Hypomethylation of glycine dehydrogenase promoter in peripheral blood mononuclear cells is a new diagnostic marker of hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: Glycine dehydrogenase (GLDC) plays an important role in the initiation and proliferation of several human cancers. In this study, we aimed to detect the methylation status of GLDC promoter and its diagnostic value for hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). METHODS: We enrolled 197 patients, 111 with HBV-HCC, 51 with chronic hepatitis B (CHB), and 35 healthy controls (HCs). The methylation status of GLDC promoter in peripheral mononuclear cells (PBMCs) was identified by methylation specific polymerase chain reaction (MSP). The mRNA expression was examined using real-time quantitative polymerase chain reaction (qPCR). RESULTS: The methylation frequency of the GLDC promoter was significantly lower in HBV-HCC patients (27.0%) compared to that in CHB patients (68.6%) and HCs (74.3%) (P < 0.001). The methylated group had lower alanine aminotransferase level (P = 0.035) and lower rates of tumor node metastasis (TNM) III/IV (P = 0.043) and T3/T4 (P = 0.026). TNM stage was identified to be an independent factor for GLDC promoter methylation. GLDC mRNA levels in CHB patients and HCs were significantly lower than those in HBV-HCC patients (P = 0.022 and P < 0.001, respectively). GLDC mRNA levels were significantly higher in HBV-HCC patients with unmethylated GLDC promoters than those with methylated GLDC promoters (P = 0.003). The diagnostic accuracy of alpha-fetoprotein (AFP) combined with GLDC promoter methylation for HBV-HCC was improved compared with that of AFP alone (AUC: 0.782 vs. 0.630, P < 0.001). In addition, GLDC promoter methylation was an independent predictor for overall survival of HBV-HCC patients (P = 0.038). CONCLUSIONS: The methylation frequency of GLDC promoter was lower in PBMCs from HBV-HCC patients than that from patients with CHB and HCs. The combination of AFP and GLDC promoter hypomethylation significantly improved the diagnostic accuracy of HBV-HCC.

Serum glycine dehydrogenase is associated with increased risk of lung cancer and promotes malignant transformation by regulating DNA methyltransferases expression.

Identification of novel risk factors that are critical to the initiation of lung cancer will be key for its prevention. Recently, it has been reported that glycine dehydrogenase (GLDC) can drive the formation of lung cancer initiating cells. However, there have been no perspective studies on the association between circulating GLDC and lung cancer until now. To identify whether serum GLDC is a risk factor for lung cancer, the present study conducted a nested case­control study within a Chinese cohort. Using ELISAs, serum GLDC was measured in 300 case subjects, who were subsequently diagnosed with lung cancer during follow­up, and in 600 matched healthy controls. The results revealed that serum GLDC was associated with increased lung cancer risk [odds ratio=1.48; 95% confidence intervals (1.01­2.04)]. Spearman correlation was employed to analyze the associations between age, body mass index, years of smoking and the serum concentration of GLDC. It was demonstrated that years of smoking was associated with serum GLDC (spearman's correlation, ρ=0.81) in patients with lung cancer. However, the association was attenuated in the serum of matched controls (ρ=0.48). In addition, overexpression of GLDC protein contributed to malignant transformation and inhibited microRNA (miR)­29 family expression in normal human bronchial epithelial (NHBE) cells. Aberrant methylation of tumor suppressive gene (TSG) is an early event in the development of lung cancer, which is controlled by DNA methyltransferases (DNMTs). The present study demonstrated that GLDC promoted the expression of DNMT proteins; however, the miR­29 family inhibited their expression in NHBE cells. Thus, it was concluded that elevated serum GLDC may increase lung cancer risk, and that smoking, GLDC, the miR­29 family and DNMT signaling pathways may serve an important role in early malignant transformation during the development of lung cancer.

Enfermedad de la orina de jarabe de arce: trastornos metabólicos y limitaciones en Honduras / Maple Syrup Urine Disease: Metabolic Disorders and Limitations in Honduras

Antecedentes: La Enfermedad de la Orina con olor a Jarabe de Arce es un error innato del metabolismo causada por deficiencia de actividad de la deshidrogenasa de los cetoácidos, que lleva acumular aminoácidos de cadena ramificada que produce una encefalopatía neonatal y al no ser tratada tempranamente, deja secuelas neurológicas permanentes hasta la muerte. Caso Clínico: Recién nacida producto de parto eutocico, a término, respiración espontanea, llanto vigoroso y buen tono muscular, alimentación exclusiva con lactancia materna. Antecedentes maternos de 2 hijos muertos en período neonatal. Paciente se presenta a los 7 días con pobre succión, vómitos, hipoactividad y fiebre. Examen físico: hipoactivo, reflejo de moro incompleto, llanto débil y constante. Posteriormente movimientos en extremidades superiores que simulan "boxeo" e hipertonicos, y pedaleo en extremidades inferiores, fontanela tensa y abombada, respiración irregular y bradipnea, se realiza intubación endotraqueal, ventilación mecánica y manejo en UCIN, EEG actividad eléctrica convulsiva, TAC cerebral normal. Se investiga enfermedad metabólica y se solicita tamizaje neonatal . Se inicia tiamina/levocarnitina ante sospecha de un error innato del metabolismo. A los 23 días de vida los resultados revelan niveles elevados de los aminoácidos específicos de la MSUD. Discusión: MSUD es una entidad rara en el mundo, que cursa con secuelas neurológicas permanentes y muerte de no ser tratada. Honduras no realiza métodos de Tamizaje Neonatal, es importante que el médico sospeche de manera temprana estas enfermedades, realizar un diagnóstico oportuno, se conduzca un tratamiento multidisciplinario y exista una mayor accesibilidad a las fórmulas especializadas..(AU)

Feocronocitoma: diagnóstico y tratamiento / Feocromocytoma: Diagnosis And Treatment

Antecedentes. El Feocromocitoma es un tumor de baja prevalencia que se origina en las células cromafines de la médula de las glándulas suprarrenales. Estos tumores como el tejido simpático normal se originan del neuroectodermo, siendo su principal locali - zación intraadrenal. A nivel extra adrenal se conocen también como parangangliomas que son de 3 tipos 1, 3, 4, y se deben a mutaciones génicas que codifican para las subunidades B, C, y D de la succinato deshidrogenasa, enzima mitocondrial que interviene en el ciclo de Krebs, siendo frecuentes en enfermedad de Von Hippel Lindau, neoplasia endocrina múltiple tipo 2Ay 2B y neurofibromatosis tipo 1. Ob- jetivo : El presente escrito se enfoca en mostrar al lector la evidencia acumulada hasta la actualidad mediante una revisión exhaustiva del Feocromocitoma para orientar clínicamente a estos pacientes y así llegar a un diagnóstico y tratamiento adecuado. Métodos: Se realizó una búsqueda de artículos originales, revisiones sistemáticas, y artículos de revisión en bases de revista PUBMED, SCIELO, e HINARI con años de cobertura de 2010 a 2016. Desarrollo y Conclusión. El feocromocitoma en una enfermedad amenazante por su morbilidad cardiovascular, estas manifestaciones están asociadas a las hormonas que producen las catecolaminas. El tratamiento quirúrgico por vía abierta o laparoscópica representa la cura definitiva a este padecimiento dependiendo de las características del tumor y del paciente, tiene resultados satisfactorios y comparables para diagnosticar a tiempo el tumor y evitar complicaciones...(AU)

Mutation analysis of GLDC, AMT and GCSH in cataract captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive inborn error of glycine metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. METHODS: This study describes the first screening of NKH in cataract captive-bred vervet monkeys (Chlorocebus aethiops). Glycine dehydrogenase (GLDC), aminomethyltransferase (AMT) and glycine cleavage system H protein (GCSH) were prioritized. RESULTS: Mutation analysis of the complete coding sequence of GLDC and AMT revealed six novel single-base substitutions, of which three were non-synonymous missense and three were silent nucleotide changes. CONCLUSION: Although deleterious effects of the three amino acid substitutions were not evaluated, one substitution of GLDC gene (S44R) could be disease-causing because of its drastic amino acid change, affecting amino acids conserved in different primate species. This study confirms the diagnosis of NKH for the first time in vervet monkeys with cataracts.

Osmotic stress response in Acinetobacter baylyi: identification of a glycine-betaine biosynthesis pathway and regulation of osmoadaptive choline uptake and glycine-betaine synthesis through a choline-responsive BetI repressor.

Acinetobacter baylyi, a ubiquitous soil bacterium, can cope with high salinity by uptake of choline as precursor of the compatible solute glycine betaine. Here, we report on the identification of a choline dehydrogenase (BetA) and a glycine betaine aldehyde dehydrogenase (BetB) mediating the oxidation of choline to glycine betaine. The betAB genes were found to form an operon together with the potential transcriptional regulator betI. The transcription of the betIBA operon and the two recently identified choline transporters was upregulated in response to choline and choline plus salt. The finding that the osmo-independent transporter BetT1 undergoes a higher upregulation in response to choline alone than betT2 suggests that BetT1 does not primarily function in osmoadaptation. Electrophoretic mobility shift assays led to the conclusion that BetI mediates transcriptional regulation of both, the betIBA gene operon and the choline transporters. BetI was released from the DNA in response to choline which together with the transcriptional upregulation of the bet genes in the presence of choline suggests that BetI is a choline sensing transcriptional repressor.

Epigenetic Silencing of the Putative Tumor Suppressor Gene GLDC (Glycine Dehydrogenase) in Gastric Carcinoma.

BACKGROUND: The metabolic enzyme, glycine dehydrogenase (GLDC), involved in glycine metabolism, is known to be involved in non-ketotic hyperglycinemia but not in cancer. Herein, we investigated GLDC expression and its promoter methylation in gastric cancer (GC). MATERIALS AND METHODS: GLDC expression and epigenetics were investigated using GC cell lines and tissues. Functional studies were also performed for identification of a correlation between methylated GLDC genes and gastric cancer progression. RESULTS: The results of the study can be summarized as follows: (i) GLDC was silenced in GC cell lines and tissues. The down-regulation of GLDC was closely linked to promoter methylation. (ii) Knockdown of GLDC increased cell proliferation, migration, invasion, colony formation and reduced apoptosis. (iii) In GC tissues, hypermethylation of GLDC had a significant correlation with down-regulation of the GLDC protein compared to normal gastric tissues. CONCLUSION: GLDC is a putative tumor suppressor gene involved in gastric cancer progression and hypermethylation of the GLDC promoter regulates its transcriptional silencing.

Merging pharmacometabolomics with pharmacogenomics using '1000 Genomes' single-nucleotide polymorphism imputation: selective serotonin reuptake inhibitor response pharmacogenomics.

OBJECTIVE: We set out to test the hypothesis that pharmacometabolomic data could be efficiently merged with pharmacogenomic data by single-nucleotide polymorphism (SNP) imputation of metabolomic-derived pathway data on a 'scaffolding' of genome-wide association (GWAS) SNP data to broaden and accelerate 'pharmacometabolomics-informed pharmacogenomic' studies by eliminating the need for initial genotyping and by making broader SNP association testing possible. METHODS: We previously genotyped 131 tag SNPs for six genes encoding enzymes in the glycine synthesis and degradation pathway using DNA from 529 depressed patients treated with citalopram/escitalopram to pursue a glycine metabolomics 'signal' associated with selective serotonine reuptake inhibitor response. We identified a significant SNP in the glycine dehydrogenase gene. Subsequently, GWAS SNP data were generated for the same patients. In this study, we compared SNP imputation within 200 kb of these same six genes with the results of the previous tag SNP strategy as a rapid strategy for merging pharmacometabolomic and pharmacogenomic data. RESULTS: Imputed genotype data provided greater coverage and higher resolution than did tag SNP genotyping, with a higher average genotype concordance between genotyped and imputed SNP data for '1000 Genomes' (96.4%) than HapMap 2 (93.2%) imputation. Many low P-value SNPs with novel locations within genes were observed for imputed compared with tag SNPs, thus altering the focus for subsequent functional genomic studies. CONCLUSION: These results indicate that the use of GWAS data to impute SNPs for genes in pathways identified by other 'omics' approaches makes it possible to rapidly and cost efficiently identify SNP markers to 'broaden' and accelerate pharmacogenomic studies.

ald of Mycobacterium tuberculosis encodes both the alanine dehydrogenase and the putative glycine dehydrogenase.

The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown.

Central metabolism in Mycobacterium smegmatis during the transition from O2-rich to O2-poor conditions as studied by isotopomer-assisted metabolite analysis.

Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O(2) transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD(+); (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways.

Treatment from birth of nonketotic hyperglycinemia due to a novel GLDC mutation.

OBJECTIVE: To determine whether the devastating outcome of neonatal-onset glycine encephalopathy (NKH) could be improved by instituting treatment immediately at birth rather than after symptoms are already well established. METHODS: A newborn with NKH diagnosed prenatally following the neonatal death of a previous affected sibling was treated from birth with oral sodium benzoate (250 mg/kg/day) and the NMDA receptor antagonist ketamine (15 mg/kg/day) immediately after sampling cord blood and cerebrospinal fluid (CSF) for glycine determination. Glycine cleavage system (CGS) activity was determined in placental tissue. Mutation analysis was performed by sequencing all GLDC, GCSH and AMT exons. RESULTS: CSF glycine (99 micromol/L, reference 3.8-8.0) was already markedly elevated at birth. GCS activity in placental tissue was severely reduced (2.6% of controls). A novel homozygous GLDC c.482A-->G(Y161C) missense mutation was identified. Neonatal hypotonia and apnea did not occur but the long-term outcome was poor, with intractable seizures and severe psychomotor retardation. This contrasts with the favorable outcome with early treatment in variant NKH with mild GCS deficiency (Ann Neuol 2004;56:139-143). INTERPRETATION: Prospective treatment with this regimen can favorably modify the early neonatal course of severe NKH but does not prevent the poor long-term outcome, suggesting glycine-induced prenatal injury and/or ongoing postnatal damage.

Heterozygous GLDC and GCSH gene mutations in transient neonatal hyperglycinemia.

Transient neonatal hyperglycinemia is clinically or biochemically indistinguishable from nonketotic hyperglycinemia at onset. In the case of transient neonatal hyperglycinemia, the elevated plasma and cerebrospinal fluid glycine levels are normalized within 2 to 8 weeks. To elucidate the pathogenesis of transient neonatal hyperglycinemia, we studied three patients by screening mutations in the genes that encode three components of the glycine cleavage system. Heterozygous mutations were identified in all of the three patients, suggesting that transient neonatal hyperglycinemia develops in some heterozygous carriers for nonketotic hyperglycinemia.

Glycine and alanine dehydrogenase activities are catalyzed by the same protein in Mycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation.

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.

Oxygen depletion-induced dormancy in Mycobacterium bovis BCG.

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and alpha-crystallin-like protein and is sensitive to metronidazole.

An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence.

It was demonstrated previously that abrupt transfer of vigorously aerated cultures of Mycobacterium tuberculosis to anaerobic conditions resulted in their rapid death, but gradual depletion of available O2 permitted expression of increased tolerance to anaerobiosis. Those studies used a model based on adaptation of unagitated bacilli as they settled through a self-generated O2 gradient, but the model did not permit examination of homogeneous populations of bacilli during discrete stages in that adaptation. The present report describes a model based on culture of tubercle bacilli in deep liquid medium with very gentle stirring that keeps them in uniform dispersion while controlling the rate at which O2 is depleted. In this model, at least two stages of nonreplicating persistence were seen. The shift into first stage, designated NRP stage 1, occurred abruptly at a point when the declining dissolved O2 level approached 1% saturation. This microaerophilic stage was characterized by a slow rate of increase in turbidity without a corresponding increase in numbers of CFU or synthesis of DNA. However, a high rate of production of glycine dehydrogenase was initiated and sustained while the bacilli were in this state, and a steady ATP concentration was maintained. When the dissolved O2 content of the culture dropped below about 0.06% saturation, the bacilli shifted down abruptly to an anaerobic stage, designated NRP stage 2, in which no further increase in turbidity was seen and the concentration of glycine dehydrogenase declined markedly. The ability of bacilli in NRP stage 2 to survive anaerobically was dependent in part on having spent sufficient transit time in NRP stage 1. The effects of four antimicrobial agents on the bacilli depended on which of the different physiologic stages the bacilli occupied at a given time and reflected the recognized modes of action of these agents. It is suggested that the ability to shift down into one or both of the two nonreplicating stages, corresponding to microaerophilic and anaerobic persistence, is responsible for the ability of tubercle bacilli to lie dormant in the host for long periods of time, with the capacity to revive and activate disease at a later time. The model described here holds promise as a tool to help clarify events at the molecular level that permit the bacilli to persist under adverse conditions and to resume growth when conditions become favorable. The culture model presented here is also useful for screening drugs for the ability to kill tubercle bacilli in their different stages of nonreplicating persistence.

Glyoxylate metabolism and adaptation of Mycobacterium tuberculosis to survival under anaerobic conditions.

Tuberculosis is characterized by periods in which the disease may be quiescent or even clinically inapparent, but in which tubercle bacilli persist and retain the potential to reactivate the disease. The present study was carried out in pursuit of an in vitro model which might contribute to the understanding of the physiology of nonreplicating persisters, with oxygen limitation used as the means of inducing this state. When actively growing aerated cultures of Mycobacterium tuberculosis were suddenly placed under anaerobic conditions the bacilli died rapidly, with a half-life of 10 h. When the bacilli were grown in liquid medium without agitation, they adapted to the microaerophilic conditions encountered in the sediment; the adapted bacilli in the sediment did not replicate there but were tolerant of anaerobiosis, exhibiting a half-life of 116 h. Among the early events associated with the adaptation were the synthesis of an antigen designated URB, the function of which is not known, and a fourfold increase in isocitrate lyase activity. The bacilli later exhibited a 10-fold increase in synthesis of a glycine dehydrogenase that catalyzes the reductive amination of glyoxylate, concomitantly oxidizing NADH to NAD. Specific activities of other enzymes studied were either not affected or moderately diminished in the sedimented bacilli. It is proposed that the glyoxylate synthesis in this model serves mainly to provide a substrate for the regeneration of NAD that may be required for the orderly completion of the final cycle of bacillary replication before oxygen limitation stops growth completely. This orderly shutdown is essential to continued survival of M. tuberculosis in a quiescent form.