Mike Farwick

Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pull‐down assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR.

Bioactive tetrapeptide GEKG boosts extracellular matrix formation: in vitro and in vivo molecular and clinical proof

Abstract:  The ‘matrikine’ concept claims that processing of the precursors for collagen results in the formation of peptides such as KTTKS which in turn augments extracellular matrix (ECM) production. In the present study, we show the development of an anti‐ageing active from an in silico approach by molecular design resulting in the tetrapeptide GEKG derived from ECM proteins. The efficacy of the peptide to significantly induce collagen production of the protein level and mRNA level has been demonstrated in vitro in human dermal fibroblasts and in vivo in a double‐blind, randomized, placebo‐controlled study enroling 10 volunteers with an average age of 48.2 years. The effect of GEKG on facial wrinkles was studied in 30 volunteers using state of the art fringe projection, which allows determination of surface roughness in three‐dimensions. Here, only GEKG but not the placebo was able to significantly decrease skin roughness as a measure for wrinkles.

Novel sphingolipid derivatives promote keratinocyte differentiation

Abstract:  Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence‐induced differentiation model of normal human keratinocytes was established to allow evaluation of pro‐ and anti‐differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (–C6), stearoyl (–C18) and salicyl (–SLC) derivatives, C12‐alkylamine‐salicylate (C12‐SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA‐microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation‐related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment‐induced transcriptional changes were analysed by the ExPlain™ software (BIOBASE GmbH). Five of the assayed substances (SA, SO‐C6, PS‐C6, SO‐SLC, PS‐SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12‐SLC revealed potential anti‐differentiation properties. ExPlain™ analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their –C6 and especially –SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications.

Metabolic engineering of the non-conventional yeast Pichia ciferrii for production of rare sphingoid bases

The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ(4)-desaturase (PcDES1) and sphingolipid Δ(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL(-1) triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.

Effects of sphingoid bases on the sphingolipidome in early keratinocyte differentiation

Keratinocyte sphingolipids are structural elements of epidermal permeability barrier and potential regulators of epidermal functions. We tested the influence of sphingoid bases sphinganine, sphingosine and phytosphingosine on in vitro keratinocyte differentiation. Lipidomic and transcriptomic analysis after treatment emphasizes sphinganine and phytosphingosine as potent modulators of keratinocyte differentiation and lipid metabolism. Sphinganine treatment regulated differentiation and sphingolipid metabolism‐related genes, and also increased all major ceramide species. Sphingosine treatment increased ceramide and phytoceramide pools without changes in dihydroceramides. Phytosphingosine treatment markedly increased phytoceramide pools without raising ceramide or dihydroceramide levels. Sphinganine treatment increased specifically very long chain ceramides essential for intact barrier function. In summary, sphingoid bases, especially sphinganine, promote differentiation and ceramide production in keratinocytes. Free sphinganine may serve as a dermatological and cosmetic agent by enhancing formation and maintenance of an intact epidermal lipid barrier, with beneficial effects for skin and hair care applications.

Draft Genome Sequence of Wickerhamomyces ciferrii NRRL Y-1031 F-60-10

ABSTRACT Wickerhamomyces ciferrii is a microorganism characterized by the production and secretion of large amounts of acetylated sphingoid bases, in particular tetraacetyl phytosphingosine. Here, we present the 15.90-Mbp draft genome sequence of W. ciferrii NRRL Y-1031 F-60-10 generated by pyrosequencing and de novo assembly. The draft genome sequence comprising 364 contigs in 150 scaffolds was annotated and covered 6,702 protein-coding sequences. This information will contribute to the metabolic engineering of this yeast to improve the yield and spectrum of acetylated sphingoid bases in biotechnological production.

Modulation of skin pigmentation by the tetrapeptide PKEK: in vitro and in vivo evidence for skin whitening effects

Abstract:  Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro‐Lys‐Glu‐Lys) can exert skin whitening effects based on one in vitro and four double‐blinded vehicle‐controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB‐stimulated mRNA expression of interleukin (IL)‐6, IL‐8 and TNF‐α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation‐inducing soluble mediator α‐ (α‐MSH). (ii) PKEK treatment significantly inhibited UVB‐induced upregulation of genes encoding for IL‐1α, IL‐6, IL‐8, TNF‐α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP‐containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP‐containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB‐induced skin pigmentation and may be suited to serve as a skin tone–modulating agent in cosmetic products.

Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes

Please cite this paper as: Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes. Experimental Dermatology 2010; 19: 100–107.

Facial skin-lightening benefits of the tetrapeptide Pro-Lys-Glu-Lys on subjects with skin types V–VI living in South Africa

Background  Irregular skin pigmentation may be a substantial contributor to the signs of aging and to a person’s lack of psychological well‐being. Although a large number of skin‐lightening agents are available, the opportunity exists to identify more efficacious agents, agents that target alternative biological mechanisms.

Effect of the multifunctional cosmetic ingredient sphinganine on hair loss in men and women with diffuse hair reduction

Sphingolipids are well known to promote keratinocyte differentiation and to induce ceramide production. In addition, they show anti-inflammatory and antimicrobial activities. Thus, the aim of this study is to investigate the potential effect of sphinganine on prolonging the hair anagen rate and improving the overall hair quality and scalp health. The inhibitory potential of sphinganine toward 5-α-reductase was studied using an in vitro assay. The stimulation of the antimicrobial peptide HBD2 by sphinganine was measured by real-time polymerase chain reaction and immunostaining. Sphinganine bioavailability was studied ex vivo using a pig skin model. A placebo-controlled, double-blind study was designed to evaluate the efficacy of sphinganine on hair loss and hair/scalp quality in vivo. In vitro results showed that sphinganine is a potent inhibitor of 5-α-reductase type I that prevents the conversion of testosterone to dihydrotestosterone, a key factor of androgenetic male baldness. In vivo results demonstrated efficacy in reducing non-illness-related hair loss among males. In terms of expert rating, all hair quality and scalp parameters improved after application of sphinganine. Improved scalp health might be linked to the observed increase of the antimicrobial peptide HBD2. Thus, sphinganine is well suited as a topical alternative for the improvement of scalp health and hair quality and anti-hair loss application.