1. Optimization and validation of a chiral GC-MS method for the determination of free D-amino acids ratio in human urine: application to a gestational diabetes mellitus study
Ma Paz Lorenzo, Danuta Dudzik, Elena Varas, Manuel Gibellini, Mariusz Skotnicki, Marcin Zorawski, Wieslaw Zarzycki, Federica Pellati, Antonia García J Pharm Biomed Anal. 2015 Mar 25;107:480-7. doi: 10.1016/j.jpba.2015.01.015. Epub 2015 Jan 15.
Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. It is affecting approximately up to 14% of all pregnancies with an increasing tendency. GDM has been related to relevant short-term and long-term health complications for both mother and offspring. Recent studies strongly emphasized the role of several essential amino acids in the pathogenesis of obesity and highlighted their strong correlation with insulin resistance, but there are no references related to modifications in D-AAs in biological fluids. As D-AA elimination proceeds mainly by renal excretion, urine was the selected sample to evaluate the alterations in free D-AAs ratio in a GDM study. Only 1 mL of first void urine or standard solution was required for purification, by using a Discovery DSC-SCX SPE cartridge (500 mg/3 mL) and derivatization into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomeric separation was carried out by GC-MS on a Chirasil-L-Val N-propionyl-L-valine-tert-butylamide polysiloxane fused-silica capillary column (25 m×0.25 mm I.D., 0.12 μm film thickness, Agilent Technologies, Waldbronn, Germany), under programmed temperature elution. Detection was performed with an ion trap mass analyzer, operating in the full scan mode in the m/z 50-350 range. 14 pairs of derivatives of D-and L-AAs were separated. The steps of sample preparation, derivatization and GC-MS conditions were optimized for both urine and standards. Several conditions affecting the SPE procedure, such as sorbent mass/volume ratio of the cartridge, sample dilution and pH, were optimized. Volume of reagents and solvents and reaction temperature and time were also tested for the derivatization. Regarding the GC-MS parameters, split ratio, temperature program and mass range were optimized. The final method was validated in terms of linearity, sensitivity, accuracy and precision for D-Ala, D-Pro, D-Ser, D-Met, D-Phe, D-Glu, D-Orn and D-Lys. Identification of AAs in urine samples was based on retention time and mass spectra. Urine from 20 women with GDM and 20 pregnant women with normal glucose tolerance (after 2-h 75-g oral glucose tolerance test), matched according to the week of gestation and age (22-28 week of gestation and age 24-37 years), were enrolled into the study. %D-Relative amounts were determined for Ala, Val, Thr, Ser, Leu, Asx (Asp+Asn), Glx (Glu+Gln), Met, Phe, Tyr, Orn and Lys. Statistically significant differences (p<0.05) were observed only for D-Phe and higher values were found in the GDM group. It is possible that D-Phe could be involved in metabolic/signaling pathways to compensate early stages of insulin resistance, although further work is necessary to confirm this hypothesis.
2. Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare
Nguyen-Hung Le, et al. Sci Adv. 2020 Jul 22;6(30):eabb5614. doi: 10.1126/sciadv.abb5614. eCollection 2020 Jul.
Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacterium Acinetobacter baumannii strain ATCC 17978 produces, secretes, and incorporates the noncanonical d-amino acid d-lysine into its PG during stationary phase. We show that PG editing increases the competitiveness of A. baumannii during bacterial warfare by providing immunity against peptidoglycan-targeting T6SS effectors from various bacterial competitors. In contrast, we found that d-Lys production is detrimental to pathogenesis due, at least in part, to the activity of the human enzyme d-amino acid oxidase (DAO), which degrades d-Lys producing H2O2 toxic to bacteria. Phylogenetic analyses indicate that the last common ancestor of A. baumannii had the ability to produce d-Lys. However, this trait was independently lost multiple times, likely reflecting the evolution of A. baumannii as a human pathogen.
3. Ca2+ transport through lipid membrane by diastereomer cyclic octapeptides
S Kimura, E Ozeki, Y Imanishi Biopolymers. 1989 Jul;28(7):1247-57. doi: 10.1002/bip.360280706.
Effects of the nature and orientation of a side chain in cyclic octapeptides on Ca2+ transport were examined by using cyclo[L-Lys(Z)-Sar-L-Leu-Sar]2 (C8-L), cyclo[L-Lys(Z)-Sar]4 (C8KS), and their diastereomer cyclic octapeptides, cyclo[L-Lys(Z)-Sar-D-Leu-Sar]2 (C8-D) and cyclo[L-Lys(Z)-Sar-D-Lys(Z)-Sar]2 (C8Kk). All these cyclic octapeptides were found to take a single conformation in CDCl3, and the conformation was C2-symmetric for C8-L and C8-D, and C4-symmetric for C8KS and C8Kk. They formed a complex with Ca2+. Upon complexation, C8KS accompanied isomerization of peptide bonds, but C8-D retained the arrangement of peptide bonds. The amount of Ca2+ extracted from an aqueous solution to a chloroform solution by all L cyclic octapeptide C8-L or C8KS was about twice that of Na+, but 6-8-fold smaller than that by C8-D or C8Kk including D units. These cyclic octapeptides were capable of transporting Ca2+ through a lipid membrane above the phase transition temperature, and the transport rate decreased in the order of C8Kk-C8KS greater than C8-D greater than C8-L.
