Mechanistic Insight into the Attenuation of Initial Pilocarpine-Induced Seizures in Young Rats by a Non-Convulsive MSO Dose

Pretreatment with non-convulsive dose of methionine sulfoximine (MSO) attenuated lithi-um-pilocarpine-induced (Li-Pilo) seizures in young rats [1]. We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was admin-istered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (onset) or 15 min before sacrifice (final phase). Their conversion to Glu and Gln in hippocampus and entorhinal cortex was followed us-ing [13C] gas chromatography-mass spectrometry. Release of in vitro loaded [3H]D-Asp from ex vi-vo brain slices was measured in continuously collected superfusates. Protein and mRNA expres-sion were measured by Western Blot and real-time PCR techniques, respectively. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]D-Asp release and reduced by ~50% the synaptic vesicular Glu transporter VGLUT1 protein, but not mRNA content in the hippo-campus. The results indicate that MSO at non-convulsive dose mitigates the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.

Network-Related Changes in Neurotransmitters and Seizure Propagation During Rodent Epileptogenesis

Objective:To test the hypothesis that glutamate and GABA are linked to the formation of epilepsy networks and the triggering of spontaneous seizures, we examined seizure initiation/propagation characteristics and neurotransmitter levels during epileptogenesis in a translationally relevant rodent model of mesial temporal lobe epilepsy.Methods:The glutamine synthetase (GS) inhibitor methionine sulfoximine was infused into one of the hippocampi in laboratory rats to create a seizure focus. Long-term video-intracranial EEG recordings and brain microdialysis combined with mass spectrometry were used to examine seizure initiation, seizure propagation, and extracellular brain levels of glutamate and GABA.Results:All seizures (n = 78 seizures, n = 3 rats) appeared first in the GS-inhibited hippocampus of all animals, followed by propagation to the contralateral hippocampus. Propagation time decreased significantly from 11.65 seconds early in epileptogenesis (weeks 1-2) to 6.82 seconds late in epileptogenesis (weeks 3 – 4, paired t-test, p = 0.025). Baseline extracellular glutamate levels were 11.6-fold higher in the hippocampus of seizure propagation (7.3 µM) vs. the hippocampus of seizure onset (0.63 µM, ANOVA/Fisher’s LSD, p = 0.01), even though the concentrations of the major glutamate transporter proteins EAAT1, EAAT2 and xCT were unchanged between the brain regions. Finally, extracellular GABA in the seizure focus decreased significantly from baseline several hours before a spontaneous seizure (paired t-test/FDR).Conclusion:The changes in glutamate and GABA suggest novel and potentially important roles of the amino acids in epilepsy network formation and in the initiation and propagation of spontaneous seizures.

Amino acid derivatives of natural chlorins as a platform for the creation of targeted photosensitizers in oncology

Objectives. This study aims to obtain the amino acid derivatives of chlorophyll a and bacteriochlorophyll a for the targeted delivery of pigments to tumor foci. This will increase biocompatibility and, as a result, reduce toxic side effects. In addition to photodynamic efficiency, an additional cytotoxic effect is expected for the obtained conjugates of photosensitizers (PSs) with amino acids. This is owing to the participation of the latter in intracellular biochemical processes, including interaction with the components of the glutathione antioxidant system, leading to the vulnerability of tumor cells to oxidative stress.Methods. In this work, we have implemented the optimization of the structure of a highly efficient infrared PS based on O-propyloxim-N-propoxybacteriopurpurinimide (DPBP), absorbing at 800 nm and showing photodynamic efficacy for the treatment of deep-seated and pigmented tumors, by introducing L-lysine, L-arginine, methionine sulfoximine (MSO), and buthionine sulfoximine (BSO) methyl esters. The structure of the obtained compounds was proved by mass spectrometry and nuclear magnetic resonance spectroscopy, and the photoinduced cytotoxicity was studied in vitro on the HeLa cell line.Results. Conjugates of DPBP with amino acids and their derivatives, such as lysine, arginine, MSO, and BSO have been prepared. The chelating ability of DPBP conjugate with lysine was shown, and its Sn(IV) complex was obtained.Conclusions. Biological testing of DPBP with MSO and BSO showed a 5–6-fold increase in photoinduced cytotoxicity compared to the parent DPBP PS. Additionally, a high internalization of pigments by tumor cells was found, and the dark cytotoxicity (in the absence of irradiation) of DPBP-MSO and DPBP-BSO increased fourfold compared to the initial DPBP compound. This can be explained by the participation of methionine derivatives in the biochemical processes of the tumor cell.

A human expression system based on HEK293 for the stable production of recombinant erythropoietin

Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

Potential Metabolites with Diagnostic Value in Plasma for Angioimmunoblastic T-Cell Lymphoma By LC-MS Based Untargeted Metabonomics Study

Background: Angioimmunoblastic T-cell lymphoma (AITL) is an uncommon subtype of mature peripheral T-cell lymphoma (PTCL). and its characteristics is not fully understood. Because it is a rare form of lymphoma, there is difficulty in diagnosis based on current traditional method. Metabolic reprogramming is a major cancer hallmark, which provides cancer cells with vital energy and metabolites. Mass spectrometry-based metabolomics has emerged as an informative technique for profiling metabolic features associated with cancers. Objective: This study aim to discover metabolites with potential diagnostic value in plasma of AITL patient. Methods: Plasma was collected from 27 patients newly diagnosed with AITL in Zhejiang Cancer Hospital and 30 age- and gender- matched healthy controls. An aliquot of 50 μL plama was added with 150 μL of chilled acetonitrile, vortexed and centrifuged. the supernatant was dried, reconstituted for metabolomics. Mass spectrometry analysis was performed on a UPLC coupled with a Q Exactive Orbitrap mass spectrometer. "XCMS" and "MetaX" were used to analyze LC-MS data. Significant features were selected as candidate for identification by matching their MS and MS/MS data with online databases HMDB and METLIN. Results: Eighteen altered metabolites (choline, betaine, L-leucine, creatine, 2-butyl-1H-benzimidazole, hypoxanthine, methionine sulfoximine, L-carnitine, 2-hydroxycinnamic acid, L-tyrosine, D-tryptophan, indoleacrylic acid, lysoPC(P-18:0), 5-aminopentanoic acid, uric acid, sucrose, L-lactic) were indentified (VIP value>1, p<0.05) in AITL patient plasma. The altered metabolites contributed to different metabolic pathways including glycine, serine and threonine metabolism (P=0.003), glycerophospholipid metabolism (P=0.024), Phenylalanine metabolism (P=.030). Conclusion: This study revealed metabolic reprogram and potential diagnostic metabolites in plasma for AITL, and gave more metabolic information for carcinogenesis of AITL. Disclosures No relevant conflicts of interest to declare.

L-methionine sulfoximine interacted with propofol and showed toxicity in PC12 cells

Background: L-Methionine sulfoximine (MSO) inhibits glutamine synthesis in a rodent animal model, and its limited clinical use is implicitly associated with glutamate deprivation and neurotoxicity. The purpose of this experiment was to determine the effect of MSO on pheochromocytoma (PC12) cells and its interaction with propofol-induced neuro-apoptosis. Objective: To study the effects of MSO on cell viability following 100 μM propofol treatment and the impact of ribosomal S6 kinase 1 (RSK1) signaling on the PC12 cell line. Methods: PC12 cells were exposed to propofol-triggered neurotoxicity for 6 h and then subjected to MSO treatment. The gene and protein expression levels of members of the RSK1 signaling pathway were determined by real-time polymerase chain reaction, Western blot and histological analyses. The CCK8 test was used to assess cell viability, and cell proliferation and apoptosis were evaluated by flow cytometric analysis. Results: Propofol, a gamma-aminobutyric acid (GABA) agonist widely used in general anesthesia, significantly changed the expr ession level of cAMP response element-binding protein (CREB) and B cell lymphoma 2 (Bcl2) and solute carrier family 1 member 3 (Slc1a3), but not extracellular signal-regulated kinase 1/2 (ERK1/2). PC12 cells that were exposed to propofol for more than 6 h exhibited downregulation of RSK1. MSO aggravated the toxicity of propofol in PC12 cells via inhibition of the p90RSK1/CREB/Bcl2 signaling pathway.

L-methionine sulfoximine interacted with propofol and showed toxicity in PC12 cells

Background: L-Methionine sulfoximine (MSO) inhibits glutamine synthesis in a rodent animal model, and its limited clinical use is implicitly associated with glutamate deprivation and neurotoxicity. The purpose of this experiment was to determine the effect of MSO on pheochromocytoma (PC12) cells and its interaction with propofol-induced neuro-apoptosis. Objective: To study the effects of MSO on cell viability following 100 μM propofol treatment and the impact of ribosomal S6 kinase 1 (RSK1) signaling on the PC12 cell line. Methods: PC12 cells were exposed to propofol-triggered neurotoxicity for 6 h and then subjected to MSO treatment. The gene and protein expression levels of members of the RSK1 signaling pathway were determined by real-time polymerase chain reaction, Western blot and histological analyses. The CCK8 test was used to assess cell viability, and cell proliferation and apoptosis were evaluated by flow cytometric analysis. Results: Propofol, a gamma-aminobutyric acid (GABA) agonist widely used in general anesthesia, significantly changed the expr ession level of cAMP response element-binding protein (CREB) and B cell lymphoma 2 (Bcl2) and solute carrier family 1 member 3 (Slc1a3), but not extracellular signal-regulated kinase 1/2 (ERK1/2). PC12 cells that were exposed to propofol for more than 6 h exhibited downregulation of RSK1. MSO aggravated the toxicity of propofol in PC12 cells via inhibition of the p90RSK1/CREB/Bcl2 signaling pathway.

Copper-Catalyzed N-Arylation of Sulfoximines with Arylboronic Acids under Mild Conditions

N-Arylation of sulfoximines with different arylboronic acids, including sterically hindered boronic acids, is achieved using copper(I) iodide and 4-DMAP at room temperature. Moreover, N-arylation of biologically relevant l-methionine sulfoximine is demonstrated for the first time. All these reactions provided the desired products in excellent yields within a short span of time. The optimized reaction conditions are well suited to the task of N-vinylation of sulfoximine with trans-2-phenylvinylboronic acid.