Search

Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur ® , Jin Sai Heng ® , Follitrope ® , Folisurge ® , and Corneumon ® ) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N -glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N -glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N -glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N -glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed.

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N-aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N-aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role as peripheral mediators with multiple actions on blood cell function.