CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.     

Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

Stereoselective reduction towards pharmaceutically potent products with multi‐chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH‐dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various alpha‐hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl₂. Detailed analysis of the pH‐dependent activity and stability yielded a broad pH‐optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half‐life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio‐ and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2‐hydroxypropiophenone in more detail. Biotechnol. Bioeng. 2013; 110: 1838–1848. © 2013 Wiley Periodicals, Inc.     

Chemerin Is an Antimicrobial Agent in Human Epidermis

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val⁶⁶-Pro⁸⁵, which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.     

A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction

DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy.     

Anticonvulsant Activity of Enantiomeric N‐trans‐Cinnamoyl Derivatives of 2‐Aminopropan‐1‐ols and 2‐Aminobutan‐1‐ols

Epilepsy, one of the most frequent neurological disorders, is still insufficiently treated in about 30% of patients. As a consequence, identification of novel anticonvulsant agents is an important issue in medicinal chemistry. In the present article we report synthesis, physicochemical, and pharmacological evaluation of N‐trans‐cinnamoyl derivatives of R and S‐2‐aminopropan‐1‐ol, as well as R and S‐2‐aminobutan‐1‐ol. The structures were confirmed by spectroscopy and for derivatives of 2‐aminopropan‐1‐ols the configuration was evaluated by means of crystallography. The investigated compounds were tested in rodent models of seizures: maximal electroshock (MES) and subcutaneous pentetrazol test (scPTZ), and also in a rodent model of epileptogenesis: pilocarpine‐induced status prevention. Additionally, derivatives of 2‐aminopropan‐1‐ols were tested in benzodiazepine‐resistant electrographic status epilepticus rat model as well as in vitro for inhibition of isoenzymes of cytochrome P450. All of the tested compounds showed promising anticonvulsant activity in MES. For R(–)‐(2E)‐N‐(1‐hydroxypropan‐2‐yl)‐3‐phenylprop‐2‐enamide pharmacological parameters were found as follows: ED₅₀ = 76.7 (68.2–81.3) mg/kg (MES, mice i.p., time = 0.5 h), ED₅₀ = 127.2 (102.1–157.9) mg/kg (scPTZ, mice i.p., time = 0.25 h), TD₅₀ = 208.3 (151.4–230.6) mg/kg (rotarod, mice i.p., time = 0.25 h). Evaluation in pilocarpine status prevention proved that all of the reported compounds reduced spontaneous seizure activity and act as antiepileptogenic agents. Both enantiomers of 2‐aminopropan‐1‐ols did not influence cytochrome P450 isoenzymes activity in vitro and are likely not to interact with CYP substrates in vivo. Chirality 28:482–488, 2016. © 2016 Wiley Periodicals, Inc.     

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C‐terminal, N‐terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H?PheCysTrp?OH, H?PheCysTyr?OH, H?PheTyrMet?OH, and H?TrpTyr?OH.     

SOD-Like Activity of Copper(II) Containing Metallopeptides Branched By 2,3-Diaminopropionic Acid: What the N-Termini Elevate,the C-Terminus Ruins

International Journal of Peptide Research and Therapeutics - Relations between structural modifiactions and SOD-like activity of four branched CuII-metallopeptides based on l-2,3-diaminopropionic...