MITOCHONDRIAL VITIATION CONGRUENTLY APTLY WITH AUTISM SPECTRUM DISORDER.

Mitochondrial dysfunction in autism leads to impair the mitochondria's ability to synthesis adenosine triphosphate (ATP) by impairment citric acid cycle as well as increase anaerobic glycolysis. Aim - measuring and evaluating the levels of mitochondrial markers; including glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), malate dehydrogenase, and pyruvate kinase) in the autistic group and knowing the possibility of using these markers to diagnose children with autism spectrum disorder. A case-control study was done in the Al-Zahraa Teaching Hospital (Kut City, Iraq) on 100 Iraqi children (male and female), between (April 2023 and January 2024). Their ages ranged between 3 and 9 years. Among them were 50 patients enrolled as autistic group and 50 healthy enrolled as control group. Blood samples were collected and bioassays for GOT, GPT, pyruvate kinase, and malate dehydrogenase were measured by ELISA technique. The autistic group showed that the urine GOT, urine GPT, serum malate, and serum pyruvate levels in the ASD group was significantly higher (P<0.001) than the control group. The ROC analysis showed that urine GOT, urine GOT, serum malate and serum pyruvate had an accuracy level of (81%,71%,77%, and 80 %) and the area under the curve (AUC) was > 0.7 (0.8),0.7, 0.7(0.76), and 0.7(0.8) thus urine GOT, urine GPT, serum, malate, and serum pyruvate are a valid diagnostic marker. There was a significant difference in the mean urine and serum concentrations of mitochondrial markers (GOT, GPT, malate dehydrogenase, and pyruvate kinase) between autistic children and the control group due to mitochondrial dysfunction.

Effect of Storage Conditions on the Volatilome, Biochemical Composition and Quality of Golden Delicious and Red Delicious Apple (Malus domestica) Varieties.

The effects of normal (NA) and controlled atmosphere (CA) storage and postharvest treatment with 1-methylcyclopropene (1-MCP) before CA storage for 5 months on the volatilome, biochemical composition and quality of 'Golden Delicious' (GD) and 'Red Delicious' (RD) apples were studied. Apples stored under NA and CA maintained and 1-MCP treatment increased firmness in both cultivars. NA storage resulted in a decrease of glucose, sucrose and fructose levels in both cultivars. When compared to CA storage, 1-MCP treatment caused a more significant decrease in sucrose levels and an increase in glucose levels. Additionally, 1-MCP-treated apples exhibited a significant decrease in malic acid content for both cultivars. All storage conditions led to significant changes in the abundance and composition of the volatilome in both cultivars. GD and RD apples responded differently to 1-MCP treatment compared to CA storage; higher abundance of hexanoate esters and (E,E)-α-farnesene was observed in RD apples treated with 1-MCP. While 1-MCP was effective in reducing (E,E)-α-farnesene abundance in GD apples, its impact on RD apples was more limited. However, for both cultivars, all storage conditions resulted in lower levels of 2-methylbutyl acetate, butyl acetate and hexyl acetate. The effectiveness of 1-MCP is cultivar dependent, with GD showing better results than RD.

Respiratory Function of Mitochondria in Patients with Coronary Heart Disease with Implantable Cardioverter-Defibrillator.

We studied the respiratory activity of mitochondria in peripheral blood leukocytes from 36 patients with coronary heart disease (CHD) and a history of ventricular tachyarrhythmias required cardioverter-defibrillator implantation. The measurements were carried out in incubation buffers with different oxidation substrates (succinate and pyruvate-malate mixture). In pyruvate-malate incubation buffer, oxygen consumption rate and respiratory control coefficients in patients with triggered device did not differ significantly from those in patients without cardioverter-defibrillator triggering. At the same time, respiratory control coefficients were below the reference values. In succinate buffer, values of mitochondrial parameters were significantly lower in patients with triggered devices. Our findings indicate that mitochondria of patients with non-triggered cardioverters-defibrillators have better functional and metabolic plasticity. It was concluded that activity of respiratory processes in mitochondria could be an indicator that should be taken into the account when assessing the risk of developing ventricular tachyarrhythmias.

Impact of low molecular weight organic acids on heavy metal(loid) desorption in biochar-amended paddy soil.

Low molecular weight organic acids (LMWOAs) are important soil components and play a key role in regulating the geochemical behavior of heavy metal(loid)s. Biochar (BC) is a commonly used amendment that could change LMWOAs in soil. Here, four LMWOAs of oxalic acid (OA), tartaric acid (TA), malic acid (MA), and citric acid (CA) were evaluated for their roles in changing Cd and SB desorption behavior in contaminated soil with (S1-BC) or without BC (S1) produced from Paulownia biowaste. The results showed that OA, TA, MA, and CA reduced soil pH with rising concentrations, and biochar partially offset the pH reduction by LMWOAs. The LMWOAs reduced Cd desorption from the soil at low concentrations but increased Cd desorption at high concentrations, and CA was the most powerful in this regard. The LMWOAs had a similar effect on Sb desorption, and CA was the most effective species of LMWOAs. Adding BC to the soil affects Cd and Sb dynamics by reducing the Cd desorption but increasing Sb desorption from the soil and increasing the distribution coefficient (Kd) values of Cd but lowering the Kd values of Sb. This study helped understand the effects of LMWOAs on the geochemical behavior of Cd and Sb in the presence of biochar, as well as the potential risks of biochar amendment in enhancing Sb desorption from contaminated soil.

Effects of 3 days of citrulline malate supplementation on short-duration repeated sprint running performance in male team sport athletes.

Citrulline malate (CM) is purported to be an ergogenic aid during various types of exercise performance. However, the effects of CM on repeated sprint performance (RSP) are under-explored. In a placebo-controlled, double-blind, counterbalanced cross-over design, male university-level team sport athletes (n = 13) performed two familiarization trials, after which CM or placebo (PLA) (8 × 1 g tablets each day) were taken on the 2 days prior to, and with breakfast on the morning of, each main experimental trial. The main experimental trials employed a RSP protocol consisting of 10 repetitions of 40 m maximal shuttle run test (MST) with a 30 s interval between the start of each sprint. Sprint times and heart rate were recorded throughout the MST, and blood lactate concentrations were measured before, immediately after, and 5 min after completing the MST. CM resulted in better RSP compared to PLA, as indicated by a lower sprint performance decrement (Sdec: CM, 4.68% ± 1.82% vs. PLA, 6.10% ± 1.83%; p = 0.03; ES = 0.77), which was possibly influenced by the fastest sprint time being faster in CM (CM, 8.16 ± 0.34 s vs. PLA, 8.29 ± 0.39 s; p = 0.011; ES = 0.34). There were no differences between CM and PLA in average sprint time (p = 0.54), slowest sprint time (p = 0.48), blood lactate concentrations (p = 0.73) or heart rate (p = 0.18), nor was there a condition × time interaction effect across the 10 sprints (p = 0.166). Three days of CM supplementation (8 g daily) attenuated the sprint performance decrement during short-duration high-intensity exercise in the form of running RSP in male university-level team sport athletes.

BomMDH1 regulates malate-mediated oxidative stress in tobacco BY-2 suspension cells.

Programmed cell death (PCD) is a genetically regulated process of cell suicide essential for plant development. The 'malate valve' is a mechanism that ensures redox balance across different subcellular compartments. In broccoli, the BomMDH1 gene encodes malate dehydrogenase in mitochondria, a critical enzyme in the 'malate circulation' pathway. This study investigates the functional role of BomMDH1 in malate (MA)-induced apoptosis in bright yellow-2 (BY-2) suspension cells. Findings revealed that transgenic cells overexpressing BomMDH1 showed enhanced viability under MA-induced oxidative stress compared to wild-type (WT) cells. Overexpression of BomMDH1 also reduced levels of reactive oxygen species (ROS), hydrogen peroxide (H2O2), and malondialdehyde (MDA), while increasing the expression of antioxidant enzyme genes such as NtAPX, NtAOX1a, NtSOD, and NtMDHAR. Additionally, treatment with salicylhydroxamic acid (SHAM), a characteristic inhibitor of mitochondrial respiration, further improved the anti-apoptotic activity of BY-2 cells. Overall, these results highlighted the function of the BomMDH1 gene and the potential of SHAM treatment in mitigating oxidative stress in BY-2 suspension cells.

γ-Aminobutyric acid (GABA) priming alleviates acid-aluminum toxicity to roots of creeping bentgrass via enhancements in antioxidant defense and organic metabolites remodeling.

MAIN CONCLUSION: γ-Aminobutyric acid alleviates acid-aluminum toxicity to roots associated with enhanced antioxidant metabolism as well as accumulation and transportation of citric and malic acids. Aluminum (Al) toxicity has become the main limiting factor for crop growth and development in acidic soils and is further being aggravated worldwide due to continuous industrial pollution. The current study was designed to examine effects of GABA priming on alleviating acid-Al toxicity in terms of root growth, antioxidant defense, citrate and malate metabolisms, and extensive metabolites remodeling in roots under acidic conditions. Thirty-seven-day-old creeping bentgrass (Agrostis stolonifera) plants were used as test materials. Roots priming with or without 0.5 mM GABA for 3 days were cultivated in standard nutrient solution for 15 days as control or subjected to nutrient solution containing 5 mM AlCl3·6H2O for 15 days as acid-Al stress treatment. Roots were sampled for determinations of root characteristics, physiological and biochemical parameters, and metabolomics. GABA priming significantly alleviated acid-Al-induced root growth inhibition and oxidative damage, despite it promoted the accumulation of Al in roots. Analysis of metabolomics showed that GABA priming significantly increased accumulations of organic acids, amino acids, carbohydrates, and other metabolites in roots under acid-Al stress. In addition, GABA priming also significantly up-regulated key genes related to accumulation and transportation of malic and citric acids in roots under acid-Al stress. GABA-regulated metabolites participated in tricarboxylic acid cycle, GABA shunt, antioxidant defense system, and lipid metabolism, which played positive roles in reactive oxygen species scavenging, energy conversion, osmotic adjustment, and Al ion chelation in roots.

Metabolic engineering of Aspergillus niger for accelerated malic acid biosynthesis by improving NADPH availability.

Microbial production of L-malic acid from renewable carbon sources has attracted extensive attention. The reduced cofactor NADPH plays a key role in biotransformation because it participates in both biosynthetic reactions and cellular stress responses. In this study, NADPH or its precursors nicotinamide and nicotinic acid were added to the fermentation medium of Aspergillus niger RG0095, which significantly increased the yield of malic acid by 11%. To further improve the titer and productivity of L-malic acid, we increased the cytoplasmic NADPH levels of A. niger by upregulating the NAD kinases Utr1p and Yef1p. Biochemical analyses demonstrated that overexpression of Utr1p and Yef1p reduced oxidative stress, while also providing more NADPH to catalyze the conversion of glucose into malic acid. Notably, the strain overexpressing Utr1p reached a malate titer of 110.72 ± 1.91 g L-1 after 108 h, corresponding to a productivity of 1.03 ± 0.02 g L-1 h-1. Thus, the titer and productivity of malate were increased by 24.5% and 44.7%, respectively. The strategies developed in this study may also be useful for the metabolic engineering of fungi to produce other industrially relevant bulk chemicals.

Enantiospecific crystallisation behaviour of malic acid in mechanochemical reactions with vinpocetine.

We report an intriguing example of enantioselectivity in the formation of new multicomponent crystalline solid containing vinpocetine and malic acid. Several experimental data sets confirmed that the multicomponent system presents a clear enantiospecific crystallisation behaviour both in the solid-state and in solution: only the system consisting of vinpocetine and L-malic acid produces a free-flowing solid consisting of a new crystalline form, while the experiments with D-malic acid produced an amorphous and often deliquescent material. The new vinpocetine-L-malic system crystallizes in the monoclinic space group of P21 and in a 1:1 molar ratio, where the two molecules are linked through intermolecular hydrogen bonds in the asymmetric unit. The vinpocetine-DL-malic system was partially crystalline (with also traces of unreacted vinpocetine) with diffraction peaks corresponding to those of vinpocetine-L-malic acid. Solid-state NMR experiments revealed strong ionic interactions in all the three systems. However, while vinpocetine-L-malic acid system was a pure and crystalline phase, the other two systems persistently showed the presence of unreacted vinpocetine. This resulted in a significant worsening of the dissolution profile with respect to the pure vinpocetine-L-malic crystalline salt, whose dissolution kinetics appeared superior.

Antidiabetic Properties of Caffeoylmalic Acid, a Bioactive Natural Compound Isolated from Urtica dioica.

The uncontrolled hyperglycemia that characterizes diabetes mellitus (DM) causes several complications in the organism. DM is among the major causes of deaths, and the limited efficacy of current treatments push the search for novel drug candidates, also among natural compounds. We focused our attention on caffeoylmalic acid, a phenolic derivative extracted from Urtica dioica, a plant investigated for its potential against type 2 DM. This compound was tested for its antidiabetic activity in vitro through a glucose uptake assay, in vivo in a mouse DM model and through molecular docking towards α-amylase and α-glucosidase. The effects on glucose blood level, liver enzymes, insulin and creatinine levels as well as on lipid and blood parameters, considered biochemical markers of diabetes, were also evaluated. The results showed an antidiabetic activity in vitro and in vivo, as the compound stimulates glucose absorbtion and reduces blood glucose levels. Moreover, it ameliorates lipid profile, liver and blood parameters, with moderate effect on insulin secretion. Taken together, these findings pave the way for the compounds from this class of caffeoylmalic acid as potential antidiabetic compounds.

Quantification of Free Radicals from Vaping Electronic Cigarettes Containing Nicotine Salt Solutions with Different Organic Acid Types and Concentrations.

Electronic (e-) cigarette formulations containing nicotine salts from a range of organic acid conjugates and pH values have dominated the commercial market. The acids in the nicotine salt formulations may alter the redox environment in e-cigarettes, impacting free radical formation in e-cigarette aerosol. Here, the generation of aerosol mass and free radicals from a fourth-generation e-cigarette device was evaluated at 2 wt % nicotine salts (pH 7, 30:70 mixture propylene glycol to vegetable glycerin) across eight organic acids used in e-liquids: benzoic acid (BA), salicylic acid (SLA), lactic acid (LA), levulinic acid (LVA), succinic acid (SA), malic acid (MA), tartaric acid (TA), and citric acid (CA). Furthermore, 2 wt % BA nicotine salts were studied at the following nicotine to acid ratios: 1:2 (pH 4), 1:1 (pH 7), and 2:1 (pH 8), in comparison with freebase nicotine (pH 10). Radical yields were quantified by spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. The EPR spectra of free radicals in the nicotine salt aerosol matched those generated from the Fenton reaction, which are primarily hydroxyl (OH) radicals and other reactive oxygen species (ROS). Although the aerosol mass formation was not significantly different for most of the tested nicotine salts and acid concentrations, notable ROS yields were observed only from BA, CA, and TA under the study conditions. The e-liquids with SLA, LA, LVA, SA, and MA produced less ROS than the 2 wt % freebase nicotine e-liquid, suggesting that organic acids may play dual roles in the production and scavenging of ROS. For BA nicotine salts, it was found that the ROS yield increased with a higher acid concentration (or a lower nicotine to acid ratio). The observation that BA nicotine salts produce the highest ROS yield in aerosol generated from a fourth-generation vape device, which increases with acid concentration, has important implications for ROS-mediated health outcomes that may be relevant to consumers, manufacturers, and regulatory agencies.

Efficient synthesis of L-malic acid by malic enzyme biocatalysis with CO2 fixation.

The malic enzyme (ME) catalyzes the synthesis of L-malic acid (L-MA) from pyruvic acid and CO2 with NADH as the reverse reaction of L-MA decarboxylation. Carboxylation requires excess pyruvic acid, limiting its application. In this study, it was determined that CO2 was the carboxyl donor by parsing the effects of HCO3- and CO2, which provided a basis for improving the L-MA yield. Moreover, the concentration ratio of pyruvic acid to NADH was reduced from 70:1 to 5:1 using CO2 to inhibit decarboxylation and to introduce the ME mutant A464S with a 2-fold lower Km than that of the wild type. Finally, carboxylation was coupled with NADH regeneration, resulting in a maximum L-MA yield of 77 % based on the initial concentration of pyruvic acid. Strategic modifications, including optimal reactant ratios and efficient mutant ME, significantly enhanced L-MA synthesis from CO2, providing a promising approach to the biotransformation process.

Targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for malate-aspartate shuttle and tumour progression.

BACKGROUND: A series of studies have demonstrated the emerging involvement of transfer RNA (tRNA) processing during the progression of tumours. Nevertheless, the roles and regulating mechanisms of tRNA processing genes in neuroblastoma (NB), the prevalent malignant tumour outside the brain in children, are yet unknown. METHODS: Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test. RESULTS: c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients. CONCLUSIONS: These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.

Addition of sodium malonate alters the morphology and increases the critical flux during tangential flow filtration of precipitated immunoglobulins.

Recent studies have demonstrated that one can control the packing density, and in turn the filterability, of protein precipitates by changing the pH and buffer composition of the precipitating solution to increase the structure/order within the precipitate. The objective of this study was to examine the effect of sodium malonate, which is known to enhance protein crystallizability, on the morphology of immunoglobulin precipitates formed using a combination of ZnCl2 and polyethylene glycol. The addition of sodium malonate significantly stabilized the precipitate particles as shown by an increase in melting temperature, as determined by differential scanning calorimetry, and an increase in the enthalpy of interaction, as determined by isothermal titration calorimetry. The sodium malonate also increased the selectivity of the precipitation, significantly reducing the coprecipitation of DNA from a clarified cell culture fluid. The resulting precipitate had a greater packing density and improved filterability, enabling continuous tangential flow filtration with minimal membrane fouling relative to precipitates formed under otherwise identical conditions but in the absence of sodium malonate. These results provide important insights into strategies for controlling precipitate morphology to enhance the performance of precipitation-filtration processes for the purification of therapeutic proteins.

A Nuclear Magnetic Resonance (NMR)- and Mass Spectrometry (MS)-Based Saturation Kinetics Model of a Bryophyllum pinnatum Decoction as a Treatment for Kidney Stones.

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5-10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation.

Combined full-length transcriptomic and metabolomic analysis reveals the molecular mechanisms underlying nutrients and taste components development in Primulina juliae.

BACKGROUND: Primulina juliae has recently emerged as a novel functional vegetable, boasting a significant biomass and high calcium content. Various breeding strategies have been employed to the domestication of P. juliae. However, the absence of genome and transcriptome information has hindered the research of mechanisms governing the taste and nutrients in this plant. In this study, we conducted a comprehensive analysis, combining the full-length transcriptomics and metabolomics, to unveil the molecular mechanisms responsible for the development of nutrients and taste components in P. juliae. RESULTS: We obtain a high-quality reference transcriptome of P. juliae by combing the PacBio Iso-seq and Illumina sequencing technologies. A total of 58,536 cluster consensus sequences were obtained, including 28,168 complete protein coding transcripts and 8,021 Long Non-coding RNAs. Significant differences were observed in the composition and content of compounds related to nutrients and taste, particularly flavonoids, during the leaf development. Our results showed a decrease in the content of most flavonoids as leaves develop. Malate and succinate accumulated with leaf development, while some sugar metabolites were decreased. Furthermore, we identified the different accumulation of amino acids and fatty acids, which are associated with taste traits. Moreover, our transcriptomic analysis provided a molecular basis for understanding the metabolic variations during leaf development. We identified 4,689 differentially expressed genes in the two developmental stages, and through a comprehensive transcriptome and metabolome analysis, we discovered the key structure genes and transcription factors involved in the pathways. CONCLUSIONS: This study provides a high-quality reference transcriptome and reveals molecular mechanisms associated with the development of nutrients and taste components in P. juliae. These findings will enhance our understanding of the breeding and utilization of P. juliae as a vegetable.

The synergistic effect of typical chiral organic acids and solution chemistry conditions on the transport of 2-arylpropionic acid chiral derivatives in porous media.

The hazards of man-made chiral compounds are of great public concern, with reports of worrying stereoselective compounds and an urgent need to assess their transport. This study evaluated the transport of 2-arylpropionic acid derivatives enantiomers (2-APA) in porous media under a variety of solution chemistry conditions via column packing assays. The results revealed the introduction of Malic acid (MA) enantiomers enhanced the mobility of 2-APA enantiomers, but the enhancement effect was different for different 2-APA enantiomers. Batch sorption experiments confirmed that the MA enantiomers occupied the sorption site of the quartz sand, thus reducing the deposition of the 2-APA enantiomer. Homo- or heterochirality between 2-APA and MA dominates the transport of 2-APA enantiomers, with homochirality between them triggering stronger retention and vice versa. Further evaluating the effect of solution chemistry conditions on the transport of 2-APA enantiomers, increased ionic strength attenuated the mobility of 2-APA enantiomers, whereas introduced coexisting cations enhanced the retention of 2-APA enantiomers in the column. The redundancy analyses corroborated these solution chemistry conditions were negatively correlated with the transport of 2-APA enantiomers. The coupling of pH and these conditions reveals electrostatic forces dominate the transport behavior and stereoselective interactions of 2-APA enantiomers. Distinguishing the transport of enantiomeric pair helps to understand the difference in stereoselectivity of enantiomers and promises to remove the more hazardous one.

Characterization of unique EDTA-insensitive methylthioalkylmalate synthase from Eutrema japonicum and its potential application in synthetic biology.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), a derivative of glucosinolate with a six-carbon chain, is a compound found in wasabi and has diverse health-promoting properties. The biosynthesis of glucosinolates from methionine depends on a crucial step catalyzed methylthioalkylmalate synthases (MAMs), which are responsible for the generation of glucosinolates with varying chain lengths. In this study, our primary focus was the characterization of two methylthioalkyl malate synthases, MAM1-1 and MAM1-2, derived from Eutrema japonicum, commonly referred to as Japanese wasabi. Eutremajaponicum MAMs (EjMAMs) were expressed in an Escherichiacoli expression system, subsequently purified, and in vitro enzymatic activity was assayed. We explored the kinetic properties, optimal pH conditions, and cofactor preferences of EjMAMs and compared them with those of previously documented MAMs. Surprisingly, EjMAM1-2, categorized as a metallolyase family enzyme, displayed 20% of its maximum activity even in the absence of divalent metal cofactors or under high concentrations of EDTA. Additionally, we utilized AlphaFold2 to generate structural homology models of EjMAMs, and used in silico analysis and mutagenesis studies to investigate the key residues participating in catalytic activity. Moreover, we examined in vivo biosynthesis in E. coli containing Arabidopsis thaliana branched-chain amino acid transferase 3 (AtBCAT3) along with AtMAMs or EjMAMs and demonstrated that EjMAM1-2 exhibited the highest conversion rate among those MAMs, converting l-methionine to 2-(2-methylthio) ethyl malate (2-(2-MT)EM). EjMAM1-2 shows a unique property in vitro and highest activity on converting l-methionine to 2-(2-MT)EM in vivo which displays high potential for isothiocyanate biosynthesis in E. coli platform.

The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport.

AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons. METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate. RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.

Alternative Splicing Underpins the ALMT9 Transporter Function for Vacuolar Malic Acid Accumulation in Apple.

Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum-activated malate transporter 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 (Ma1α) drastically decreases fruit acidity in "Royal Gala" apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1ß is 68 amino acids shorter with much lower expression than the full-length protein Ma1α. Ma1ß does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1ß/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1ß level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1ß or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9-mediated malate transport underlying apple fruit acidity.