Labeling Cancer Stem Cells with 13C6 Glucose and 13C5 Glutamine for Metabolic Flux Analysis.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are a population of cells present within tumor that have increased self-renewal, chemoresistance, and aggressiveness, thereby contributing to tumor relapse. Literature shows that CSCs or TICs typically originate within the hypoxic niches of the tumor, making hypoxia one of the driving factors for generation of this population. Hypoxic stress promotes adaptation to low oxygen tension in the tissues by altering metabolic properties of the CSCs. This leads to a number of altered enzymatic activities in the CSC population that further contribute to the survival of the CSCs leading to resistance to standard therapy. Hence, understanding this altered metabolic pathways as well as targeting key nodes in these may pave the way for cancer management.Glucose and glutamine are the major substrates utilized by cancer cells and feed into multiple biosynthetic pathways. Hence, labeling and tracking these compounds may reveal some novel metabolic pathways exploited by cancer stem cells to acquire survival advantage. In these current book chapters, we elaborately summarized the basic steps required for isolation, characterization, and metabolic labeling (13C6 glucose and 13C5 glutamine) of CSC for flux analysis.

Enantioseparation of N-acetyl-glutamine enantiomers by LC-MS/MS and its application to a plasma protein binding study.

A novel chiral method was developed and validated to determine N-acetyl-glutamine (NAG) enantiomers by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Enantioseparation was achieved on a Chiralpak QD-AX column (150 × 4.6 mm i.d., 5 µm) using methanol-water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 µL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N-acetyl-l-glutamine and N-acetyl-d-glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02-20 µg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within-run and between-run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.

Rapid quantification of glutaminase 2 (GLS2)-related metabolites by HILIC-MS/MS.

Glutamine, glutamate and glutathione are key modulators of excessive oxidative stress in tumor cells. In this study, we developed a rapid and accurate HILIC-MS/MS method to simultaneously determine concentrations of cellular glutamine, glutamate and glutathione. A bared silica HILIC column was employed to analyze these polar metabolites. The LC-MS parameters were optimized to achieve high sensitivity and selectivity. The analysis can be completed within 4 min under optimal conditions. The method was validated in terms of accuracy, precision, and linearity. Intra-day (n = 9) precision was within 2.68-6.24% among QCs. Inter-day precision (n = 3) was below 12.4%. The method accuracy was evaluated by the recovery test, and the accuracy for three analytes were between 91.6 and 110%. The developed method was applied to study antioxidant function of GLS2 in non-small cell lung cancer cells. Changes in concentrations of glutamine, glutamate and glutathione revealed that the overexpression of GLS2 could effectively decrease oxidative stress. In summary, this study developed a rapid HILIC-MS/MS method for quantification of GLS2-related metabolites that could facilitate elucidation of the role of GLS2 in tumor development.

Guhong injection protects against focal cerebral ischemia-reperfusion injury via anti-inflammatory effects in rats.

Guhong injection (GHI), composed of aceglutamide and safflower aqueous extract, has been used clinically for the treatment of cerebrovascular diseases such as cerebral embolism, hemorrhage and mental deterioration. In this paper, we reported the results of the first study on the anti-inflammatory effects of GHI in murine focal cerebral ischemia-reperfusion (I/R) injury. Adult male SD rats were randomly divided into six groups: Sham group, I/R group, GHI-L group (2.5 mL/kg), GHI-M group (5 mL/kg), GHI-H group (10 mL/kg) and Nimodipine group. I/R injury was induced by middle cerebral artery occlusion (MCAO) for 1.5 h followed by reperfusion for 24 h. Compared with I/R group, rats treated with GHI showed dose dependent reductions in neurological defect scores and cerebral infarct volume. GHI obviously down-regulated nitric oxide (NO), inducible NO synthase (iNOS), myeloperoxidase (MPO), interleukin-1ß (IL-1ß), TNF-α (tumor necrosis factor-α) and C reactive protein (CRP) levels in serum. Moreover, histological examination by H&E staining showed that clear cell outline, less vacuolated spaces and largely surviving neurons were observed in GHI-treated rats. The immunohistochemical staining revealed that GHI administration significantly diminished the positive expressions of intercellular cell adhesion molecule-1 (ICAM-1) and nuclear factor-κB p65 (NF-κB p65) in brain tissues. Western blot analysis for ICAM, NF-κB p65 and iNOS further solidified the above findings. All these results demonstrate that GHI exerts a strong and ameliorative effect on cerebral I/R injury in rats possibly through the inhibition of inflammation.

Optimal excitation and emission wavelengths to analyze amino acids and optimize neurotransmitters quantification using precolumn OPA-derivatization by HPLC.

We describe an analytical methodology to obtain high sensitivity and better resolution through the study of fluorometric excitation (λex) and emission (λem) spectrum wavelengths of OPA-amino acids. The spectrum emission study revealed a maximum signal peak at 450 nm for aspartate and glutamine. For glycine, taurine, and GABA, the maximum signal peak was at 448 and for glutamate at 452 nm. The remaining amino acids analyzed showed a maximum emission around 450 nm. The best signal obtained within the spectrum excitation experiments was using 229- to 450-nm λex-λem. The drawbacks observed at these wavelengths were a baseline drift and negative peaks occurrence. Thus, the excitation wavelength of 240 nm was chosen (240- to 450-nm λex-λem) as a compromise between a very good signal response and a baseline stability to resolve the 18 amino acids studied. Furthermore, this protocol was properly validated. On the other hand, the elution gradient program used for neuroactive amino acids (aspartate, glutamate, glycine, taurine and GABA) showed separation to the baseline, in a 15-min run in all of them. Other amino acids, up to 18, also exhibited a very good separation in a 25-min run. In conclusion, we propose the use of 240- to 450-nm λex-λem wavelengths, in OPA-amino acids analysis, as the most suitable protocol to obtain the best signal response, maintaining an optimum chromatographic resolution.

Morphological and molecular identification of ticks infesting Boa constrictor (Squamata, Boidae) in Manaus (Central Brazilian Amazon) / Identificação morfológica e molecular de carrapatos coletados de jiboias (Boa constrictor) (Squamata, Boidae) da zona urbana de Manaus

The Boa constrictor is one of the world's largest vertebrate carnivores and is often found in urban areas in the city of Manaus, Brazil. The morphological identification of ticks collected from 27 snakes indicated the occurrence of Amblyomma dissimile Koch 1844 on all individuals sampled. In contrast, Amblyomma rotundatum Koch was found on only two snakes. An analysis of the 16S rRNA molecular marker confirmed the morphological identification of these ectoparasites.

A jiboia (Boa constrictor), vertebrado carnívoro, tem sido encontrada em abundância na área urbana de Manaus. A identificação morfológica dos carrapatos coletados em 27 dessas serpentes verificou a ocorrência de Amblyomma dissimile Koch 1844, em todos os exemplares avaliados e a presença de Amblyomma rotundatum Koch 1844, em duas dessas serpentes. A análise do marcador 16S rRNA confirma a identificação morfológica das espécies A. rotundatum e A. dissimile e apresenta novas sequências destes organismos.

A rare glutamine derivative from the flower buds of daylily.

A rare glutamine derivative, hemerocallisamine I (1), was isolated from the methanolic extract of the flower buds of daylily, together with a new pyrrole alkaloid hemerocallisamine II (2) and a new γ-lactam derivative, hemerocallisamine III (3). The chemical structures of the new compounds were elucidated on the basis of chemical and physicochemical evidence. For hemerocallisamine I (1), the absolute configuration was determined by Mo-Kα X-ray crystallographic analysis. This is the first report of a glutamine derivative with a pyrrole ring from natural plants.

Microchip CE-LIF method for the hydrolysis of L-glutamine by using L-asparaginase enzyme reactor based on gold nanoparticle.

L-Asparaginase (L-Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, L-glutamine (L-Gln) and L-asparagine (L-Asn). To study the cytotoxic effect and the secondary complications of L-Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of L-Asnase for the hydrolysis of L-Gln, employing the enzyme-gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)-LIF was established for the separation of L-amino acids (L-Gln and L-glutamic acid) and studying the hydrolysis of L-Gln by using L-Asnase enzyme reactor. Then, using L-Gln as target analyte, the enzyme kinetics of L-Asnase in free solution, enzyme-gold nanoparticle conjugates (E-GNP), and the enzyme-gold nanoparticle conjugates immobilized in capillary (E-GNP-C) were investigated in detail with the proposed MCE-LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of L-Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E-GNP-C enzyme reactor exhibited the best performance for the hydrolysis of L-Gln.

Fatty acid-amino acid conjugates diversification in lepidopteran caterpillars.

Fatty acid amino acid conjugates (FACs) have been found in noctuid as well as sphingid caterpillar oral secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and its biochemical precursor, N-linolenoyl-L-glutamine, are known elicitors of induced volatile emissions in corn plants. These induced volatiles, in turn, attract natural enemies of the caterpillars. In a previous study, we showed that N-linolenoyl-L-glutamine in larval Spodoptera litura plays an important role in nitrogen assimilation which might be an explanation for caterpillars synthesizing FACs despite an increased risk of attracting natural enemies. However, the presence of FACs in lepidopteran species outside these families of agricultural interest is not well known. We conducted FAC screening of 29 lepidopteran species, and found them in 19 of these species. Thus, FACs are commonly synthesized through a broad range of lepidopteran caterpillars. Since all FAC-containing species had N-linolenoyl-L-glutamine and/or N-linoleoyl-L-glutamine in common, and the evolutionarily earliest species among them had only these two FACs, these glutamine conjugates might be the evolutionarily older FACs. Furthermore, some species had glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is limited mostly to larger and more developed macrolepidopteran species.

Artificial liver support system using large buffer volumes removes significant glutamine and is an ideal bridge to liver transplantation.

BACKGROUND: Fulminant hepatitis is an intractable disease of varying etiology. Artificial liver support (ALS) is used to control serious symptoms of fulminant hepatitis, such as brain edema, which may induce postoperative neurological deficits. PATIENTS AND METHODS: ALS was evaluated in 12 patients with fulminant hepatitis who had been placed on an ALS system comprising plasma exchange and online hemodiafiltration. Six of 12 patients fell into an ahepatic state, the absolute indication for liver transplantation. The effects of ALS were evaluated on the basis of improvements in clinical symptoms, removal of glutamine (Gln), and brain computed tomography. RESULTS: All 12 patients regained consciousness with ALS and 5 survived; 7 died despite recovering from hepatic coma. The ALS systems sustained patients in an ahepatic state for more than 2 weeks. The median estimated plasma equivalent volume of removed Gln was 17.9 L (range, 6.7-64.3 L). There was a significant relationship between total buffer volume and the plasma equivalent volume of removed Gln. CONCLUSION: Plasma exchange combined with hemodiafiltration using large buffer volumes is a promising and effective bridging method to liver transplantation.

Citrinamides, new potentiators of antifungal miconazole activity, produced by Penicillium sp. FKI-1938.

Two new aromatic alkaloids, designated citrinamides A and B, were isolated from the culture broth of Penicillium sp. FKI-1938 by solvent extraction, silica gel column chromatography and HPLC. Their structures were elucidated by spectroscopic analysis, including NMR and amino acid analysis. Citrinamides A and B showed moderate potentiation of miconazole activity against Candida albicans.

N-(17-Phosphonooxylinolenoyl)glutamine and N-(17-phosphonooxylinoleoyl)glutamine from insect gut: the first backbone-phosphorylated fatty acid derivatives in nature.

N-(17-Phosphonooxylinolenoyl)glutamine (1) and N-(17-phosphonooxylinoleoyl)glutamine (2) were isolated from the regurgitate of Spodoptora exigua and identified as the first natural alkyl chain-phosphorylated fatty acid derivatives. The compounds were characterized by HPLC-MS/MS and the assigned structures confirmed by synthesis via a dissymmetric bis-Wittig approach as the key reaction. Rearing of the larvae on a diet enriched with inorganic phosphate increased the amount of the phosphorylated N-acyl glutamines in the regurgitate.

Differential activity and degradation of plant volatile elicitors in regurgitant of tobacco hornworm (Manduca sexta) larvae.

Plants respond to insect herbivory by emitting volatile compounds that attract natural enemies of the herbivores. Biosynthesis of many of these volatiles in plants is induced by herbivore-produced compounds. Components of tobacco hornworm (THW) regurgitant were investigated for their efficacy as elicitors of corn seedling volatiles. Two components that elicited the strongest release of volatiles were isolated and identified as N-linolenoyl-L-glutamine (18:3-GLN) and N-linolenoyl-L-glutamic acid (18:3-GLU). The approximately 10 times more active 18:3-GLN, which also is found in the regurgitant of several other Lepidopteran larvae, was rapidly degraded when THW regurgitant was left at room temperature, while 18:3-GLU degraded at a much slower rate. Different dietary sources of THW and tobacco bud worm larvae, including both host and nonhost plants, did not affect the amino acid composition of the fatty acid-amino acid conjugates in the regurgitant.

Identification of phenylbutyrylglutamine, a new metabolite of phenylbutyrate metabolism in humans.

Phenylbutyrate is used in humans for treating inborn errors of ureagenesis, certain forms of cancer, cystic fibrosis and thalassemia. The known metabolism of phenylbutyrate leads to phenylacetylglutamine, which is excreted in urine. We have identified phenylbutyrylglutamine as a new metabolite of phenylbutyrate in human plasma and urine. We describe the synthesis of phenylbutyrylglutamine and its assay by gas chromatography/mass spectrometry as a tert-butyldimethylsilyl or methyl derivative, using standards of [(2)H(5)]phenylbutyrylglutamine and phenylpropionylglutamine. After administration of phenylbutyrate to normal humans, the cumulative urinary excretion of phenylacetate, phenylbutyrate, phenylacetylglutamine and phenylbutyrylglutamine amounts to about half of the dose of phenylbutyrate. Thus, additional metabolites of phenylbutyrate are yet to be identified.

Industrial production of L-glutamine.

The industrial production of L-glutamine (L-Gln) started with its fermentation in the late 1960s. Currently, it is manufactured for use as pharmaceuticals and health foods at the worldwide annual production of approximately 2000 metric tons. To manufacture high quality L-Gln at a low cost, it is of prime importance to obtain a strain of microorganism with good production efficiency and minimum by-products. Furthermore, to obtain the final crystalline powder product, the efficient removal of impurities contained in the fermentation broth becomes paramount. Therefore, the industrial process is designed to take into account characteristics of the fermentation broth as well as chemical, physical and biological properties of L-Gln. Points that should be considered in the process design and typical industrial production of L-Gln are described in this report.

Study of zone broadening in optically gated high-speed capillary electrophoresis.

Fluorescein isothiocyanate (FITC) labeled compounds are separated by capillary zone electrophoresis (CZE) in seconds rather than minutes while high separation efficiency is maintained. These rapid analysis times are achieved through high electric fields applied over short separation distances and the use of a unique optical-gating injection system. The optical-gating injection is based on photodecomposition of FITC induced by an argon ion laser beam. A fraction of this same laser beam is also used for detection of the analyte zones by on-column laser-induced fluorescence. Four FITC-labeled amino acids and fluorescein dye were analyzed to compare system performance to that predicted by theory. To do so, the total variance for each analyte zone was measured experimentally. This was compared to the sum of the theoretically expected variance contributions from the injection and detection systems and longitudinal diffusion. The variance due to longitudinal diffusion was calculated from diffusion constants measured experimentally with a traditional CZE system. After accounting for these three known sources of variance, this method is found to achieve better than 80% of the performance predicted by theory in analysis times as short as 2-3 s.

Isolation and structure determination of a novel compatible solute from the moderately halophilic purple sulfur bacterium Ectothiorhodospira marismortui.

The halophilic phototrophic bacterium Ectothiorhodospira marismortui produces three organic osmolytes to counterbalance the osmotic pressure of the surrounding medium: glycine betaine, sucrose, and a novel compound. This new compound, which accounts for approximately 30% of the cells' compatible solutes, was isolated and identified by mass spectrometry and nuclear magnetic resonance. It was characterized as N alpha-carbamoyl-L-glutamine 1-amide, an unusual amino acid derivative with no previous reference in the chemical literature. The relatively high cytoplasmic concentration of this compound (approximately 0.5 M) observed at all growth conditions suggests that it may serve a vital function as an osmoticum and/or protectant for Ectothiorhodospira marismortui in a saline environment.

Isolation of glutamyltaurine from bovine brains and proof of its gamma-linkage by the B/E linked scan SIMS technique.

We isolated a glutamyltaurine from bovine brains and determined its structure as gamma-glutamyltaurine (gamma-Glu-Tau; glutaurine) by use of a new mass spectrometric technique [B/E linked scan sputtered ion mass spectrometry (SIMS)], which we have recently shown to be useful for distinguishing the gamma- from the alpha-isomer of glutamyl-dipeptides. Neither the alpha-isomer of glutamyltaurine nor any aspartyltaurines could be detected in bovine brain.