Characteristics of amino acid metabolism in myeloid-derived suppressor cells in septic mice / 北京大学学报(医学版)

OBJECTIVE@#To explore the amino acid metabolomics characteristics of myeloid-derived suppressor cells (MDSCs) in mice with sepsis induced by the cecal ligation and puncture (CLP).@*METHODS@#The sepsis mouse model was prepared by CLP, and the mice were randomly divided into a sham operation group (sham group, n = 10) and a CLP model group (n = 10). On the 7th day after the operation, 5 mice were randomly selected from the surviving mice in each group, and the bone marrow MDSCs of the mice were isolated. Bone marrow MDSCs were separated to measure the oxygen consumption rate (OCR) by using Agilent Seahorse XF technology and to detect the contents of intracellular amino acids and oligopeptides through ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) technology. Different metabolites and potential biomarkers were analyzed by univariate statistical analysis and multivariate statistical analysis. The major metabolic pathways were enriched using the small molecular pathway database (SMPDB).@*RESULTS@#The proportion of MDSCs in the bone marrow of CLP group mice (75.53% ± 6.02%) was significantly greater than that of the sham group (43.15%± 7.42%, t = 7.582, P < 0.001), and the basal respiratory rate [(50.03±1.20) pmol/min], maximum respiration rate [(78.07±2.57) pmol/min] and adenosine triphosphate (ATP) production [(25.30±1.21) pmol/min] of MDSCs in the bone marrow of CLP group mice were significantly greater than the basal respiration rate [(34.53±0.96) pmol/min, (t = 17.41, P < 0.001)], maximum respiration rate [(42.57±1.87) pmol/min, (t = 19.33, P < 0.001)], and ATP production [(12.63±0.96) pmol/min, (t = 14.18, P < 0.001)] of sham group. Leucine, threonine, glycine, etc. were potential biomarkers of septic MDSCs (all P < 0.05). The increased amino acids were mainly enriched in metabolic pathways, such as malate-aspartate shuttle, ammonia recovery, alanine metabolism, glutathione metabolism, phenylalanine and tyrosine metabolism, urea cycle, glycine and serine metabolism, β-alanine metabolism, glutamate metabolism, arginine and proline metabolism.@*CONCLUSION@#The enhanced mitochondrial oxidative phosphorylation, malate-aspartate shuttle and alanine metabolism in MDSCs of CLP mice may provide raw materials for mitochondrial aerobic respiration, thereby promoting the immunosuppressive function of MDSCs. Blocking the above metabolic pathways may reduce the risk of secondary infection in sepsis and improve the prognosis.

Lactobacillus plantarum as a malolactic starter culture in winemaking: a new (old) player?

Malolactic fermentation (MLF) is a process in winemaking responsible for the conversion of L-malic acid to L-lactic acid and CO2, which reduces the total acidity, improves the biological stability, and modifies the aroma profile of wine. MLF takes place during or after alcoholic fermentation and is carried out by one or more species of lactic acid bacteria (LAB), which are either present in grapes and cellars or inoculated with malolactic starters during the winemaking process. Although the main bacterium among LAB used in commercial starter cultures for MLF has traditionally been Oenococcus oeni, in the last decade, Lactobacillus plantarum has also been reported as a malolactic starter, and many works have shown that this species can survive and even grow under harsh conditions of wine (i.e., high ethanol content and low pH values). Furthermore, it has been proved that some strains of L. plantarum are able to conduct MLF just as efficiently as O. oeni. In addition, L. plantarum exhibits a more diverse enzymatic profile than O. oeni, which could play an important role in the modification of the wine aroma profile. This enzymatic diversity allows obtaining several starter cultures composed of different L. plantarum biotypes, which could result in distinctive wines. In this context, this review focuses on showing the relevance of L. plantarum as a MLF starter culture in winemaking.

Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

Effect of malate on docosahexaenoic acid production from Schizochytrium sp. B4D1

Background: Malate involves in the citrate/malate and transhydrogenase cycles to provide precursors for docosahexaenoic acid (DHA) synthesis. The optimal strategy was investigated for increasing DHA production in Schizochytrium species during fermentation. Results: DHA production increased by 47% and reached 5.51 g/L when 4 g malate/L was added during the rapid lipid accumulation stage in shake-flasks culture. Inducing effects of malate was further investigated through the analysis of three kinetic parameters, including specificcell growth rate(μ), specific glucose consumption rate (qGlu)and DHA formation rate (qDHA). DHA concentration was enhanced through a novel fed-batch strategy to a maximum value of 30.7 g/L, giving a yield of 0.103 g DHA/g glucose and a productivity of 284 mg L-1 h-1. Conclusion: A novel malate feeding strategy was developed that enhanced DHA yield and productivity of Schizochytrium species which may offer a desirable method for industrial applications.

Effect of glutamine on the mRNA level of key enzymes of malate-aspartate shuttle in the rat intestine subjected to ischemia reperfusion / Efeito da glutamina sobre o nível de RNA Mensageiro das enzimas-chave do ciclo malato-aspartato no intestino de ratos submetidos à isquemia e reperfusão

PURPOSE: To determine the effects of oral L-glutamine (L-Gln) and the dipeptide l-alanyl-glutamine (L-Ala-Gln) upon the activity of the malate-aspartate shuttle in the rat distal small intestine following ischemia and reperfusion. METHODS: Seventy-two Wistar rats (350-400g), were randomized in 2 groups (n = 36): group S (Sham) and Group T (Treatment) and divided into 12 subgroups (n = 6): A-A6, and B1-B6. The subgroups A1-A3 were subjected to sham procedures at 30 and 60 minutes. Thirty minutes before the study, rats were treated with calcium caseinate, 0.5g/Kg (subgroups A1, A4, B1, B4), L-Gln, 0.5g / kg (subgroups A2, A5, B2 and B5) or L-Ala-Gln, 0.75g/Kg (subgroups A3, A6, B3, B6), administered by gavage. Ischemia was achieved by clamping the mesenteric vessels, delimiting a segment of bowel 5 cm long and 5 cm apart from the ileocecal valve. Samples were collected 30 and 60 minutes after start of the study for real-time PCR assay of malate dehydrogenases (MDH1-2) and aspartate-aminotransferases (GOT1-2) enzymes. RESULTS: Tissue MDH and GOT mRNA expression in intestinal samples from rats preconditioned with either L-Gln or L-Ala-Gln showed no significant differences both during ischemia and early reperfusion. CONCLUSION: Activation of the malate-aspartate shuttle system appears not to be the mechanism of glutamine-mediated elevation of glucose oxidation in rat intestine during ischemia/reperfusion injury.

OBJETIVO: Determinar os efeitos da administração oral de L-glutamina (L-Gln) e do dipeptídeo L-alanil-glutamina (L-Ala-Gln) sobre a atividade do ciclo malato-aspartato no intestino delgado distal de ratos após isquemia/reperfusão. MÉTODOS: Setenta e dois ratos Wistar (350-400g) foram randomizados em 2 grupos (n = 36): T grupo S (Sham) e grupo (Tratamento) e distribuídos em 12 subgrupos (n = 6): A-A6, e B1-B6. Os subgrupos A1-A3 foram submetidos a procedimentos "sham" aos 30 e 60 minutos. Trinta minutos antes do estudo, os ratos foram tratados com caseinato de cálcio, 0,5 g/kg (subgrupos A1, A4, B1 e B4), L-Gln, 0,5 g/kg (subgrupos A2, A5, B2 e B5) ou L-Ala -Gln, 0,75g/kg (subgrupos A3, A6, B3, B6), administrado por gavagem. A isquemia foi obtida por pinçamento dos vasos mesentéricos, delimitando um segmento do intestino cinco centímetros de comprimento e 5 cm da válvula ileocecal. Amostras foram coletadas aos 30-60 minutos para ensaio de PCR em tempo real das enzimas malato desidrogenases (MDH1-2), aspartato-aminotransferase (GOT1-2). RESULTADOS: A expressão de MDH e GOT mRNA nas amostras provenientes do intestino delgado de ratos pré-condicionados com L-Gln ou L-Ala-Gln não apresentou diferenças significativas, tanto durante a isquemia como na fase inicial de reperfusão. CONCLUSÃO: Ativação do ciclo malato-aspartato não parece ser o mecanismo de elevação glutamina-mediada da oxidação da glicose no intestino de ratos durante a isquemia / reperfusão.

Dietary restriction and triiodothyronine (T3) regulation of malate-aspartate shuttle enzymes in the liver and kidney of mice.

The activities of malate-aspartate shuttle enzymes viz., cytosolic and mitochondrial aspartate aminotransferase (c- and m-AsAT) and malate dehydrogenase (c- and m-MDH) were measured in liver and kidney of ad libitum (AL) and dietary-restricted (DR) mice and also on triiodothyronine (T3) treatment. The results show that the activity (U/mg protein) of c-AsAT is increased significantly in liver and the activities of c-MDH and m-AsAT are increased significantly in kidney during DR. On T3 treatment, the activities of both the isoenzymes (c- and m-) of MDH and AsAT are increased significantly in the liver of AL- and DR-fed mice. In the kidney, m-MDH showed no effect by T3 treatment, however, c-MDH increased significantly in both AL- and DR-fed mice. In contrast, m-AsAT is increased significantly in the kidney in AL-fed mice, but was not affected in DR-fed animals. In vitro reconstitution of malate-aspartate shuttle showed a higher activity in the liver and kidney of DR-fed mice, as compared to AL-fed ones and also in the T3-treated mice, compared to untreated ones. These findings suggest that malate-aspartate shuttle enzymes are differentially regulated during DR in mice, in order to adapt to the metabolic need of liver and kidney. T3 potentially regulates the shuttle enzymes, albeit to a varying degree in the liver and kidney of AL- and DR-fed mice.

Modification of maize NADP-malic enzyme by Woodward's reagent 'K'.

Maize leaf NADP-malic enzyme was rapidly inactivated by micromolar concentrations of Woodward's reagent K (WRK). The inactivation followed pseudo-first order reaction kinetics. The order of reaction with respect to WRK was 1, suggesting that inactivation was a consequence of the modification of a single residue per active site. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification and also exhibited altered surface charge as seen from the elution profile on "Mono Q" anion exchange column and the mobility on native polyacrylamide gel electrophoresis. Substrate NADP and NADP + Mg2+ strongly protected the enzyme against WRK inactivation indicating that the modified residue may be located at or near the active site. Binding affinity of NADPH to the malic enzyme was studied by the fluorescence technique. The native enzyme binds NADPH strongly resulting in enhancement of the fluorescence emission and also causes a blue shift in the emission maximum of NADPH from 465 nm to 450 nm, however, the modified enzyme neither exhibited the enhancement of fluorescence emission nor the blue shift, indicating loss of NADPH binding site on modification. The essential carboxyl group may be involved in NADPH binding during catalysis by the enzyme.