Enhanced protection for interfacial lipid ozonolysis by sulfur-containing amino acids.

Sulfur-containing amino acids have been proposed as drugs for lipid oxidation associated with diseases for a long time, but the molecular-level mechanism on the effectiveness of sulfur-containing amino acids against lipid oxidation remains elusive. In this work, with the interfacial sensitivity mass spectrometry method, oxidation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), a widely used model lipid, was significantly inhibited on hung droplet surface in presence of sulfur-containing amino acids, such as cysteine (Cys) and methionine (Met). Both the Cys and Met showed a self-sacrificing protection. The amino acids with -S-R tails (R referring to methyl or t-butyl group) showed more effective against POPG oxidation than those with -SH tails, and this process was not related to the conformations of amino acids. The low effectiveness of Cys during the interfacial chemistry was proved to arise from the formation of disulfide bond. This study extends the current understanding of chemistry of sulfur-containing amino acids and provides insights to aid the sulfur-containing amino acids against cell oxidation.

Alleviating effect of methionine on intestinal mucosal injury induced by heat stress.

Climate change is an increasing concern of stakeholders worldwide. The intestine is severely impacted by the heat stress. This study aimed to investigate the alleviating effects of methionine on the intestinal damage induced by heat stress in mice. The mice were divided into four groups: control group (C), methionine deficiency group (MD), methionine + heat stress group (MH), and methionine deficiency + heat stress group (MDH). Histopathological techniques, PAS-Alcian blue staining, immunohistochemistry method, biochemical quantification method, ELISA, and micro method were used to study the changes in the intestinal mucosal morphology, the number of goblet cells, the expression of tight junction proteins, the peroxide product contents and antioxidant enzyme activities, the intestinal mucosal damage, the content of immunoglobulins and HSP70, the activity of Na+/K+-ATPase. The results showed that methionine can improve intestinal mucosal morphology (increase the villi height, V/C value, and muscle layer thickness, decrease crypt depth), increase the expression of tight junction proteins (Claudin-1, Occludin, ZO-1) and the content of DAO, decrease the content of intestinal mucosa damage markers (ET, FABP2) and peroxidation products (MDA), increase the activity of antioxidant enzymes (GR, GSH-Px, SOD), the number of goblet cells, the contents of immunoglobulins (sIgA, IgA, IgG, IgM) and stress protein (HSP70), and the activity of Na+/K+-ATPase. It is suggested that methionine can alleviate intestinal damage in heat-stressed mice.

Protection of Membrane Contact Protein by the Methionine Sulfoxide Reductases.

In this News and Views, I discuss our recent publication that established how steroidogenic acute regulatory-related lipid transfer domain-3 (STARD3), a membrane contact protein situated at lysosomal membranes, plays a role in the detoxification of cholesterol hydroperoxide. STARD3's methionine residues can be oxidized to methionine sulfoxide by cholesterol hydroperoxide, after which methionine sulfoxide reductases reduce the methionine sulfoxide residues back to methionine. The reaction also results in the reduction of the cholesterol hydroperoxide to an alcohol. The cyclic oxidation and reduction of methionine residues in STARD3 at membrane contact sites creates a catalytically efficient mechanism for detoxification of cholesterol hydroperoxide during cholesterol transport, thus protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Comprehensive insight into exploring the potential of microbial enzymes in cancer therapy: Progress, challenges, and opportunities: A review.

Microbial enzymes are crucial catalysts in various industries due to their versatility and efficiency. The microbial enzymes market has recently expanded due to increased demand for many reasons. Among them are eco-friendly solutions, developing novel microbial strains with enhanced enzymes that perform under harsh conditions, providing sustainability, and raising awareness about the benefits of enzyme-based products. By 2030, the global enzyme market is expected to account for $525 billion, with a growth rate of 6.7 %. L-asparaginase and L-glutaminase are among the leading applied microbial enzymes in antitumor therapy, with a growing market share of 16.5 % and 9.5 %, respectively. The use of microbial enzymes has opened new opportunities to fight various tumors, including leukemia, lymphosarcoma, and breast cancer, which has increased their demand in the pharmaceutical and medicine sectors. Despite their promising applications, commercial use of microbial enzymes faces challenges such as short half-life, immunogenicity, toxicity, and other side effects. Therefore, this review explores the industrial production, purification, formulation, and commercial utilization of microbial enzymes, along with an overview of the global enzyme market. With ongoing discoveries of novel enzymes and their applications, enzyme technology offers promising avenues for cancer treatment and other therapeutic interventions.

Simultaneous determination of total homocysteine, methionine, methylmalonic acid and 2-methylcitric acid in dried blood spots by ultra-performance liquid chromatography-tandem mass spectrometry.

Homocysteine, methionine, methylmalonic acid and 2-methylcitric acid are clinically relevant markers in the methionine, propionate, and cobalamin metabolism. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneously determining total homocysteine, methionine, methylmalonic acid and 2-methylcitric acid in dried blood spots. Three 3.2 mm discs were punched from each calibrator, quality control, and sample dried blood spot into a 96-well U-plate. Each sample was spiked with internal standards and extracted. Then the supernatant was transferred to another 96-well U-plate. After nitrogen drying, the dried residues were reconstituted, centrifuged, and the resulting supernatant was transferred to another 96-well plate for analysis. The method was performed using UPLC-MS/MS within 3 min, validated according to guidance documents, and applied to 72 samples from confirmed patients with methionine, propionate, and cobalamin metabolism disorders. The UPLC-MS/MS method provided satisfactory separation of the four analytes. The R2 values were ≥ 0.9937 for all analytes. The recoveries ranged from 94.17 to 114.29 %, and the coefficients of variation for intraday and interday precision were 0.19 % to 5.23 % and 1.02 % to 6.89 %, respectively. No significant carry-over was detected for the four analytes, and most of confirmed samples exhibited biomarker patterns characteristic of the relevant disorders. A simple and fast UPLC-MS/MS method was successfully developed, validated, and applied to clinical samples for the simultaneous determination of total homocysteine, methionine, methylmalonic acid, and 2-methylcitric acid in dried blood spots.

Methionine aminopeptidases: Potential therapeutic target for microsporidia and other microbes.

Methionine aminopeptidases (MetAPs) have emerged as a target for medicinal chemists in the quest for novel therapeutic agents for treating cancer, obesity, and other disorders. Methionine aminopeptidase is a metalloenzyme with two structurally distinct forms in humans, MetAP-1 and MetAP-2. The MetAP2 inhibitor fumagillin, which was used as an amebicide in the 1950s, has been used for the successful treatment of microsporidiosis in humans; however, it is no longer commercially available. Despite significant efforts and investments by many pharmaceutical companies, no new MetAP inhibitors have been approved for the clinic. Several lead compounds have been designed and synthesized by researchers as potential inhibitors of MetAP and evaluated for their potential activity in a wide range of diseases. MetAP inhibitors such as fumagillin, TNP-470, beloranib, and reversible inhibitors and their analogs guide new prospects for MetAP inhibitor development in the ongoing quest for new pharmacological indications. This perspective provides insights into recent advances related to MetAP, as a potential therapeutic target in drug discovery, bioactive small molecule MetAP2 inhibitors, and data on the role of MetAP-2 as a therapeutic target for microsporidiosis.

Alterations in metabolome and lipidome in patients with in-stent restenosis.

CONTEXT: In-stent restenosis (ISR) can lead to blood flow obstruction, insufficient blood supply to the brain, and may even result in serious complications such as stroke. Endothelial cell hyperproliferation and thrombosis are the primary etiologies, frequently resulting in alterations in intravascular metabolism. However, the metabolic changes related to this process are still undermined. OBJECTIVE: We tried to characterize the serum metabolome of patients with ISR and those with non-restenosis (NR) using metabolomics and lipidomics, exploring the key metabolic pathways of this pathological phenomenon. RESULTS: We observed that the cysteine and methionine pathways, which are associated with cell growth and oxidative homeostasis, showed the greatest increase in the ISR group compared to the NR group. Within this pathway, the levels of N-formyl-l-methionine and L-methionine significantly increased in the ISR group, along with elevated levels of downstream metabolites such as 2-ketobutyric acid, pyruvate, and taurocholate. Additionally, an increase in phosphatidylcholine (PC) and phosphatidylserine (PS), as well as a decrease in triacylglycerol in the ISR group, indicated active lipid metabolism in these patients, which could be a significant factor contributing to the recurrence of blood clots after stent placement. Importantly, phenol sulfate and PS(38:4) were identified as potential biomarkers for distinguishing ISR, with an area under the curve of more than 0.85. CONCLUSIONS: Our study revealed significant metabolic alterations in patients with ISR, particularly in the cysteine and methionine pathways, with phenol sulfate and PS(38:4) showing promise for ISR identification.

Dietary methionine functions in proliferative zone maintenance and production of eggs via sams-1 in Caenorhabditis elegans.

The maintenance of germ cells is critical for the prosperity of offspring. The amount of food consumption is known to be closely related to reproduction, i.e., the number of eggs decreases under calorie-restricted conditions in various organisms. Previous studies in Caenorhabditis elegans have reported that calorie restriction reduces the number of eggs and the reduction can be rescued by methionine. However, the effect of methionine on the reproductive process has not been fully understood. In this study, to assess the gonadal function of methionine metabolism, we firstly demonstrated that a depletion in dietary methionine resulted in reduced levels of S-adenosyl-l-methionine (SAM) and S-adenosyl homocysteine (SAH) in wild-type N2, but not in glp-1 mutants, which possess only a few germ cells. Second, we found no recovery in egg numbers upon methionine administration in SAM synthase (sams)-1 mutants. Furthermore, a reduced number of proliferative zone nuclei exhibited in the sams-1 mutants was not rescued via methionine. Thus, our results have shown that dietary methionine is required for the normal establishment of both the germline progenitor pool and fecundity, mediated by sams-1.

Citrullus mucosospermus Extract Exerts Protective Effects against Methionine- and Choline-Deficient Diet-Induced Nonalcoholic Steatohepatitis in Mice.

In recent years, there has been increasing interest in exploring the potential therapeutic advantages of Citrullus mucosospermus extracts (CME) for nonalcoholic steatohepatitis (NASH). In this study, we investigated the therapeutic effects of CME on NASH using a mice model. High-performance liquid chromatography (HPLC) was employed to identify cucurbitacin E and cucurbitacin E-2-O-glucoside from the CME. Although CME did not significantly alter the serum lipid levels in methionine- and choline-deficient (MCD) mice, it demonstrated a protective effect against MCD diet-induced liver damage. CME reduced histological markers, reduced alanine transaminase (ALT) and aspartame transaminase (AST) levels, and modulated key NASH-related genes, including C/EBPα, PPARγ, Fas, and aP2. In addition, CME was found to restore hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) activity, both crucial for fat catabolism, and reduced the levels of pro-inflammatory cytokines. Furthermore, CME demonstrated the potential to mitigate oxidative stress by maintaining or enhancing the activation and expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and superoxide dismutase (SOD), both pivotal players in antioxidant defense mechanisms. These findings underscore the promising therapeutic potential of CME in ameliorating liver damage, inflammation, and oxidative stress associated with NASH.

Single-center, double-blind, randomized, placebo-controlled pilot study of Canoxidin® for prevention of catheter encrustation in patients with indwelling catheters.

INTRODUCTION AND OBJECTIVES: Long-term use of an indwelling catheter is associated with complications such as catheter encrustation and infection. Canoxidin® is a novel oral treatment that can potentially prevent catheter encrustation, as it contains a urine acidifier and a combination of two crystallization inhibitors. This study aimed to evaluate the effects of Canoxidin® on catheter encrustation in patients with indwelling Foley catheters. PATIENTS AND METHODS: This was a single-center, double-blind, randomized, placebo-controlled study. Neuro-urology patients aged ≥18 years with an indwelling catheter (urethral or suprapubic) were randomized to treatment consisting of either Canoxidin® or placebo for one month. Foley catheters (two per patient, one before treatment and one after treatment) were removed for analysis of the presence and degree of encrustation. RESULTS: A total of 40 patients were enrolled and randomized, 28 of whom had analyzable catheters (13 assigned to Canoxidin® and 15 assigned to placebo). The patients had a mean age of 51.8 years, and eight (28.6%) were female. Two patients (13.3%) in the placebo group and eight patients (61.5%) in the Canoxidin® group experienced an improvement (less encrustation). There was a significant association between Canoxidin® and improvement (odds ratio: 10.4, 95% confidence interval: 1.6 to 66.9, P = 0.016). No adverse effects attributable to the treatment were reported. CONCLUSIONS: The overall rate of catheter encrustation was high among those with indwelling Foley catheters. One-month treatment with Canoxidin® reduced the formation of these encrustations, with an excellent short-term safety profile.

Glutamine enhances pneumococcal growth under methionine semi-starvation by elevating intracellular pH.

Introduction: Bacteria frequently encounter nutrient limitation in nature. The ability of living in this nutrient shortage environment is vital for bacteria to preserve their population and important for some pathogenic bacteria to cause infectious diseases. Usually, we study how bacteria survive after nutrient depletion, a total starvation condition when bacteria almost cease growth and try to survive. However, nutrient limitation may not always lead to total starvation. Methods: Bacterial adaptation to nutrient shortage was studied by determining bacterial growth curves, intracellular pH, intracellular amino acid contents, gene transcription, protein expression, enzyme activity, and translation and replication activities. Results: No exogenous supply of methionine results in growth attenuation of Streptococcus pneumoniae, a human pathogen. In this paper, we refer to this inhibited growth state between ceased growth under total starvation and full-speed growth with full nutrients as semi-starvation. Similar to total starvation, methionine semi-starvation also leads to intracellular acidification. Surprisingly, it is intracellular acidification but not insufficient methionine synthesis that causes growth attenuation under methionine semi-starvation. With excessive glutamine supply in the medium, intracellular methionine level was not changed, while bacterial intracellular pH was elevated to ~ 7.6 (the optimal intracellular pH for pneumococcal growth) by glutamine deamination, and bacterial growth under semi-starvation was restored fully. Our data suggest that intracellular acidification decreases translation level and glutamine supply increases intracellular pH to restore translation level, thus restoring bacterial growth. Discussion: This growth with intracellular pH adjustment by glutamine is a novel strategy we found for bacterial adaptation to nutrient shortage, which may provide new drug targets to inhibit growth of pathogenic bacteria under semi-starvation.

Redox-dependent plasticity of oxMIF facilitates its interaction with CD74 and therapeutic antibodies.

MIF is a ubiquitous protein involved in proinflammatory processes, which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid, produced by neutrophil-released myeloperoxidase (MPO) under inflammatory conditions, effectively oxidizes MIF into the oxMIF isoform, which is specifically recognized by the anti-oxMIF therapeutic antibody, ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure, including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation, whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA, SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor, CD74, which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor, and a route for therapeutic intervention targeting the oxMIF isoform.

Methionine Source and Level Modulate Gut pH, Amino Acid Transporters and Metabolism Related Genes in Broiler Chickens.

This study determined the effects of two methionine (Met) sources at three total sulfur amino acids (TSAA) to lysine ratios (TSAA/Lys) on gut pH, digestive enzyme activity, amino acid transporter expression, and Met metabolism of broilers. The birds were randomly assigned to a 2 × 3 factorial arrangement with Met sources (dl-Met and dl-2-hydroxy-4-(methylthio)-butanoic acid (OH-Met)) and TSAA/Lys (0.58, 0.73, and 0.88) from 1 to 21 days. The results demonstrated that dl-Met and OH-Met supported the same growth performance, but high TSAA/Lys ratio reduced the feed intake and body weight (P < 0.05). OH-Met reduced the crop chyme pH and enhanced the jejunal lipase activity (P < 0.05). ATB0,+ expression decreased with increased dl-Met levels in the duodenum; the low TSAA/Lys ratio induced a stronger mRNA expression of basolateral Met transporters. OH-Met resulted in an increase of cystathionine ß-synthase expression in the liver and a decrease in serum homocysteine levels at middle TSAA/Lys ratio compared with dl-Met treatment (P < 0.05). In conclusion, two Met sources support the same growth, but OH-Met acidified the crop chyme. The investigated transporter transcripts differed significantly along the small intestine. At the middle TSAA/Lys ratio, OH-Met showed a higher metabolic tendency of the trans-sulfuration pathway compared with dl-Met.

Recombinant Methioninase Is Selectively Synergistic With Doxorubicin Against Wild-type Fibrosarcoma Cells Compared to Normal Cells and Overcomes Highly-Doxorubicin-resistant Fibrosarcoma.

BACKGROUND/AIM: Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro. MATERIALS AND METHODS: The 50% inhibitory concentrations (IC50) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope. RESULTS: The IC50 for doxorubicin was 3.3 µM for HT1080 cells, 12.4 µM for DR-HT1080 cells, and 7.25 µM for Hs27 cells. The IC50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells. CONCLUSION: The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma.

Effects of L-Methionine and DL-Methionine on Growth Performance, Methionine-Metabolizing Enzyme Activities, Feather Traits, and Intestinal Morphology of Medium-Growing, Yellow-Feathered Chickens between 1 and 30 Days of Age.

This experiment investigated the effects of L-Methionine (L-Met) on growth performance, Met-metabolizing enzyme activity, feather traits, and small intestinal morphological characteristics, and compared these with DL-Methionine (DL-Met) for medium-growing, yellow-feathered broilers during the starter phase. Furthermore, the aim was to provide recommendations for the appropriate dietary Met levels in feed. A total of 1584 1-d broilers were randomly divided into 11 treatment groups with six replicates of 24 birds each: basal diet (CON, Met 0.28%), basal diet + L-Met (0.04%, 0.08%, 0.12%, 0.16%, 0.20%), and basal diet + DL-Met (0.04%, 0.08%, 0.12%, 0.16%, 0.20%). The total trial period was 30 days. Compared with broilers fed the basal diet, those fed 0.04 to 0.20% supplemental Met had higher final body weight (FBW), average daily feed intake (ADFI), average daily gain (ADG), and lower feed-to-gain ratio (F: G) (p < 0.05). Compared with DL-Met groups, the L-Met group had higher FBW and ADG (p < 0.05). The relative bioavailability (RBV) of L-Met in ADG of 1-30 d was 142.5%. Chicks fed diets supplemented with L-Met had longer fourth primary feather lengths compared to birds fed the control and diets supplemented with DL-Met (p < 0.05). Compared to the control, birds supplemented with DL-Met or L-Met had an increased moulting score (p ≤ 0.05). Chicks fed diets supplemented with L-Met had lower activities of methionine adenosyl transferase (MAT) compared to those fed the basal diet or supplemented with DL-Met (p < 0.05). Chicks supplemented with either DL-Met or L-Met had higher activities of cystathionine ß-synthase (CBS) than those fed the basal diet (p < 0.05). Compared with the control, chicks fed diets supplemented with either DL-Met or L-Met had an enhanced level of albumin in plasma (p < 0.05). There were no obvious differences in the plasma content of uric acid and total protein among the treatments (p > 0.05). Chicks fed diets supplemented with either DL-Met or L-Met had higher villus height and V/C in the duodenal than chicks fed the basal diet (p < 0.05). The jejunum morphology was not affected by either L-Met or DL-Met supplementation (p > 0.05). Therefore, dietary supplementation with DL-Met or L-Met improved the growth performance, feather traits, and intestinal morphological characteristics of medium-growing, yellow-feathered broiler chickens aged 1 to 30 d by decreasing the enzyme activities of Met methylation (MAT) and increasing the enzyme activities of the sulfur transfer pathway (CBS), and supplementation with L-Met showed a better improvement compared with DL-Met. The relative efficacy of L-Met to DL-Met was 142.5% for ADG of yellow-feathered broilers. The appropriate Met levels for medium-growing, yellow-feathered broilers are between 0.36~0.38% (supplementation with DL-Met) or 0.32~0.33% (supplementation with L-Met) when based on ADG and feed-to-gain ratio.

The Effects of Seleno-Methionine in Cadmium-Challenged Human Primary Chondrocytes.

Cadmium (Cd) is a potentially toxic element able to interfere with cellular functions and lead to disease or even death. Cd accumulation has been demonstrated in cartilage, where it can induce damage in joints. The aim of this study was to evaluate the effect of CdCl2 on primary cultures of human chondrocytes and the possible protective effect of seleno-methionine (Se-Met). Human primary articular chondrocytes were cultured and treated as follows: control groups, cells challenged with 7.5 µM and 10 µM CdCl2 alone, and cells pretreated with 10 and 20 µM Se-Met and then challenged with 7.5 µM and 10 µM CdCl2. Twenty-four hours after incubation, cell viability, histological evaluation with hematoxylin-eosin stain, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed. Furthermore, reverse transcription-PCR was carried out to evaluate mRNA levels of BAX, BAK1, CASP-3, and CASP-9. After CdCl2 challenge at both doses, a reduced cell viability and an overexpression of BAX, BAK1, CASP-3, and CASP-9 genes, as well as a high number of TUNEL-positive cells, were demonstrated, all parameters becoming higher as the dose of CdCl2 was increased. The pretreatment with Se-Met lowered the expression of all considered genes, improved cell viability and morphological changes, and reduced the number of TUNEL-positive cells. It was concluded that Se-Met plays a protective role against CdCl2-induced structural and functional changes in chondrocytes in vitro, as it improved cell viability and showed a positive role in the context of the apoptotic pathways. It is therefore suggested that a translational, multifaceted approach, with plant-based diets, bioactive functional foods, nutraceuticals, micronutrients, and drugs, is possibly advisable in situations of environmental pollution caused by potentially toxic elements.

Congenital Heart Disease and Genetic Changes in Folate/Methionine Cycles.

Congenital heart disease is one of the most common congenital malformations and thus represents a considerable public health burden. Hence, the identification of individuals and families with an increased genetic predisposition to congenital heart disease (CHD) and its possible prevention is important. Even though CHD is associated with the lack of folate during early pregnancy, the genetic background of folate and methionine metabolism perturbations and their influence on CHD risk is not clear. While some genes, such as those coding for cytosolic enzymes of folate/methionine cycles, have been extensively studied, genetic studies of folate transporters (de)glutamation enzymes and mitochondrial enzymes of the folate cycle are lacking. Among genes coding for cytoplasmic enzymes of the folate cycle, MTHFR, MTHFD1, MTR, and MTRR have the strongest association with CHD, while among genes for enzymes of the methionine cycle BHMT and BHMT2 are the most prominent. Among mitochondrial folate cycle enzymes, MTHFD2 plays the most important role in CHD formation, while FPGS was identified as important in the group of (de)glutamation enzymes. Among transporters, the strongest association with CHD was demonstrated for SLC19A1.

Use of the Saccharomycopsis schoenii MET17 promoter for regulated heterologous gene expression.

The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for specific processes or the effect of genes expressed only under specific conditions. Saccharomycopsis species show a unique property of necrotrophic mycoparasitism that is activated upon starvation. Here we describe the use of the MET17 promoter of S. schoenii as a tool to regulate gene expression based on the availability of methionine. Conditional expression was tested using lacZ and GFP reporter genes. Gene expression could be strongly down-regulated by the addition of methionine or cysteine to the growth medium and upregulated by starvation for methionine. We used X-gal (5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside) to detect lacZ-expression in plate assays and ONPG (ortho-nitrophenyl-ß-galactopyranoside) as a substrate for ß-galactosidase in liquid-phase assays. For in vivo expression analyses we used fluorescence microscopy for the detection and localization of a MET17-driven histone H4-GFP reporter gene. With these assays we demonstrated the usefulness of the MET17 promoter to regulate expression of genes based on methionine availability. In silico analyses revealed similar promoter motifs as found in MET3 genes of Saccharomyces cerevisiae and Ashbya gossypii. This suggests a regulation of the MET17 promoter by CBF1 and MET31/MET32 in conjunction with the transcriptional activator MET4, which were also identified in the S. schoenii genome.

This article describes the characterization of the S. schoenii MET17 promoter for regulated gene expression.

The Combination of Methionine Restriction and Docetaxel Synergistically Arrests Androgen-independent Prostate Cancer But Not Normal Cells.

Background/Aim: Androgen-independent prostate cancer (AIPC) is resistant to androgen-depletion therapy and is a recalcitrant disease. Docetaxel is the first-line treatment for AIPC, but has limited efficacy and severe side-effects. All cancers are methionine-addicted, which is termed the Hoffman effect. Recombinant methioninase (rMETase) targets methionine addiction. The purpose of the present study was to determine if the combination of docetaxel and rMETase is effective for AIPC. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of docetaxel and rMETase alone were determined for the human AIPC cell line PC-3 and Hs27 normal human fibroblasts in vitro. The synergistic efficacy for PC-3 and Hs27 using the combination of docetaxel and rMETase at their IC50s for PC-3 was determined. Results: The IC50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC50 of rMETase for PC-3 and for Hs27 was 0.67 U/ml and 0.76 U/ml, respectively. The combination of docetaxel and rMETase was synergistic for PC-3 but not Hs27 cells. Conclusion: The combination of a relatively low concentration of docetaxel and rMETase was synergistic and effective for AIPC. The present results also suggest that the effective concentration of docetaxel can be reduced by using rMETase, which may reduce toxicity. The present results also suggest the future clinical potential of the combination of docetaxel and rMETase for AIPC.

Synergy of Rapamycin and Methioninase on Colorectal Cancer Cells Requires Simultaneous and Not Sequential Administration: Implications for mTOR Inhibition.

Background/Aim: Rapamycin inhibits the mTOR protein kinase. Methioninase (rMETase), by degrading methionine, targets the methionine addiction of cancer cells and has been shown to improve the efficacy of chemotherapy drugs, reducing their effective doses. Our previous study demonstrated that rapamycin and rMETase work synergistically against colorectal-cancer cells, but not on normal cells, when administered simultaneously in vitro. In the present study, we aimed to further our previous findings by exploring whether  synergy exists between rapamycin and rMETase when used sequentially against HCT-116 colorectal-carcinoma cells, compared to simultaneous administration, in vitro. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line were previously determined using the CCK-8 cell viability assay (11). We then examined the efficacy of rapamycin and rMETase, both at their IC50, administered simultaneously or sequentially on the HCT-116 cell line, with rapamycin administered before rMETase and vice versa. Results: The IC50 for rapamycin and rMETase, determined from previous experiments (11), was 1.38 nM and 0.39 U/ml, respectively, of HCT-116 cells. When rMETase was administered four days before rapamycin, both at the IC50, there was a 30.46% inhibition of HCT-116 cells. When rapamycin was administered four days before rMETase, both at the IC50, there was an inhibition of 41.13%. When both rapamycin and rMETase were simultaneously administered, both at the IC50, there was a 71.03% inhibition. Conclusion: Rapamycin and rMETase have synergistic efficacy against colorectal-cancer cells in vitro when administered simultaneously, but not sequentially.