Adenosine Monophosphate Deaminase 1 Research Articles

Molecular characterization of adenosine monophosphate deaminase 1 and the correlation analysis between its mRNA expression levels and inosine monophosphate content in large yellow croaker (Larimichthys crocea)

Declining flesh quality has drawn considerable attention in the farmed large yellow croaker (LYC; Larimichthys crocea) industry. Inosine monophosphate (IMP) is the primary flavor substance in aquatic animals. Adenosine monophosphate deaminase 1 (AMPD1) plays a critical role in IMP formation by catalyzing the deamination of AMP to IMP in the purine nucleotide cycle. To further evaluate the correlation between ampd1 mRNA expression levels and IMP content in the LYC muscle tissue, the relevant open reading frame (ORF) of L. crocea (Lcampd1) was cloned, and the IMP content and Lcampd1 mRNA expression in the muscles of LYCs of different sizes were examined. The ORF cDNA of Lcampd1 was 2211 bp in length and encoded a polypeptide of 736 amino acids (AAs). The deduced protein, LcAMPD1, possesses conserved AMPD active regions (SLSTDDP) and shows high homology with AMPD proteins of other teleost fishes. The genomic DNA sequence of Lcampd1 exhibits a high degree of evolutionary conservation in terms of structural organization among species. Phylogenetic analysis of the deduced AA sequence revealed that teleost fish and mammalian AMPD1 were separate from each other and formed a cluster with AMPD3, suggesting that AMPD1 and AMPD3 arose by duplication of a common primordial gene. In healthy LYC, Lcampd1 mRNA was expressed only in the muscle tissue. The IMP content in the muscle of LYCs with different average body weights was measured by high-performance liquid chromatography; the results showed that the IMP content in the muscle of LYCs with greater body weight was significantly higher than that in LYC with lower body weight. Moreover, a similar trend in Lcampd1 expression was observed in these muscle tissues. The Pearson correlation analysis further showed that the Lcampd1 mRNA expression was positively correlated with IMP content in the muscles of different-sized LYCs. These results suggest the potential function of Lcampd1 in determining the IMP content in LYC and provide a theoretical basis for flesh quality improvement, as well as a scientific basis for the development of the molecular breeding of LYC.

The AMPD1 Gene’s rs17602729 Polymorphism and Athletic Performance in Track and Field Athletes

The aim of the current study was to determine whether the rs17602729 polymorphism in the Adenosine monophosphate deaminase-1 (AMPD1) gene is related to elite athlete performance. A total of 60 elite athletes, aged 18–35, who were split into two groups—31 sprinters/power athletes and 29 endurance athletes—as well as 20 control/sedentary individuals, willingly participated in the study. The performance levels of the athletes, based on their personal bests (PBs), were rated using the World Athletics (WA) score. Whole exome sequencing (WES) was performed on the genomic DNA that was extracted from the subjects’ blood samples. Using linear regression models, the study sought to determine the relationship between the athletes’ PB, sex, and sport type and the rs17602729 polymorphism both within and between the groups. The distribution of the GG, GA, and AA genotypes of the rs17602729 polymorphism differed significantly within and between the groups, according to the data (p < 0.05). Nonetheless, no statistically significant variations were observed in the correlation between the athletes’ PBs and the rs17602729 polymorphism among the groups (p > 0.05). In conclusion, the G allele of the ADMP1 rs17602729 polymorphism appears to provide a benefit to sprinters and power athletes. Nonetheless, to confirm this hypothesis, additional research with more participants and a multi-genetic analysis approach is required.

Early postmortem beef muscle proteome and metabolome variations due to supranutritional zinc and ractopamine hydrochloride supplementation.

It was hypothesized that the longissimus thoracis (LT) muscle proteome, phosphoproteome, and metabolome could explain postmortem metabolism and tenderness differences in muscle from cattle supplemented zinc (Zn) and/or ractopamine hydrochloride (RH). High percentage Angus steers (N = 20) were fed in a 2 × 2 factorial assigned to Zn and RH treatments: control (CON; n = 10; analyzed 36mg Zn/kg dry matter [DM]) or supranutritional Zn supplementation (SUPZN; n = 10; control diet + 60mg Zn/kg DM [from ZnSO4] + 60mg Zn/kg DM [from Zn-amino acid complex]) for the entire 89-d trial. During the 28 d before harvest, steers were blocked by body weight within Zn treatments to RH treatments of 0 (NO; n = 10) or 300mg (RAC; n = 10) per steer per day. Steers were harvested at the Iowa State Meat Laboratory, where pH decline (1, 3, 6, and 24h postmortem) was measured. At 24h postmortem, LT muscle sections were removed from carcasses, and steaks were analyzed for Warner-Bratzler shear force (WBSF) values at 1, 3, 7, and 14 d postmortem. Muscle samples were taken at 1h, 1, 3, 7, and 14 d postmortem for the following analysis: troponin-T degradation (1, 3, 7, and 14 d postmortem), myosin heavy chain analysis (1h postmortem), sarcoplasmic proteome analysis through tandem mass tagging analysis (1h and 1 d postmortem), metabolome analysis (1h and 1 d postmortem), and phosphoproteome analysis (1h postmortem). SUPZN-NO tended to have a lower (P = 0.06) pH at 6h postmortem and a lower WBSF value (P = 0.06) at 1 d postmortem. CON-RAC had a higher (P = 0.04) pH at 6h postmortem and WBSF value (P < 0.01) at 1 d postmortem. A lower pH at 6h postmortem and lower WBSF value at 1 d postmortem in the SUPZN-NO treatment was accompanied by more sorbitol and fructose at 1 d postmortem, and less myosin regulatory light chain 2 at 1h postmortem, and less adenosine monophosphate deaminase 1 (AMPD1) at 1 d postmortem than all other treatments. A higher pH at 6h postmortem and higher WBSF value at 1 d postmortem in CON-RAC and SUPZN-RAC was accompanied by more soluble structural proteins (troponin-T and myosin-7) at 1h postmortem than CON-NO. At 1h postmortem, CON-RAC had more glyceraldehyde-3-phosphate dehydrogenase than CON-NO or SUPZN-RAC. Differences in energy metabolism enzymes, metabolites, and structural proteins may affect ATP production, rigor development, and lactate buildup which may explain the differences in postmortem metabolism and tenderness development at 1 d postmortem.

Functional identification of PGM1 in the regulating development and depositing of inosine monophosphate specific for myoblasts.

Inosine monophosphate (IMP) is naturally present in poultry muscle and plays a key role in improving meat flavour. However, IMP deposition is regulated by numerous genes and complex molecular networks. In order to excavate key candidate genes that may regulate IMP synthesis, we performed proteome and metabolome analyses on the leg muscle, compared to the breast muscle control of 180-day-old Jingyuan chickens (hens), which had different IMP content. The key candidate genes identified by a differential analysis were verified to be associated with regulation of IMP-specific deposition. The results showed that the differentially expressed (DE) proteins and metabolites jointly involve 14 metabolic pathways, among which the purine metabolic pathway closely related to IMP synthesis and metabolism is enriched with four DE proteins downregulated (with higher expression in breast muscles than in leg muscles), including adenylate kinase 1 (AK1), adenosine monophosphate deaminase 1 (AMPD1), pyruvate kinase muscle isoenzyme 2 (PKM2) and phosphoglucomutase 1 (PGM1), six DE metabolites, Hypoxanthine, Guanosine, L-Glutamine, AICAR, AMP and Adenylsuccinic acid. Analysis of PGM1 gene showed that the high expression of PGM1 promoted the proliferation and differentiation of myoblasts and inhibited the apoptosis of myoblasts. ELISA tests have shown that PGM1 reduced adenosine triphosphate (ATP) and IMP and uric acid (UA), while enhancing the biosynthesis of hypoxanthine (HX). In addition, up-regulation of PGM1 inhibited the expression of purine metabolism pathway related genes, and promoted the IMP de novo and salvage synthesis pathways. This study preliminarily explored the mechanism of action of PGM1 in regulating the growth and development of myoblasts and specific IMP deposition in Jingyuan chickens, which provided certain theoretical basis for the development and utilization of excellent traits in Jingyuan chickens.

High fructose-induced skeletal muscle insulin resistance could be alleviated by berberine via AMPD1 and ADSL.

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis that is activated in response to an elevated intracellular AMP/ATP ratio. Although many studies have shown berberine is an AMPK activator widely used in metabolic syndrome, how to properly control AMPK activity remains obscure. Our present study aimed to examine the protective effect of berberine against fructose-induced insulin resistance in rats and L6 cells, as well as its potential activation mechanism on AMPK. The results showed that berberine effectively reversed body weight gain, Lee's index, dyslipidemia and insulin intolerance. Moreover, berberine alleviated inflammatory response, antioxidant capacity and promoted glucose uptake in vivo and in vitro. The beneficial effect was associated with upregulation of both Nrf2 and AKT/GLUT4 pathways, which were regulated by AMPK. Notably, berberine could increase the level of AMP and the ratio of AMP/ATP, then further activate AMPK. Mechanistic experiments revealed that berberine suppressed the expression of adenosine monophosphate deaminase 1 (AMPD1) and promoted the expression of adenylosuccinate synthetase (ADSL). Taken together, berberine exerted excellent therapeutic effect on insulin resistance. And its mode of action may be related to the AMP-AMPK pathway by regulating AMPD1 and ADSL.

Metformin alleviates long-term high-fructose diet-induced skeletal muscle insulin resistance in rats by regulating purine nucleotide cycle

Nutrient excess caused by excessive fructose intake can lead to insulin resistance and dyslipidemia, which further causes the development of metabolic syndrome. Metformin is a well-known AMPK activator widely used for the treatment of metabolic syndrome, while the mechanism of AMPK activation remains unclear. The present study aimed to investigate the pharmacological effects of metformin on fructose-induced insulin resistance rat, and the potential mechanism underlying AMPK activation in skeletal muscle tissue. Results indicated that metformin significantly ameliorated features of insulin resistance, including body weight, Lee's index, hyperinsulinemia, dyslipidemia, insulin intolerance and pancreatic damage. Moreover, treatment with metformin attenuated the inflammatory response in serum and enhanced the antioxidant capacity in skeletal muscle tissue. The therapeutic effects of metformin on fructose-induced insulin resistance may be related to the activation of AMPK to regulate Nrf2 pathway and mitochondrial abnormality. Additionally, metformin suppressed the expression of adenosine monophosphate deaminase 1 (AMPD1) and up-regulated the expression of adenylosuccinate synthetase (ADSS) in the purine nucleotide cycle (PNC), which facilitated the increase of AMP level and the ratio of AMP/ATP. Therefore, we proposed a novel mechanism that metformin activated AMPK via increasing AMP by regulating the expression of AMPD1 and ADSS in PNC pathway.

Molecular characterization of adenosine monophosphate deaminase 1 and its regulatory mechanism for inosine monophosphate formation in triploid crucian carp.

Inosine monophosphate (IMP) is the main flavoring substance in aquatic animal, and adenosine monophosphate deaminase1 (AMPD1) gene is a key gene in IMP formation. At present, the research on the mechanism of AMPD1 regulating IMP formation in aquatic animal is still blank. In this study, in order to study the mechanism of AMPD1 regulating IMP formation in fish, the full open reading frame (ORF) of AMPD1 which was 2160bp was obtained for the first time in triploid crucian carp (Carassius auratus). It encoded 719 amino acids with a molecular mass of 82.97 kDa, and the theoretical isoelectric point value was 6.31. The homology analysis showed that the homology of triploid crucian carp and diploid Carassius auratus was the highest, up to 99%. And the phylogenetic tree showed that triploid crucian carp was grouped with diploid Carassius auratus, Culter alburnus, and Danio rerio. And real-time fluorescence quantitative results showed that AMPD1 was expressed specifically in muscle of triploid crucian carp (p < 0.05). The results of detection the localization of AMPD1 in cells indicated that the AMPD1 was mainly localized in cytoplasm and cell membrane. Further, we examined the effects of glutamate which was the promotor of IMP formation on the expression of AMPD1 and the formation of IMP in vivo and in vitro experiments, the results showed that 3% glutamate and 2 mg/ml glutamate could significantly promote AMPD1 expression and IMP formation in triploid crucian carp muscle tissue and muscle cells (p < 0.05). Then we inhibited the expression of AMPD1 in vivo and in vitro experiments, we found the formation of IMP in muscle tissue and muscle cells of triploid crucian carp all were inhibited and they affected the gene expression of AMPK-mTOR signaling pathway. The all results showed that AMPD1 mediated glutamate through AMPK-mTOR signaling pathway to regulate the formation of fish IMP.

Comparative Analysis of Skeleton Muscle Proteome Profile between Yak and Cattle Provides Insight into High-Altitude Adaptation

Background::Mechanisms underlying yak adaptation to high-altitude environments have been investigated at the levels of morphology, anatomy, physiology, genome and transcriptome, but have not been explored at the proteome level.Objective:The protein profiles were compared between yak and cattle to explore molecular mechanisms underlying yak adaptation to high altitude conditions.Methods:In the present study, an antibody microarray chip was developed, which included 6,500 mouse monoclonal antibodies. Immunoprecipitation and mass spectrometry were performed on 12 selected antibodies which showed that the chip was highly specific. Using this chip, muscle tissue proteome was compared between yak and cattle, and 12 significantly Differentially Expressed Proteins (DEPs) between yak and cattle were identified. Their expression levels were validated using Western blot.Results:ompared with cattle, higher levels of Rieske Iron-Sulfur Protein (RISP), Cytochrome C oxidase subunit 4 isoform 1, mitochondrial (COX4I1), ATP synthase F1 subunit beta (ATP5F1B), Sarcoplasmic/ Endoplasmic Reticulum Calcium ATPase1 (SERCA1) and Adenosine Monophosphate Deaminase1 (AMPD1) in yak might improve oxygen utilization and energy metabolism. Pyruvate Dehydrogenase protein X component (PDHX) and Acetyltransferase component of pyruvate dehydrogenase complex (DLAT) showed higher expression levels and L-lactate dehydrogenase A chain (LDHA) showed lower expression level in yak, which might help yak reduce the accumulation of lactic acid. In addition, higher expression levels of Filamin C (FLNC) and low levels of AHNAK and Four and a half LIM domains 1 (FHL1) in yak might reduce the risks of pulmonary arteries vasoconstriction, remodeling and hypertension.Conclusion:Overall, the present study reported the differences in protein profile between yak and cattle, which might be helpful to further understand molecular mechanisms underlying yak adaptation to high altitude environments.

A functional mutation in the AMPD1 promoter region affects promoter activity and breast meat freshness in chicken.

Freshness is an important index to determine the quality deterioration (protein degradation and changes in appearance) of chilled chicken meat and is a primary consideration of consumers. Adenosine monophosphate deaminase 1 (AMPD1) catalyzes the deamination of adenosine monophosphate to inosine monophosphate in skeletal muscle and is the rate-limiting step in the purine nucleotide cycle. Inosine monophosphate is regarded as an important indicator of meat freshness in chicken. This study investigated the association of polymorphisms in the chicken AMPD1 promoter region with meat freshness during freezing storage. An SNP (c. -905G>A) was found to be associated with the freshness (K-value) of chicken breast meat. Chickens with the AA genotype had significantly lower K-values than those with GG and AG genotypes (P<0.01). Individuals with the AA genotype also had higher breast meat AMPD1 mRNA levels than did those with the GG and AG genotypes (P<0.01, P<0.05). A luciferase assay revealed that genotype AA had greater transcriptional activity than genotype GG. Transcription factor binding site analysis identified distinct putative transcription factor binding sites in the two alleles of mutation site c. -905. In summary, we identified an SNP (c. -905G>A) in the promoter region of the AMPD1 gene that may modulate the binding affinity of different transcription factors to control AMPD1 expression and affect the freshness K-value of chicken meat.

Targeting the Plasma Cell Specific Purine Metabolic Enzyme, AMPD1, Induces Multiple Myeloma Cell Death Accompanying NAD Depletion

Introduction. Novel agents have improved the prognosis of multiple myeloma (MM) patients. However, MM is still an incurable disease. In order to achieve a cure, it is necessary to develop novel therapeutic agents that are highly specific to MM cells and target different pathways from the present anti-MM agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and MM cells, and to examine whether it can be a novel target for MM therapy. Materials and methods. Public available gene expression websites, GenomicScape and Genevestigator were utilized to study genes specifically expressed in human plasma cells and MM cells. To examine gene expression in MM cell lines, gene expression data set from the Cancer Cell Line Encyclopedia (CCLE) was analyzed. AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression in MM cell lines and patient derived MM cells were examined by western blot and immunohistochemistry. Genes co-expressed with AMPD1 in MM cells were identified using public available gene expression datasets. Molecular pathway analysis was conducted using Molecular Signatures Database. Cell viability of MM cell lines, peripheral blood mononuclear cells (PBMCs) and patient derived bone marrow mononuclear cells treated by AMPD1 inhibitor (compound #3) (Admyre T et al. Chemistry &amp; Biology. 2014; 21: 1486-1496.) were analyzed by flow-cytometry after staining with 7AAD. Intracellular NAD and NADH concentrations in MM cell lines were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Z-VAD-FMK (Caspase inhibitor) and Nec-1 (RIP1 kinase inhibitor) were used in combination with compound #3 to study the mechanism of AMPD1 inhibition induced MM cell death. Results. We identified several genes specifically expressed in human plasma cells and MM cells using public available gene expression websites. Among the identified genes, we focused on AMPD1, a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), since this gene has not been previously studied in MM. We found that AMPD1 gene expression was limited to MM cell lines and patient derived MM cells through CCLE analysis and RT-PCR. AMPD1 protein expression was detected only in MM cell lines, bone marrow MM cells and extramedullary plasmacytomas. Genes associated with AMPD1 expression were related to hypoxic pathways. MM cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to those cultured under normoxia, indicating that AMPD1 plays a significant role in MM cell surviving under hypoxic condition such as the bone marrow microenvironment. Compound #3 induced cell death in MM cell lines and patient derived MM cells, while toxicity against PBMCs and non-MM cells were minimal. Compound #3 was more effective against MM cell lines cultured under hypoxia compared to those under normoxia, reflecting the higher AMPD1 expression under hypoxia. Since, AMPD1 is associated with purine metabolism, we analyzed the intracellular concentration of NAD and NADH, which are major cellular metabolites, in MM cell lines post compound #3 treatment. Marked decrease of NAD concentration and NAD / NADH ratio was observed in compound #3 treated MM cell lines compared to control, demonstrating that AMPD1 inhibition depletes intracellular NAD leading to MM cell death. Compound #3 treated MM cell lines showed increase in Annexin V- PI+ and Annexin V+ PI+ fractions. Additionally, Z-VAD-FMK and Nec-1 treatment did not reverse cell death induced by compound #3, indicating a distinct mechanism of cytotoxicity by compound #3 from other anti-MM agents inducing apoptosis and necroptosis. Conclusions. This is the first report so far that AMPD1 is specifically expressed in MM cells. AMPD1 inhibitor showed specific cytotoxicity against MM cells, while toxicities against non-MM cells were minimal. Additionally, AMPD1 inhibitor was more effective under hypoxic condition, suggesting that AMPD1 inhibition works more efficiently in the bone marrow microenvironment. Considering the specificity against MM cells and its distinct mechanism of action from the present anti-MM agents, AMPD1 inhibition is a potent novel therapeutic strategy for MM. Disclosures Matsuoka: Bristol-Myers Squibb Corp.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

The Purine Metabolic Enzyme AMPD1 Is a Novel Therapeutic Target for Multiple Myeloma

Introduction. Novel agents have improved the prognosis of multiple myeloma. However, side effects of novel agents have been a huge issue for especially elderly and frail patients. Additionally, despite the high remission rate by novel agents, multiple myeloma is still an incurable disease. In order to improve those issues, it is necessary to develop a new therapeutic strategy which is highly specific to myeloma cells and which targets a different pathway from the present anti-myeloma agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and myeloma cells, and to examine whether it can be a novel target for multiple myeloma therapy.Materials and methods. A public available gene expression website GenomicScape (http://www.genomicscape.com/) and Genevestigator (https://genevestigator.com/gv/index.jsp) were utilized in order to study genes specifically expressed in human plasma cells and myeloma cells. To examine gene expression in myeloma cell lines, we utilized the gene expression data set of multiple cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE: http://www.broadinstitute.org/ccle). AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression was analyzed by westernblot and immunohistochemistry. Genes co-expressed with AMPD1 in human myeloma cells were identified using public available gene expression datasets (GSE 4581, GSE 9782). Molecular pathways associated with genes co-expressed with AMPD1 were analyzed using Molecular Signatures Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp). Cell viability of myeloma cell lines and peripheral blood mononuclear cells (PBMCs) treated by AMPD inhibitors (compound #3, #4) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed using 7AAD dye and flow-cytometry.Results. Public gene expression screening analysis identified several genes specifically expressed in human plasma cells and myeloma cells. Among the identified genes, we focused on AMPD1, which has not been previously studied in multiple myeloma. CCLE analysis and RT-PCR analysis showed that AMPD1 is specifically expressed in bone marrow plasma cells, myeloma cell lines and patient derived myeloma cells. AMPD1 protein expression was limited to myeloma cell lines, human bone marrow myeloma cells and extramedullary plasmacytomas. Genes co-expressed with AMPD1 in human myeloma cells were associated with hypoxic pathways. Myeloma cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to cell lines cultured under normoxic condition. AMPD inhibitors induced cell death in myeloma cell lines from around 50 uM, while the effect against PBMCs were minimal. AMPD inhibitors were more effective against myeloma cell lines under hypoxic condition compared to normoxic condition, reflecting the higher AMPD1 expression under hypoxia.Conclusions. AMPD1 is a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), freeing ammonia during the process. AMPD1 has been previously reported that its expression is limited to skeletal muscles. This is the first report so far that among leukocytes and hematological malignancies, AMPD1 is specifically expressed in bone marrow plasma cells and myeloma cells. We also showed that AMPD1 expression in myeloma cells are increased under hypoxia. This indicates that AMPD1 plays a significant role in myeloma cells surviving under hypoxic conditions such as the bone marrow microenvironment. AMPD inhibitors showed cytotoxicity on myeloma cell lines in vitro, while PBMCs were not affected. Additionally, AMPD inhibitors were more effective under hypoxic condition, suggesting that AMPD1 inhibition works more specifically in the bone marrow microenvironment. Our report raise the possibility that AMPD1 inhibition can be a novel therapeutic strategy for multiple myeloma. Detailed analysis of myeloma cell death by AMPD1 inhibition is now undergoing. DisclosuresMatsuoka:Bristol Myers Squibb: Research Funding.

GENETIC POLYMORPHISMS INFLUENCING EFFICACY AND SAFETY OF METHOTREXATE IN RHEUMATOID ARTHRITIS

Objective: This review will summarize pharmacogenetic studies of single nucleotide polymorphisms in genes coding enzymes of methotrexate (MTX) pathway, related to its response and toxicity in rheumatoid arthritis (RA). In addition, this review focuses on the racial and ethnic differences in distribution of the polymorphisms in genes related to efficacy and toxicity of MTX in RA.Methods: Articles were searched using Pubmed database using the search term “pharmacogenetics and MTX and arthritis.” The search revealed 72 articles, of which 27 were given special importance, due to open-access.Results: Many genes and single nucleotide polymorphisms are investigated in this context, and the highlighting genes are ATP-binding cassette proteins (ABCB1) reduced folate carrier (RFC), methylenetetrahydrofolatereductase (MTHFR), gamma-glutamyl hydrolase (GGH), serine hydroxyl methyltransferase (SHMT), 5-aminoimidazole-4-carboxamide ribonucleotidetransformylase (ATIC), methionine synthase reductase (MTRR), methionine synthase (MS), adenosine monophosphate deaminase1 (AMPD1), inosine triphosphate pyrophosphatase (ITPA). The study highlighted RFC-1 80AA, ITPA 94CC, and AMPD1 347CC as responders. The allelic types prone to toxicity were MTHFR 677TT, MTRR 2756AA, MS 66GG, SHMT 1420CC, ATIC 347GG, and thymidylate synthase *3/*2. The genotypes reported as non-responders were ABCB1 3434TT, MTHFR 1298AA, DHFR A317G, GGH 16CC, and GGH 401TT.Conclusion: Although these studies highlight inconsistency in results, due to the difference in sample size and assessment parameters and racial and ethnic differences, larger prospective studies are essential to reach the cornerstone of the concept of personalized medicine.

Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer.

Altered re-wiring of cell metabolism and transcriptional programs are both hallmarks of cancer that sustain rapid proliferation and metastasis1. However mechanisms controlling the interplay between metabolic reprogramming and transcriptional regulation remain elusive. Here we show that metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). We employed a method for identifying potential kinases that modulate coactivator functions by integrating kinome-wide RNA interference (RNAi)-based screening coupled to intrinsic SRC-3-transcriptional response. PFKFB4, a regulatory enzyme that synthesizes an allosteric stimulator of glycolysis2, was found to be a robust stimulator of SRC-3 that co-activates estrogen receptor (ER). PFKFB4 phosphorylates SRC-3 at serine 857 (S857) enhancing its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient SRC-3 mutant S857A (SRC-3S857A) significantly abolishes SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway enabling purine synthesis by transcriptionally upregulating the expression of enzyme transketolase (TKT). In addition, two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH) involved in purine metabolism were identified as SRC-3 targets which may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation at S857 increases coactivator interaction with the transcription factor ATF4 stabilizing SRC-3/ATF4 recruitment to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses in vivo breast tumor growth and prevents metastasis to the lung from an orthotopic setting as does an SRC-3S857A mutant. PFKFB4 and pSRC-3-S857 levels are elevated and significantly correlate in ER positive tumors whereas, in patients with basal subtype, PFKFB4-SRC-3 drives a common protein signature that positively correlates with the poor survival of breast cancer patients. These findings suggest that the Warburg-pathway enzyme PFKFB4 acts as a molecular fulcrum coupling sugar metabolism to transcriptional activation by stimulating SRC-3 critical to promote aggressive metastatic tumors.

Expression of serum AMPD1 in thyroid carcinoma and its clinical significance.

This study investigated the expression of adenosine monophosphate deaminase 1 (AMPD1) in serum of patients with papillary thyroid carcinoma (PTC) and its clinical significance. The expression levels of AMPD1 mRNA in serum of 157 patients with PTC and 100 normal controls were detected by real-time fluorescent quantitative polymerase chain reaction (PCR), and the relationships between expression level of AMPD1 in serum of PTC patients and clinicopathological factors as well as prognosis were analyzed. The results of real-time fluorescent quantitative PCR showed that the expression of AMPD1 mRNA in serum of PTC patients was lower than that in normal human serum (P<0.01). The expression of AMPD1 in serum of PTC patients was not significantly different from the clinicopathological features such as sex, age, lymph node metastasis and the number of lesions (P>0.05); there were distinct differences between its expression and tumor-node-metastasis (TNM) staging and tumor diameter (P<0.05). The single factor Cox analysis revealed that sex, age, number of lesions, TNM staging and the occurrence of lymph node metastasis were significantly correlated with the prognosis of patients (P<0.05). Multivariate Cox analysis showed that TNM staging hazard ratio (HR)=2.93, 95% confidence interval (CI): 1.52–7.04, P=0.015 was an independent prognostic factor in PTC patients. Survival analysis indicated that there was a statistically significant difference in the 5-year overall survival rate between patients with high expression of AMPD1 and those with low expression (P=0.007). In conclusion, the expression of AMPD1 in serum of patients with PTC is closely related to the malignant evolution of PTC and clinical prognosis of patients. AMPD1 is expected to become an important molecule in judging the clinical prognosis of PTC patients, and may become a new target for molecular targeted therapy of PTC.

Clinical Pharmacogenetic Models of Treatment Response to Methotrexate Monotherapy in Slovenian and Serbian Rheumatoid Arthritis Patients: Differences in Patient's Management May Preclude Generalization of the Models.

Objectives: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but nevertheless 30% of patients experience MTX inefficacy. Our aim was to develop a clinical pharmacogenetic model to predict which RA patients will not respond to MTX monotherapy. We also assessed whether this model can be generalized to other populations by validating it on a group of Serbian RA patients.Methods: In 110 RA Slovenian patients, data on clinical factors and 34 polymorphisms in MTX pathway were analyzed by Least Absolute Shrinkage and Selection Operator (LASSO) penalized regression to select variables associated with the disease activity as measured by Disease Activity Score (DAS28) score after 6 months of MTX monotherapy. A clinical pharmacogenetic index was constructed from penalized regression coefficients with absolute value above 0.05. This index was cross-validated and also independently validated on 133 Serbian RA patients.Results: A clinical pharmacogenetic index for prediction of DAS28 after 6 months of MTX monotherapy in Slovenian RA patients consisted of DAS28 score at diagnosis, presence of erosions, MTX dose, Solute Carrier Family 19 Member 1 (SLC19A1) rs1051266, Solute Carrier Organic Anion Transporter Family Member 1B1 (SLCO1B1) rs2306283, Thymidylate Synthase (TYMS), and Adenosine Monophosphate Deaminase 1 (AMPD1) rs17602729. It correctly classified 69% of Slovenian patients as responders or nonresponders and explained 30% of variability in DAS28 after 6 months of MTX monotherapy. Testing for validity in another population showed that it classified correctly only 22.5% of Serbian RA patients.Conclusions: We developed a clinical pharmacogenetic model for DAS28 after 6 months of MTX monotherapy in Slovenian RA patients by combining clinical and genetic variables. The clinical pharmacogenetic index developed for Slovenian patients did not perform well on Serbian patients, presumably due to the differences in patients' characteristics and clinical management between the two groups.

Abstract P3-05-01: Increased glycolysis directly activates transcriptional coactivator SRC-3 to promote aggressive metastatic breast cancer

Abstract Reprogramming of metabolic pathways is one of the central hallmarks of cancer cell growth and survival to support anabolic and energetic demands. Rapidly proliferating tumor cells utilize increased amounts of glucose for energy generation and macromolecule biosynthesis, yet it is still poorly understood how increased glucose-flux accentuates aggressive metastatic tumor. Understanding the molecular and genetic regulators of these metabolic-programs is critical to discover new drug targets for cancer therapy. Here, we report that the oncogenic transcriptional coregulator steroid receptor coactivator-3 (SRC-3) is a critical sensor of increased tumor glycolysis and re-programs transcriptional responses to support breast cancer growth and metastasis. From an unbiased kinome-wide RNAi library screen, we identified the glycolytic enzyme PFKFB4 as a direct stimulator of SRC-3 activity. PFKFB4 is a bifunctional metabolic enzyme that contains both 'kinase' and 'phosphatase' domain, yet the functional role of these regulatory domains in tumor metabolism is not clear. Glucose stimulation promotes PFKFB4-dependent phosphorylation of SRC-3 (pSRC-3) which then enhances transcriptional coactivation by this coregulator. Mechanistic studies revealed that SRC-3 and PFKFB4 mutually cooperate to maintain high levels of purine biosynthesis by transcriptionally upregulating rate limiting enzymes transketolase (TKT), adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH). Knockdown of PFKFB4 or SRC-3 significantly decreased the expression of these enzymes resulting in reduced proliferation of breast tumor cells, and this functional deficiency could be rescued by exogenous addition of purines. Chromatin immunoprecipitation (ChIP) studies identified increased occupancy of pSRC-3 on target gene promoters, which overlapped with activating transcription factor 4 (ATF4) binding sites in the proximal promoter region. Immunoprecipitation of ATF4 identified increased interaction with pSRC-3 in glycolytic breast tumor cells, whereas knockdown of PFKFB4 or expression of a phospho-deficient SRC-3 mutant significantly reduced the SRC-3/ATF4 association. These findings suggest that upon enhanced glycolysis, PFKFB4 induced activation of SRC-3 by phosphorylation promotes expression of purine biosynthetic genes primarily by coactivating ATF4. Importantly, ablation of either SRC-3 or PFKFB4 suppresses in vivo breast tumor growth and metastasis, and these tumorigenic-effects cannot be simulated by a phospho-deficient SRC-3 mutant suggesting the PFKFB4-SRC-3 axis may be therapeutically exploited for treating breast cancer patients. [S.D. is supported by Susan G. Komen Award PDF14300468]. Citation Format: Dasgupta S, Zhu B, Nikolai BC, Putluri N, Foulds CE, Tsai SY, Tsai M-J, O'Malley BW. Increased glycolysis directly activates transcriptional coactivator SRC-3 to promote aggressive metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-01.

Genetic polymorphisms of the AMPD1 gene and their correlations with IMP contents in Fast Partridge and Lingshan chickens

The object of this study was to evaluate associations between the adenosine monophosphate deaminase 1 (AMPD1) gene polymorphisms and inosine monophosphate acid (IMP) contents of chicken to provide a molecular marker for breeding. Three single nucleotide polymorphisms (SNPs), g.4064G/A, g.5573A/G and g.6805G/A were detected in exons IV, VI, and VIII of the AMPD1 gene in Fast Partridge and Lingshan chickens, respectively. All were purine conversion and caused no alteration in amino acid sequence. Statistical analysis revealed that Lingshan chicken with the homozygous genotype AA at position 4064 and 6805 had a significantly greater IMP content than those with the GG genotype (P<0.05). Fast Partridge chicken with the genotype GG at position 6805 had a significantly greater IMP content compared with those with the AA genotype (P<0.05). In conclusion, the polymorphism at g.6805A/G was correlated with IMP content (P<0.05) in both Fast Partridge and Lingshan chickens. The results in our study suggest that SNP 6805A/G can be used as a possible candidate marker of IMP content of chicken.

DNA sequence polymorphism within the bovine adenosine monophosphate deaminase 1 (AMPD1) is associated with production traits in Chinese cattle.

The objectives of the present study were to detect an 18-bp deletion mutation in the bovine adenosine monophosphate deaminase 1 (AMPD1) gene and analyze its effect on growth traits in 2 Chinese cattle breeds using DNA sequencing and agarose electrophoresis. The five 19-bp polymerase chain reaction products of the AMPD1 gene exhibited 3 genotypes and 2 alleles: WW: homozygote genotype (wild-type); DD: homozygote genotype (mutant-type); WD: heterozygote genotype. Frequencies of the W allele varied from 66.15-70.35%. The associations between the 18-bp deletion mutation in the AMPD1 gene with production traits in 226 Jia-Xian red cattle was analyzed. The animals with genotype WW showed significantly higher heart girth and body weight than those with genotypes WD and DD at 24 months (P < 0.01). Our results indicate that the deletion mutation in the AMPD1 gene is associated with production traits, and may be used for marker-assisted selection in beef cattle breeding programs.

Performance enhancing genetic variants, oxygen uptake, heart rate, blood pressure and body mass index of elite high altitude mountaineers

To analyse and compare the ACE (angiotensin-converting enzyme), ACTN3 (actinin-3) and AMPD1 (adenosine monophosphate deaminase 1) genetic variants, oxygen uptake (VO2max), heart rate (HR), blood pressure (BP) and body mass index (BMI) of elite high altitude mountaineers and average athletes. Elite Bulgarian alpinists (n = 5) and control group of athletes (n = 72) were recruited. VO2max was measured using a treadmill graded protocol. HR, BP and BMI were recorded. Genotyping was done by polymerase chain reaction (PCR) amplification followed by agarose gel electrophoresis. Chi2-test and Fisher's exact test were used for statistical analysis. Alpinists showed significantly higher frequencies of 60% ACE I allele (p = 0.002), 50% ACTN3 X allele (p = 0.032) and 30% AMPD1 T allele (p = 0.003) compared to controls - 39%, 36%, 13%, respectively. ACE ID genotype prevalence and null DD genotype were observed in mountaineers. Higher absolute VO2max, but no differences in VO2max ml kg-1 min-1, HR, oxygen pulse, blood pressure and BMI were found. The ID genotype and higher frequencies of ACE I allele could contribute to successful high altitude ascents in mountaineers. The genetic make-up of the two mountaineers who made the summit of Mt Everest was distinctive, revealing ACE ID genotype, mutant ACTN3 XX and AMPD1 TT genotypes.

Genetic polymorphisms associated with exertional rhabdomyolysis

Exertional rhabdomyolysis (ER) occurs in young, otherwise healthy, individuals principally during strenuous exercise, athletic, and military training. Although many risk factors have been offered, it is unclear why some individuals develop ER when participating in comparable levels of physical exertion under identical environmental conditions and others do not. This study investigated possible genetic polymorphisms that might help explain ER. DNA samples derived from a laboratory-based study of persons who had never experienced an episode of ER (controls) and clinical ER cases referred for testing over the past several years were analyzed for single nucleotide polymorphisms (SNPs) in candidate genes. These included angiotensin I converting enzyme (ACE), α-actinin-3 (ACTN3), creatine kinase muscle isoform (CKMM), heat shock protein A1B (HSPA1B), interleukin 6 (IL6), myosin light chain kinase (MYLK), adenosine monophosphate deaminase 1 (AMPD1), and sickle cell trait (HbS). Population included 134 controls and 47 ER cases. The majority of ER cases were men (n = 42/47, 89.4 %); the five women with ER were Caucasian. Eighteen African Americans (56.3 %) were ER cases. Three SNPs were associated with ER: CKMM Ncol, ACTN3 R577X, and MYLK C37885A. ER cases were 3.1 times more likely to have the GG genotype of CKMM (odds ratio/OR = 3.1, confidence interval/CI 1.33-7.10), 3.0 times for the XX genotype of ACTN3 SNP (OR = 2.97, CI 1.30-3.37), and 5.7 times for an A allele of MYLK (OR = 21.35, CI 2.60-12.30). All persons with HbS were also ER cases. Three distinct polymorphisms were associated with ER. Further work will be required to replicate these findings and determine the mechanism(s) whereby these variants might confer susceptibility.