WNT7A wingless-type MMTV integration site family, 3p25 member 7A 7477 WNT7B wingless-type MMTV integration site family, 22q13 member 7B 7478 WNT8A wingless-type MMTV integration site family, 5q31 member 8A 7479 WNT8B wingless-type MMTV integration site family, 10q24 member 8B 7480 WNT10B wingless-type MMTV integration site family, 12q13 member 10B 7481 WNT11 wingless-type MMTV integration site family, 11q13.5 member 11 7482 WNT2B wingless-type MMTV integration site family, 1p13 member 2B 7483 WNT9A wingless-type MMTV integration site family, 1q42 member 9A 7484 WNT9B wingless-type MMTV integration site family, 17q21 member 9B 7498 XDH xanthine dehydrogenase 2p23.1 7839 LSL Leptin, serum levels of 2p21 7855 FZD5 frizzled homolog 5 (Drosophila) 2q33-q34 7976 FZD3 frizzled homolog 3 (Drosophila) 8p21 8061 FOSL1 FOS-like antigen 1 11q13 8200 GDF5 growth differentiation factor 5 20q11.2 8215 DVL1L1 dishevelled, dsh homolog 1 (Drosophila)-like 1 22q11.21 8309 ACOX2 acyl-Coenzyme A oxidase 2, branched chain 3p14.3 8310 ACOX3 acyl-Coenzyme A oxidase 3, pristanoyl 4p15.3 8312 AXIN1 axin 1 16p13.3 8313 AXIN2 axin 2 17q23-q24 8321 FZD1 frizzled homolog 1 (Drosophila) 7q21 8322 FZD4 frizzled homolog 4 (Drosophila) 11q14.2 8323 FZD6 frizzled homolog 6 (Drosophila) 8q22.3-q23.1 8324 FZD7 frizzled homolog 7 (Drosophila) 2q33 8325 FZD8 frizzled homolog 8 (Drosophila) 10p11.21 8326 FZD9 frizzled homolog 9 (Drosophila) 7q11.23 8425 LTBP4 latent transforming growth factor beta binding 19q13.1-q13.2 protein 4 8454 CUL1 cullin 1 7q36.1 8509 NDST2 N-deacetylase/N-sulfotransferase (heparan 10q22 glucosaminyl) 2 8515 ITGA10 integrin, alpha 10 1q21 8516 ITGA8 integrin, alpha 8 10p13 8534 CHST1 carbohydrate (keratan sulfate Gal-6) 11p11.2-p11.1 sulfotransferase 1 8607 RUVBL1 RuvB-like 1 (E. coli) 3q21 8646 CHRD chordin 3q27 8649 MAPKSP1 MAPK scaffold protein 1 4q23 8702 B4GALT4 UDP-Gal:betaGlcNAc beta 1,4- 3q13.3 galactosyltransferase, polypeptide 4 8703 B4GALT3 UDP-Gal:betaGlcNAc beta 1,4- 1q21-q23 galactosyltransferase, polypeptide 3 8704 B4GALT2 UDP-Gal:betaGlcNAc beta 1,4- 1p34-p33 galactosyltransferase, polypeptide 2 8759 ADAM1 ADAM metallopeptidase domain 1 (pseudogene) 12q24.13 8785 MATN4 matrilin 4 20q13.1-q13.2 8945 BTRC beta-transducin repeat containing 10q24.32 9241 NOG noggin 17q21-q22 9348 NDST3 N-deacetylase/N-sulfotransferase (heparan 4q26 glucosaminyl) 3 9350 CER1 cerberus 1, cysteine knot superfamily, homolog 9p23-p22 (Xenopus laevis) 9370 ADIPOQ adiponectin, C1Q and collagen domain containing 3q27 9372 ZFYVE9 zinc finger, FYVE domain containing 9 1p32.3 9394 HS6ST1 heparan sulfate 6-O-sulfotransferase 1 2q21 9415 FADS2 fatty acid desaturase 2 11q12-q13.1 9435 CHST2 carbohydrate (N-acetylglucosamine-6-O) 3q24 sulfotransferase 2 9469 CHST3 carbohydrate (chondroitin 6) sulfotransferase 3 10q22.1 9475 ROCK2 Rho-associated, coiled-coil containing protein 2p24 kinase 2 9653 HS2ST1 heparan sulfate 2-O-sulfotransferase 1 1p31.1-p22.1 9672 SDC3 syndecan 3 1pter-p22.3 9765 ZFYVE16 zinc finger, FYVE domain containing 16 5q14 9899 SV2B synaptic vesicle glycoprotein 2B 15q26.1 9900 SV2A synaptic vesicle glycoprotein 2A 1q21.2 9953 HS3ST3B1 heparan sulfate (glucosamine) 3- 17p12-p11.2 Osulfotransferase 3B1 9955 HS3ST3A1 heparan sulfate (glucosamine) 3- 17p12-p11.2 Osulfotransferase 3A1 9956 HS3ST2 heparan sulfate (glucosamine) 3- 16p12 Osulfotransferase 2 9957 HS3ST1 heparan sulfate (glucosamine) 3- 4p16 Osulfotransferase 1 9978 RBX1 ring-box 1 22q13.2 10023 FRAT1 frequently rearranged in advanced T-cell 10q24.1 lymphomas 10062 NR1H3 nuclear receptor subfamily 1, group H, member 3 11p11.2 10090 UST uronyl-2-sulfotransferase 6q25.1 10135 NAMPT nicotinamide phosphoribosyltransferase 7q22.2 10164 CHST4 carbohydrate (N-acetylglucosamine 6-O) 16q22.3 sulfotransferase 4 10297 APC2 adenomatosis polyposis coli 2 19p13.3 10319 LAMC3 laminin, gamma 3 9q31-q34 10468 FST follistatin 5q11.2 10580 SORBS1 sorbin and SH3 domain containing 1 10q23.3-q24.1 10637 LEFTY1 left-right determination factor 1 1q42.1 10678 B3GNT2 UDP-GlcNAc:betaGal beta-1,3- 2p15 Nacetylglucosaminyltransferase 2 10725 NFAT5 nuclear factor of activated T-cells 5, 16q22.1 tonicityresponsive 10998 SLC27A5 solute carrier family 27 (fatty acid transporter), 19q13.43 member 5 10999 SLC27A4 solute carrier family 27 (fatty acid transporter), 9q34.11 member 4 11001 SLC27A2 solute carrier family 27 (fatty acid transporter), 15q21.2 member 2 11041 B3GNT1 UDP-GlcNAc:betaGal beta-1,3- 11q13.1 Nacetylglucosaminyltransferase 1 11197 WIF1 WNT inhibitory factor 1 12q14.3 11211 FZD10 frizzled homolog 10 (Drosophila) 12q24.33 11285 B4GALT7 xylosylprotein beta 1,4-galactosyltransferase, 5q35.2-q35.3 polypeptide 7 (galactosyltransferase I) 22798 LAMB4 laminin, beta 4 7q22-q31.2 22801 ITGA11 integrin, alpha 11 15q23 22856 CHSY1 chondroitin sulfate synthase 1 15q26.3 22927 HABP4 hyaluronan binding protein 4 9q22.3-q31 22943 DKK1 dickkopf homolog 1 (Xenopus laevis) 10q11.2 22987 SV2C synaptic vesicle glycoprotein 2C 5q13.3 23002 DAAM1 dishevelled associated activator of morphogenesis 1 14q23.1 23236 PLCB1 phospholipase C, beta 1 20p12 (phosphoinositidespecific) 23291 FBXW11 F-box and WD repeat domain containing 11 5q35.1 23305 ACSL6 acyl-CoA synthetase long-chain family member 6 5q31 23401 FRAT2 frequently rearranged in advanced T-cell 10q24.1 lymphomas 2 23500 DAAM2 dishevelled associated activator of morphogenesis 2 6p21.2 26035 GLCE glucuronic acid epimerase 15q23 26229 B3GAT3 beta-1,3-glucuronyltransferase 3 11q12.3 (glucuronosyltransferase I) 27087 B3GAT1 beta-1,3-glucuronyltransferase 1 11q25 (glucuronosyltransferase P) 27101 CACYBP calcyclin binding protein 1q24-q25 27121 DKK4 dickkopf homolog 4 (Xenopus laevis) 8p11.2-p11.1 27123 DKK2 dickkopf homolog 2 (Xenopus laevis) 4q25 28965 SLC27A6 solute carrier family 27 (fatty acid transporter), 5q23.3 member 6 29940 DSE dermatan sulfate epimerase 6q22 50509 COL5A3 collagen, type V, alpha 3 19p13.2 50515 CHST11 carbohydrate (chondroitin 4) sulfotransferase 11 12q 51129 ANGPTL4 angiopoietin-like 4 19p13.3 51176 LEF1 lymphoid enhancer-binding factor 1 4q23-q25 51206 GP6 glycoprotein VI (platelet) 19q13.4 51384 WNT16 wingless-type MMTV integration site family, 7q31 member 16 51701 NLK nemo-like kinase 17q11.2 51703 ACSL5 acyl-CoA synthetase long-chain family member 5 10q25.1-q25.2 54361 WNT4 wingless-type MMTV integration site family, 1p36.23-p35.1 member 4 55454 CSGALNACT2 chondroitin sulfate 10q11.21 Nacetylgalactosaminyltransferase 2 55501 CHST12 carbohydrate (chondroitin 4) sulfotransferase 12 7p22 55790 CSGALNACT1 chondroitin sulfate 8p21.3 Nacetylgalactosaminyltransferase 1 56548 CHST7 carbohydrate (N-acetylglucosamine 6-O) Xp11.23 sulfotransferase 7 56729 RETN resistin 19p13.2 56998 CTNNBIP1 catenin, beta interacting protein 1 1p36.22 57154 SMURF1 SMAD specific E3 ubiquitin protein ligase 1 7q22.1 57216 VANGL2 vang-like 2 (van gogh, Drosophila) 1q22-q23 57680 CHD8 chromodomain helicase DNA binding protein 8 14q11.2 58496 LY6G5B lymphocyte antigen 6 complex, locus G5B 6p21.3 59343 SENP2 SUMO1/sentrin/SMT3 specific peptidase 2 3q27.2 63923 TNN tenascin N 1q23-q24 64131 XYLT1 xylosyltransferase I 16p12.3 64132 XYLT2 xylosyltransferase II 17q21.3-q22 64321 SOX17 SRY (sex determining region Y)-box 17 8q11.23 64579 NDST4 N-deacetylase/N-sulfotransferase (heparan 4q25-q26 glucosaminyl) 4 64750 SMURF2 SMAD specific E3 ubiquitin protein ligase 2 17q22-q23 64840 PORCN porcupine homolog (Drosophila) Xp11.23 79586 CHPF chondroitin polymerizing factor 2q35 79718 TBL1XR1 transducin (beta)-like 1 X-linked receptor 1 3q26.32 79966 SCD5 stearoyl-CoA desaturase 5 4q21.22 80070 ADAMTS20 ADAM metallopeptidase with thrombospondin 12q12 type 1 motif, 20 80319 CXXC4 CXXC finger 4 4q22-q24 80326 WNT10A wingless-type MMTV integration site family, 2q35 member 10A 81029 WNT5B wingless-type MMTV integration site family, 12p13.3 member 5B 81839 VANGL1 vang-like 1 (van gogh, Drosophila) 1p11-p13.1 83439 TCF7L1 transcription factor 7-like 1 (T-cell specific, 2p11.2 HMG-box) 83729 INHBE inhibin, beta E 12q13.3 85407 NKD1 naked cuticle homolog 1 (Drosophila) 16q12 85409 NKD2 naked cuticle homolog 2 (Drosophila) 5p15.3 89780 WNT3A wingless-type MMTV integration site family, 1q42 member 3A 90161 HS6ST2 heparan sulfate 6-O-sulfotransferase 2 Xq26.2 90665 TBL1Y transducin (beta)-like 1Y-linked Yp11.2 93010 B3GNT7 UDP-GlcNAc:betaGal beta-1,3- 2q37.1 Nacetylglucosaminyltransferase 7 113189 CHST14 carbohydrate (N-acetylgalactosamine 4-0) 15q15.1 sulfotransferase 14 116519 APOA5 apolipoprotein A-V 11q23 122011 CSNK1A1L casein kinase 1, alpha 1-like 13q13.3 126129 CPT1C carnitine palmitoyltransferase 1C 19q13.33 126792 B3GALT6 UDP-Gal:betaGal beta 1,3-galactosyltransferase 1p36.33 polypeptide 6 130399 ACVR1C activin A receptor, type IC 2q24.1 135152 B3GAT2 beta-1,3-glucuronyltransferase 2 6q13 (glucuronosyltransferase S) 144165 PRICKLE1 prickle homolog 1 (Drosophila) 12q12 151449 GDF7 growth differentiation factor 7 2p24.1 166012 CHST13 carbohydrate (chondroitin 4) sulfotransferase 13 3q21.3 166336 PRICKLE2 prickle homolog 2 (Drosophila) 3p14.1 222537 HS3ST5 heparan sulfate (glucosamine) 3- 6q22.31 Osulfotransferase 5 266722 HS6ST3 heparan sulfate 6-O-sulfotransferase 3 13q32.1 283106 CSNK2A1P casein kinase 2, alpha 1 polypeptide pseudogene 11p15.3 284217 LAMA1 laminin, alpha 1 18p11.31 284541 CYP4A22 cytochrome P450, family 4, subfamily A, 1p33 polypeptide 22 337876 CHSY3 chondroitin sulfate synthase 3 5q23.3 353500 BMP8A bone morphogenetic protein 8a 1p34.2 375790 AGRN agrin 1p36.33 376497 SLC27A1 solute carrier family 27 (fatty acid transporter), 19p13.11 member 1 392255 GDF6 growth differentiation factor 6 8q22.1 642956 FABP5L9 fatty acid binding protein 5-like 9 15q25.3 728641 FABP5L7 fatty acid binding protein 5-like 7 11q12.1 728729 FABP5L8 fatty acid binding protein 5-like 8 15q25.2 729163 FABP5L2 fatty acid binding protein 5-like 2 13q14.3

(17) Human Primary Cells

(18) Human dermal fibroblasts or human subcutaneous preadipocytes in the form of in vitro cell culture systems are used as biological test systems. Both cell types are commercially available (the company Lifeline or the company CellApplications).

(19) Human dermal fibroblasts are as a rule obtained from the dermis of neonatal donors (phimoses) or from the skin of adult donors. They are always noninfectious donor materials, which were isolated by the respective supplier from the in vivo tissue dressing and were expanded singly in a corresponding cell culture medium. Cryopreserved human dermal fibroblasts can, after seeding in suitable cell culture media, go through up to 16 doubling cycles without impairment of their typical physiology and morphology. Human dermal fibroblasts occur in all connective tissue structures and are a well-characterized in vitro test system. They release extracellular matrix proteins in vitro and are suitable in in vitro research in particular also for investigating fibroblast growth, investigation of the differentiation of fibroblasts and also in particular for investigating collagen metabolism in the context of wound healing. It has been shown in various ways that human dermal fibroblasts are suitable for the population of in vitro reconstituted, three-dimensional dermis equivalents and in vitro reconstituted human skin models.

(20) Human preadipocytes (HPAd) are usually obtained from human fat tissue of noninfectious donors. Whereas human adipocytes are not suitable for cryopreservation and therefore also not for possible reseeding after extraction of the cells from the donor tissue, after corresponding extraction, human preadipocytes can be cryopreserved very well and can undergo in vitro cultivation. However, they cannot be passaged more often than twice. After that, they differentiate into human adipocytes. Human preadipocytes are characterized by a morphology that closely resembles the morphology of human dermal fibroblasts. Human adipocytes that were obtained by in vitro differentiation from human preadipocytes can be used as an in vitro test system for investigating insulin-stimulated glucose transport, for characterizing hormone-controlled lipolysis and for investigating gene expression in fat tissues. The HPAd/HAd system is an extremely well characterized in vitro test system for investigating the causes of adiposity and type II diabetes.

(21) Contacting the Human Cells with Yeast Extract

(22) Human prim. fibroblasts were expanded in medium 199 (Souto L R, Rehder J, Vassallo J, Cintra M L, Kraemer M H, Puzzi M B (2006) Sao Paulo Med J. 124(2), 71-6. Model for human skin reconstructed in vitro composed of associated dermis and epidermis; E. Pinney, K. Liu, B. Sheeman and J. Mansbridge (2001): Human three-dimensional fibroblast cultures express angiogenic activity. J Cell Physiol 183, 74-82) up to 75% subconfluence with 10% FCS (fetal calf serum) and 2% penicillin (Pen)/streptomycin (Strep). After cell harvesting, the cells were integrated into the extracellular collagen matrix (ECM) and were cultured in the presence of M199 medium with 5% FCS and 2% Pen/Strep. Exposure with the active substance (yeast extract) took place 4-5 days after seeding the cells in the ECM for a period of 24-48 h. Cell viability was monitored during cultivation using the standard MTT test (Mosmann, Tim Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65 (1-2): 55-63). Normal cell culture conditions (5% CO.sub.2; 37 C., max. humidity) were selected.

(23) Human preadipocytes were expanded in normal cell culture conditions and using a special growth medium (supplier: Cell Applications) up to 60-75% subconfluence. Differentiation of the cells to adipocytes was inhibited. After harvesting the cells and reseeding in 6-well plates (>10 000 cells/cm.sup.2), the cells were cultured for 24-48 h in the presence of the growth medium and were then differentiated using a special differentiation medium (supplier: Cell Applications). Differentiation took place for a period of 10-14 days in the presence of the yeast extract (repeated dose continuously every 5-7 days).

(24) The cells in co-culture were in each case expanded as described above. 48 h after integration of the fibroblasts into the ECM (as described above), the hum. preadipocytes were applied on the collagen matrix. The whole construct was cultured in normal cell culture conditions and using the (pre)adipocyte growth medium for a further 48 h. Then differentiation of the preadipocytes took place in the presence of the active substance using the differentiation medium. The cell viability of both cell types was once again monitored with the standard MTT test. Release of intracellular lactate dehydrogenase (LDH) was determined quantitatively to assess the integrity of the cell membranes.

(25) Human epidermal keratinocytes were expanded in Medium 154 from Cascade Biologics (Invitrogen Company). After reaching 70-80% subconfluence, the cells were harvested and were submerged-cultured in the presence of the medium described above on a polycarbonate membrane (<0.4 M pore size) for a period of >48 h. The medium was additionally supplemented with FCS, and a BSA (bovine serum albumin) : fatty acid complex and vitamin E; L-serine and L-carnitine. Then there was shift of the cells to the air/medium boundary layer and transfer to a serum-free differentiation medium with increased Ca.sup.2+ ion concentration with addition of vitamin C. The cells were cultured starting from the 7th day up to the 14th day of this airlift culture in the presence of the active substance (repeated dose continuously, daily). Normal cell culture conditions were also selected in this case.

(26) Isolation of the RNA from the Fibroblasts

(27) The total RNA is isolated from the fibroblasts using the RNeasy Mini Kit. For the analysis, the frozen fibroblasts are homogenized with a 5 mm steel ball, 300 L RLT buffer and with 10 l/mL beta mercaptoethanol in the Tissuelyser for 3 min at 16 000 Hz. The samples are centrifuged briefly, to bring down the foam in the Eppendorf cap. After addition of 590 L RNase-free water and 10 L protease K, the solution is digested for 10 min at 55 C. in the thermo-shaker. The supernatant of approx. 900 L is put in a new Eppendorf cap and 450 L of 96% ethanol is added. 700 L is withdrawn, transferred to an RNeasy Mini Spin Column and centrifuged at 10 000 rev/min for 15 s at room temperature; the liquid can be discarded. Repeat this step with the liquid that remains. Then 700 L of buffer RW1 is put in the RNeasy Mini Spin Column and centrifuged at 10 000 rev/min for 15 s at room temperature; the liquid can be discarded. The RNeasy Mini Spin Columns are put on a new tube (2.2 mL), 500 L buffer RPE is added and centrifuged again for 15 s at 10 000 rev/min; the liquid is discarded. Now 500 L of buffer RPE is added, and it is centrifuged at 10 000 rev/min at room temperature for 2 min; the liquid is discarded. The RNeasy Mini Spin Columns are put on a new tube (2.2 mL) and centrifuged at room temperature for 1 min at 13 000 rev/min until dry. The RNeasy Mini Spin Columns are put on an Eppendorf cap (1.5 mL) and 30 L of RNase-free water is put directly on the membrane. Then it is centrifuged at 10 000 rev/min for 1 min. For measurement, 3 L of RNA solution is investigated on the bioanalyser. The remaining solution is divided up (5 L portions) and stored in the freezer at 80 C.

(28) Isolation of the RNA from the Adipocytes

(29) The total RNA is isolated from the adipocytes using the RNeasy Mini Kit. For this, the differentiation/exposure medium is removed quantitatively from the adipocytes by decanting. For lysis of the cells and release of the cytosolic RNA, the adipocytes are incubated in the 6-well cell culture plates for a period of 2 min at RT in the presence of a buffer containing guanidinium hydrochloride (V=600 L; Qiagen buffer RLT). For complete disruption of the cells, the cell suspension is sheared by means of a 1 mL disposable syringe and a disposable injection cannula (0.6030 mm; 23 G1). The cell-free extract is transferred quantitatively to a 2-mL reaction vessel. The total RNA is precipitated by adding one volume of ethanol (70%). Then 700 L of the precipitated lysate is transferred to an RNeasy Mini Column and centrifuged at >8000g for 15 s. The total RNA binds to the column material. The flow-through is discarded. The same procedure is followed with the remaining volume of the lysate. Then 700 L of a washing buffer (Qiagen buffer RW1) is added and it is centrifuged at 8000g for 15 s. Once again, the flow-through is discarded. Then the columns are washed twice with 500 L of an ethanol-containing buffer (Qiagen buffer RPE). The first washing operation takes place by centrifugation for a period of 15 s at 8000g (the flow-through is discarded) and the second washing operation takes place for a period of 2 min at >8000g (the flow-through is again discarded). For complete drying of the column material, the extraction columns are transferred to new collecting vessels and centrifuged for a period of 1 min at 8000g. The extraction columns are then transferred to 1.5-mL reaction vessels and the total RNA bound to the columns is rehydrated by adding 50 L water (RNase-free). Final elution of the total RNA then takes place by another centrifugation step (1 min at >8000 x g). For measurement, 90 L water is added to 10 L RNA solution and the absorption is measured at a wavelength of 260 nm. An absorption (A260) of 1.00 corresponds to a concentration of 50 g/mL of double-stranded DNA, 33 g/mL of short, single-stranded DNA or 40 g/mL RNA. The remaining 40 L of solution is divided up (10 L portions) and stored in the freezer at 80 C.

(30) Chip Technology

(31) The isolated mRNA is investigated by biochip. For this biochip, 500 biomarkers (see Table 1) were selected and their specific pathway was investigated. The biochip was developed by the company Febit Biomed GmbH in Heidelberg. Five different samples were produced for each biomarker and six technical replicas for each sample, so that there are 30 replicas for each biomarker. The Geniom biochip was synthesized with the Geniom One Instrument using the standard kit for oligonucleotide syntheses. The light-activated in-situ oligonucleotide synthesis is connected with a digital Micromirror unit to the Geniom One Instrument on an activated three-dimensional reaction carrier on a glass-silicon-glass sandwich. The quality of the mRNA is investigated with an Agilent 2100 bioanalyser using the RNA 6000 Nano Kit.

(32) An example of two representative sets with chip data, obtained using the protocol described, is shown in FIGS. 2a and 2b. The data there were obtained by analysis of the markers of human fibroblasts that were treated with extract from Yarrowia cells that had been cultured normally at pH 5.4 (FIG. 2a) or had been exposed to stress at pH 2 (FIG. 2b).

(33) The results of the tests are summarized in Table 2.

(34) TABLE-US-00002 TABLE 2 Regulation of dermatologically relevant fibroblast markers after treatment with extracts of Saccharomyces cerevisiae cultures, which had been-stressed by increased temperature, treatment with hydrogen peroxide or exposure to medium with low pH. Total number of induced or Number of strongly Stress repressed induced or repressed Yeast strain condition markers markers (2) Saccharomyces cerevisiae T 273 1 Saccharomyces cerevisiae pH 32 14 Saccharomyces cerevisiae H.sub.2O.sub.2 292 3

(35) The different expression profiles of the cells treated with the extracts of variously stressed yeasts show that the yeast extracts have different properties and they are therefore different and distinguishable products.

EXAMPLE 3

Comparative Investigation of the Action of Extracts of pH-Stressed Cells of Different Yeast Genera on Human Fibroblasts

(36) The extent to which the cells of different yeast genera are suitable for producing dermatologically especially effective extracts was investigated.

(37) The test conditions corresponded to those stated in example 2 for the production of extracts of pH-stressed cells, apart from the fact that extracts not only from one strain, but from Pichia CBS1991, Yarrowia lipolytica and Saccharomyces cerevisiae were contacted with human fibroblasts and their effects were compared.

(38) TABLE-US-00003 TABLE 3 Regulation of dermatologically relevant fibroblast markers after treatment with extracts of cultures of different yeast strains, which had been stressed by exposure to medium with low pH. Number of induced or repressed Yeast strain pH Type of regulation markers Yarrowia lipolytica 2 Induced 148 Repressed 65 5.4 Induced Repressed Saccharomyces 2 Induced 31 cerevisiae Repressed 1 5.4 Induced Repressed Pichia CBS1991 3 Induced Repressed 5.4 Induced Repressed

(39) The results summarized in Table 3 show that the pH-stressed yeast cells of the genus Yarrowia have a particularly advantageous effect on the skin, in that in contact with human fibroblasts they regulate a particularly large number of skin markers positively.

EXAMPLE 4

Comparison of the Effect of Different Stress Conditions on Yeast Cells of the Genus Yarrowia with Respect to their Dermatological Efficacy After Processing to Extracts and Application on Human Fibroblasts

(40) After identifying yeast cells of the genus Yarrowia as a biotechnologically and dermatologically especially advantageous source of yeast extracts for skin applications, various stress conditions and combinations thereof were compared, with the following results, in order to find an especially advantageous method of production for these yeast extracts.

(41) The extracts were in each case produced using a water-based and alcohol-based solvent, so as to be able to compare the respective effects.

(42) TABLE-US-00004 TABLE 4 Regulation of dermatologically relevant fibroblast markers after treatment with extracts of Yarrowia cultures, which had been stressed under various stress conditions and combinations thereof. Number of Water (w) or induced or Stress propanediol Type of repressed Test # condition(s) (p) lysis regulation markers 1 w Induced 22 2 w Repressed 1 3 p Induced 8 4 p Repressed 5 37 C. w Induced 125 6 w Repressed 119 7 37 C. p Induced 2 8 p Repressed 1 9 pH 2 at 37 C. w Induced 30 10 w Repressed 6 11 pH 2 at 37 C. p Induced 1 12 p Repressed 13 pH 2 .fwdarw. 37 C. w Induced 110 14 w Repressed 93 15 pH 2 .fwdarw. 37 C. p Induced 4 16 p Repressed 1 17 37 C. .fwdarw. pH 2 w Induced 154 18 w Repressed 118 19 37 C. .fwdarw. pH 2 p Induced 3 20 p Repressed 1

(43) The results summarized in Table 4 show, firstly, that lysis of the cells using an aqueous solution is preferable to lysis using an alcoholic solution, as in the first case a much higher number of dermatologically relevant fibroblast markers is induced or repressed in a dermatologically advantageous direction.

(44) Furthermore, it can be seen that the combination of stress factors of exposure to medium with low pH followed by temperature increase brings about the highest positive regulation of dermatologically relevant markers and is therefore preferable to other stress conditions or combinations thereof.