PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.

Dermal peptide delivery using enhancer molecules and colloidal carrier systems. Part III: Tetrapeptide GEKG.

Dermal application of peptides as drugs is an interesting and growing field in therapeutics. Besides therapy, they are increasingly used as cosmetic agents. Peptide delivery into the skin is highly challenging since they provide disadvantageous properties like a high molecular weight, hydrophilicity, polarity and susceptibility to enzymatic degradation. The aim of the presented study was to improve the bioavailability of a dermal administered tetrapeptide GEKG (amino acid sequence in one-letter notation). A nano-sized carrier system (microemulsion, ME) of w/o type was therefore developed since ME provide excellent penetration enhancing properties. Furthermore, enhancers were used to increase the penetration capacity of GEKG. The penetration of GEKG from the ME and enhancer-containing emulsions was compared to the penetration from a standard formulation. For this purpose ex vivo penetration studies with human skin were performed, which provide insights into dermal penetration and also localization and distribution of the active substances in each skin layer as well as the influence of vehicle variations. Experiments with three incubation times (30, 100, 300 min) and 5000 ppm GEKG in the formulations clearly show that the nano-sized carrier system is significantly better suited to transport GEKG rapidly into upper and deeper vital skin layers as well as through the skin compared to the standard cream. Studies with o/w emulsions containing 5% glyceryl caprylate/caprate 2.0× or 1.5× as enhancer and 50 ppm peptide revealed only a tendency to increased penetration into the SC and for the formulation with glyceryl caprylate/caprate 1.5× into the vital epidermis compared to the standard cream without additional enhancer. Significant better penetration rates were observed for the ME with 50 ppm GEKG. It can be concluded that liquid nano-sized systems are significantly more effective as carriers for extremely hydrophilic peptides used in cosmetics and also in therapeutics than classic cosmetic emulsions or enhancer-containing emulsions. CHEMICAL COMPOUNDS: Tetrapeptide-21 (CAS: 960608-17-7) glyceryl caprylate/caprate (CAS: 73398-61-5) sorbitan oleate (CAS: 1338-43-8) polyglyceryl-4 laurate (CAS: 75798-42-4) butylene glycol (CAS: 107-88-0) isopropyl palmitate (CAS: 142-91-6).

Effect of the multifunctional cosmetic ingredient sphinganine on hair loss in males and females 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.

PIP3 induces the recycling of receptor tyrosine kinases.

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

Combinatorial chemistry and the synthesis of compound libraries.

Solid Phase Organic Synthesis (SPOS) has become a powerful tool for the preparation of compound libraries used for screening efforts in Chemical Biology. While different types of screening libraries have become commercially available through several vendors, the elaboration of a hit compound in a screening campaign to a useful chemical probe still requires the preparation of a focused library. In this report, protocols are given which allow the synthesis of a focused compound library on solid phase by the diversification of a central scaffold using reliable functionalization reactions. Diversity elements can be introduced by attaching building blocks via amidation, esterification, reductive amination, and Suzuki cross-coupling.