Guanine Phosphoribosyltransferase Research Articles

A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts

BackgroundLesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine–guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5ʹ-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells.MethodsWe prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts.ResultsLND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND.ConclusionsA complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

Preliminary X-ray Study of Crystals Obtained by Co-Crystallization of Hypoxanthine‒Guanine Phosphoribosyltransferase from Escherichia coli and Pyrazine-2-Carboxamide Derivatives

Preliminary X-ray Study of Crystals Obtained by Co-Crystallization of Hypoxanthine‒Guanine Phosphoribosyltransferase from Escherichia coli and Pyrazine-2-Carboxamide Derivatives

Preliminary X-ray Study of Crystals Obtained by Co-Crystallization of Hypoxanthine‒Guanine Phosphoribosyltransferase from Escherichia coli and Pyrazine-2-Carboxamide Derivatives

A highly effective producer strain Escherichia coli C3030/pET23d+-EcHGPRT, allowing production of recombinant hypoxanthine‒guanine phosphoribosyltransferase from E. coli (EcHGPRT) in a soluble form, has been created. A method for isolating and purifying the recombinant protein has been developed. The specific activity against the natural substrate and pyrazine-2-carboxamide derivatives has been determined. Crystals of the EcHGPRT complexes with 3-hydroxypyrazine-2-carboxamide (T-1105) and 6-fluoro-3-hydroxypyrazine-2-carboxamide (T-705), suitable for X-ray diffraction analysis, have been grown by capillary counter diffusion. X-ray diffraction sets with a resolution of up to 2.4 and 2.5 Å have been collected at the ESRF synchrotron (France, station ID23-1) at a temperature of 100 K. The crystals belong to the sp. gr. P3(1)21; the independent part of the cell contains two enzyme molecules.

Hypoxanthine guanine phosphoribosyltransferase 1, a target of miR-125b-5p, promotes cell proliferation and invasion in head and neck squamous cell carcinoma

The mechanism of hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) upregulation and its function in head and neck squamous cell carcinoma (HNSCC) remains obscure. Herein, the expression and function of HPRT1 and the mechanism underlying its upregulation in HNSCC were explored. Firstly, the expression of HPRT1 and its prognostic values were simultaneously validated using bioinformatic analysis and quantitative real-time PCR (qRT-PCR), and immunohistochemistry staining with local HNSCC samples. The effects of HPRT1 knockdown on proliferation and invasion of HNSCC cells were detected using cell counting kit-8 (CCK-8), plate clone formation, Transwell invasion, nude mouse xenograft model assays. Moreover, the miRNA targeting HPRT1 was validated using dual-luciferase report assay, qRT-PCR and Western blot analysis. The functions of miRNA targeting HPRT1 and its dependence on HPRT1 were further investigated in HNSCC. The results indicated that HPRT1 was highly expressed in HNSCC tissues and cells, which positively correlated with advanced tumor progression and predicted poor prognosis in patients with HNSCC. HPRT1 knockdown markedly inhibited proliferation and invasion of HNSCC cells both in vitro and in vivo. MiR-125b-5p, which was downregulated and positively correlated with a favorable outcome for patients, directly targeted and downregulated HPRT1 expression, and subsequently suppressed cell proliferation and invasion in HNSCC. Collectively, the present study demonstrates that HPRT1 upregulation, at least partially caused by miR-125b-5p downregulation, could promote the malignant progression of HNSCC, highlighting the potential application of the miR-125b-5p/HPRT1 axis as a novel indicator and target in the diagnosis and treatment of HNSCC.

Integrating metabolomics and network pharmacology to assess the effects of quercetin on lung inflammatory injury induced by human respiratory syncytial virus

Quercetin (QR) has significant anti-respiratory syncytial virus (RSV) effects. However, its therapeutic mechanism has not been thoroughly explored. In this study, a lung inflammatory injury model caused by RSV was established in mice. Untargeted lung tissue metabolomics was used to identify differential metabolites and metabolic pathways. Network pharmacology was used to predict potential therapeutic targets of QR and analyze biological functions and pathways modulated by QR. By overlapping the results of the metabolomics and the network pharmacology analyses, the common targets of QR that were likely to be involved in the amelioration of RSV-induced lung inflammatory injury by QR were identified. Metabolomics analysis identified 52 differential metabolites and 244 corresponding targets, while network pharmacology analysis identified 126 potential targets of QR. By intersecting these 244 targets with the 126 targets, hypoxanthine–guanine phosphoribosyltransferase (HPRT1), thymidine phosphorylase (TYMP), lactoperoxidase (LPO), myeloperoxidase (MPO), and cytochrome P450 19A1 (CYP19A1) were identified as the common targets. The key targets, HPRT1, TYMP, LPO, and MPO, were components of purine metabolic pathways. The present study demonstrated that QR effectively ameliorated RSV-induced lung inflammatory injury in the established mouse model. Combining metabolomics and network pharmacology showed that the anti-RSV effect of QR was closely associated with purine metabolism pathways.

HPRT1 Deficiency Induces Alteration of Mitochondrial Energy Metabolism in the Brain

Alterations in function of hypoxanthine guanine phosphoribosyl transferase (HPRT), one of the major enzymes involved in purine nucleotide exchange, lead to overproduction of uric acid and produce various symptoms of Lesch-Nyhan syndrome (LNS). One of the hallmarks of LNS is maximal expression of HPRT in the central nervous system with the highest activity of this enzyme in the midbrain and basal ganglia. However, the nature of neurological symptoms has yet to be clarified in details. Here, we studied whether HPRT1 deficiency changes mitochondrial energy metabolism and redox balance in murine neurons from the cortex and midbrain. We found that HPRT1 deficiency inhibits complex I-dependent mitochondrial respiration resulting in increased levels of mitochondrial NADH, reduction of the mitochondrial membrane potential, and increased rate of reactive oxygen species (ROS) production in mitochondria and cytosol. However, increased ROS production did not induce oxidative stress and did not decrease the level of endogenous antioxidant glutathione (GSH). Thus, disruption of mitochondrial energy metabolism but not oxidative stress could play a role of potential trigger of brain pathology in LNS.

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine-Guanine Phosphoribosyltransferases.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine–guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

In silico study, Synthesis and evaluation purine analogues as potential antimalarial agents

The challenges concerning the control of malaria remain due to the continuous emergence of drug resistant strains. Available treatments for this disease present several limitations such as lack of efficacy, toxic side effects and drug resistance. One of the most striking differences was found in the purine metabolism, because parasites are incapable of de novo purine biosynthesis in which enzyme HGPRT is central to the purine salvage pathway and whose activity is critical for the production of the nucleotides required for DNA/RNA synthesis within this protozoan parasite. Thus, new drugs are urgently needed. Herein we have designed, docked, synthesized and screened 9-alkyl purine derivatives for antimalarial activity against pfHGPRT. We have reported four compounds with promising activity against the highly artimisnin resistant P. falciparum namely: compounds 2,6-dichloro-9-(propan-2-yl)-9H-purine (1a), 9-Butyl-2,6-dichloro-9H-purine (1b), 2,6-dichloro-9-(2-methylbutyl)-9H-purine (1c), 2,6-dichloro-9-pentyl-9H-purine (1d) and binding energies to predictable antimalarial agents (-5.1, -5.3, -5.5 and -5.6 kcal/mole and rmsd 1.198, 1.238, 1.354 and 1.554 respectively). These analogs have acceptable binding interactions, Moreover, an in silico docking study revealed that P. falciparum and hypoxanthine guanine phosphoribosyltransferase enzymes could be the potential targets of those compounds. Our study identified novel, purine-based chemotypes that could be further optimized to generate potent and diversified anti-parasitic drugs against malaria.

HGprt deficiency disrupts dopaminergic circuit development in a genetic mouse model of Lesch–Nyhan disease

In Lesch–Nyhan disease (LND), deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase (HGprt) leads to a characteristic neurobehavioral phenotype dominated by dystonia, cognitive deficits and incapacitating self-injurious behavior. It has been known for decades that LND is associated with dysfunction of midbrain dopamine neurons, without overt structural brain abnormalities. Emerging post mortem and in vitro evidence supports the hypothesis that the dopaminergic dysfunction in LND is of developmental origin, but specific pathogenic mechanisms have not been revealed. In the current study, HGprt deficiency causes specific neurodevelopmental abnormalities in mice during embryogenesis, particularly affecting proliferation and migration of developing midbrain dopamine (mDA) neurons. In mutant embryos at E14.5, proliferation was increased, accompanied by a decrease in cell cycle exit and the distribution and orientation of dividing cells suggested a premature deviation from their migratory route. An abnormally structured radial glia-like scaffold supporting this mDA neuronal migration might lie at the basis of these abnormalities. Consequently, these abnormalities were associated with an increase in area occupied by TH+ cells and an abnormal mDA subpopulation organization at E18.5. Finally, dopaminergic innervation was disorganized in prefrontal and decreased in HGprt deficient primary motor and somatosensory cortices. These data provide direct in vivo evidence for a neurodevelopmental nature of the brain disorder in LND. Future studies should not only focus the specific molecular mechanisms underlying the reported neurodevelopmental abnormalities, but also on optimal timing of therapeutic interventions to rescue the DA neuron defects, which may also be relevant for other neurodevelopmental disorders.

Preparation of an Immunoaffinity Column Based on Bispecific Monoclonal Antibody for Aflatoxin B1 and Ochratoxin A Detection Combined with ic-ELISA.

A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B1 (AFB1) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB1 and OTA from food samples and detection of AFB1/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB1 hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB1 and OTA. The subtype of the BsMAb was IgG1 via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB2 was 37%, with AFG1 15%, with AFM1 48%, with AFM2 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB1) and Ka (OTA) was 2.43 × 108 L/mol and 1.57 × 108 L/mol, respectively. Then the anti-AFB1/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB1/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol–water (60:40, v:v, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB1 and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB1 and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB1 and OTA were applied combined with IAC. The IC50 (50% inhibiting concentration) of AFB1 was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL~0.119 ng/mL. The IC50 of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL~6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB1-OTA IAC. The recovery rates of AFB1 and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB1 and OTA in grains.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Characteristic mutations induced in the small intestine of Msh2-knockout gpt delta mice

BackgroundBase pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2−/−::gpt/0; Msh2-KO).ResultsGpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3′-side of 5′-GpG-3′), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice.ConclusionsOur previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).

Novel Purine Chemotypes with Activity against Plasmodium falciparum and Trypanosoma cruzi.

Malaria and Chagas disease, caused by Plasmodium spp. and Trypanosoma cruzi parasites, remain important global health problems. Available treatments for those diseases present several limitations, such as lack of efficacy, toxic side effects, and drug resistance. Thus, new drugs are urgently needed. The discovery of new drugs may be benefited by considering the significant biological differences between hosts and parasites. One of the most striking differences is found in the purine metabolism, because most of the parasites are incapable of de novo purine biosynthesis. Herein, we have analyzed the in vitro anti-P. falciparum and anti-T. cruzi activity of a collection of 81 purine derivatives and pyrimidine analogs. We firstly used a primary screening at three fixed concentrations (100, 10, and 1 µM) and progressed those compounds that kept the growth of the parasites < 30% at 100 µM to dose–response assays. Then, we performed two different cytotoxicity assays on Vero cells and human HepG2 cells. Finally, compounds specifically active against T. cruzi were tested against intracellular amastigote forms. Purines 33 (IC50 = 19.19 µM) and 76 (IC50 = 18.27 µM) were the most potent against P. falciparum. On the other hand, 6D (IC50 = 3.78 µM) and 34 (IC50 = 4.24 µM) were identified as hit purines against T. cruzi amastigotes. Moreover, an in silico docking study revealed that P. falciparum and T. cruzi hypoxanthine guanine phosphoribosyltransferase enzymes could be the potential targets of those compounds. Our study identified two novel, purine-based chemotypes that could be further optimized to generate potent and diversified anti-parasitic drugs against both parasites.

Abstract 1840: Isolation and evaluation of human nanobodies against hypoxanthine guanine phosphoribosyltransferase (HPRT) and their potential use for the detection and targeting of lung, breast and colon cancer cells

Abstract Since the clinical relevance of hypoxanthine guanine phosphoribosyltransferase (HPRT) as both a cancer biomarker and a potential tumor target is increasing, we isolated human nanobodies (Nbs) against HPRT and tested their potential to detect and target cytosolic and membrane associated HPRT in lung, breast and colon cancer cells. HPRT is a purine salvage pathway enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Until recently, HPRT was considered as a housekeeping gene and its clinical relevance was mainly limited to congenital central nervous disorders such as Lesch-Nyhan disease. However, recent gene expression analysis across all cancers in The Cancer Genome Atlas (TCGA) database and analysis of HPRT expression within cancer patients' tissues has revealed that the expression of HPRT is elevated in the majority of malignant tissues in comparison to normal tissues. Moreover, multiple studies have recently shown that HPRT may be associated with the cell membrane in lung, colon cancer and lymphoma cells, and not in normal cells. Thus, HPRT is a potential immunotherapeutic target for cancer. Using phage display technology we isolated 384 nanobody clones from the Christ human single domain antibody (dAb) library against human HPRT. Ten of the most sensitive Nbs were sequenced and evaluated to determine their sensitivity using dose response curves. Four parameter logistic regression analysis of the top 10 HPRT nanobodies showed R squared values ranging from 0.9388-0.9989 and a sigmoidal shape denoting a concentration-dependent antigen-ligand relationship. The anti-HPRT Nbs were able to detect minimum HPRT amounts ranging from 10-20 ng/ml. Validation of the anti-HPRT Nbs was conducted with a siRNA HPRT knockdown in A549 cells. A549 siRNA HPRT knockdown cell lysate showed at least a 5-fold reduction in the overall signal in phage ELISA compared to A549 wild type cell lysate (p&amp;lt;0.001) indicating the specificity of the Nbs to HPRT. In addition, flow cytometry analysis showed that the anti-HPRT Nbs were able to detect membrane associated HPRT on A549 (20%), NCI-H460 (23%), HT-29 (41%) , SW620 (46%) and MDA-MB-231 (32%) cancer cell lines. Anti-HPRT Nbs did not show significant binding to normal mononuclear cells compared to cancer cells (p&amp;lt;0.002). Anti-HPRT Nbs were successfully expressed and purified using the pET-scFv expression system. Human anti-HPRT Nbs may be used for the detection of both cytosolic and membrane associate HPRT. Future directions of this research will explore the uses of anti-HPRT-Nb-based therapies and their use in other applications such as chimeric antigen receptors (CARs) for cell adoptive therapies. Citation Format: Edwin J. Velazquez, Tyler B. Humpherys, Toni O. Mortimer, David M. Bellini, Kathryn R. Smith, Rachel M. Morris, Abigail Goodman, Kim L. O'Neill. Isolation and evaluation of human nanobodies against hypoxanthine guanine phosphoribosyltransferase (HPRT) and their potential use for the detection and targeting of lung, breast and colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1840.

Effect of hypoxia on purine metabolism of human skeletal muscle cells

Mammals experience some degree of hypoxia during their lifetime. In response to hypoxic challenge, mammalian cells orchestrate specific responses at transcriptional and posttranslational level which lead to changes in the purine metabolites in order to cope with threatening conditions. The aim of this study was to evaluate the response of the enzymes involved in the purine metabolism of human muscle cells to hypoxic conditions. Muscle cells in culture were exposed to hypoxia and the enzymatic activity of inosine monophosphate dehydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP) and hypoxanthine guanine phosphoribosyl transferase (HGPRT) as well as their transcript expression were quantified under normoxic and hypoxic conditions. Purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD+), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine diphosphate (GDP) and guanosine triphosphate (GTP)) concentrations were also quantified. Significant reduction of IMPDH activity and HX and IMP concentrations (p &lt; 0.05) were observed after hypoxia, suggesting a decrease of de novo synthesis of purines. After hypoxia a global reduction of transcripts was observed, suggesting a reduction of the metabolic machinery of purine metabolism to new steady states that balance ATP demand and ATP supply pathways.

Xanthine oxidase activity in thiopurine curative Chinese inflammatory bowel disease patients.

Xanthine oxidase (XO) competes with thiopurine S‐methyltransferase (TPMT) and hypoxanthine guanine phosphoribosyltransferase (HPRT) to metabolize azathioprine (AZA)/6‐mercaptopurine (6‐MP) in vivo. A retrospective investigation was performed to detect the activity of XO in thiopurine curative Chinese inflammatory bowel disease (IBD) patients. We also evaluated whether a relationship between XO activity and incidence of thiopurine‐induced adverse effects (AEs) existed. Clinical data and blood samples were collected from 140 IBD patients before receiving AZA/6‐MP therapy, and the erythrocyte XO activity was measured. The XO activities of all patients were 20.29 ± 4.43 U/g Hb. No sex difference in XO activity was observed (p = .728), and the XO activity showed no difference between the UC and CD patients (p = .082). AEs were observed in 41 (29.3%) patients including leukopenia (26, 18.57%), gastrointestinal intolerance (11, 7.86%), flu‐like symptom (5, 3.57%), alopecia (5, 3.57%), and hepatotoxicity (1, 0.71%). XO activity was significantly lower in the patients with AEs than in those without AEs (18.40 ± 3.73 vs. 21.07 ± 4.48 U/g Hb, p = .001), especially in the patients with leukopenia (18.29 ± 3.68 vs. 21.07 ± 4.48 U/g Hb, p = .004). However, no significant difference in XO activity was found between patients with and without other AEs. Decreased XO activity was observed in the patients who developed flu‐like symptoms (17.58 ± 3.50 U/g Hb) and alopecia (18.67 ± 2.91 U/g Hb) compared to those who did not, although the differences did not reach statistical significance. These findings suggested that patients with low XO expression might have a high risk of thiopurine‐induced toxicity.

Overexpression and surface localization of HPRT in prostate cancer provides a potential target for cancer specific antibody mediated cellular cytotoxicity

We chose to evaluate Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) as a possible biomarker for prostate cancer due to its involvement in nucleotide synthesis and cell cycle progression. We utilized two prostate cancer cell lines (PC3 and DU145) along with patient tissue and knockdowns to evaluate overall HPRT expression. The surface localization of HPRT was determined utilizing flow cytometry, confocal microscopy, and scanning electron microscopy followed by ADCC to evaluate targeting potential. We found significant upregulation of HPRT within malignant samples with approximately 47% of patients had elevated levels of HPRT compared to normal controls. We also observed a significant association between HPRT and the plasma membrane of DU145 cells (p = 0.0004), but found no presence on PC3 cells (p = 0.14). This was confirmed with scanning electron microscopy and confocal microscopy. ADCC experiments were performed to determine whether HPRT could be used as a target antigen for selective cell-mediated killing. We found that DU145 cells treated with HPRT antibodies had a significantly higher incidence of cell death than both isotype treated samples and PC3 cells treated with the same concentrations of HPRT antibody. Finally, we determined that p53 had a significant impact on HPRT expression both internally and on the surface of cancer cells. These results suggest HPRT as a possible biomarker target for the treatment of patients with prostate cancer.

Identification of Suitable Housekeeping Genes for Quantitative Gene Expression Analysis During Retinoic Acid-induced Differentiation of Embryonal Carcinoma NCCIT Cells

Real-time quantitative PCR (qRT-PCR) is often used as an effective experimental method for analyzing gene expression. In this method, normalization of target gene expression levels must be performed using housekeeping genes (HKGs). HKGs are used to compensate for difference between samples due to diverse quality and quality of RNAs and different reverse transcription yield. For an ideal reference gene, constant expression levels across different samples of one experiment is necessary. In the current study, expression stability of four candidate references genes including Beta actin (ACTB), glyceraldeyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase (HPRT1) and Beta-2-Microglobulin (β2M) following retinoic acid (RA) treatment in embryonal carcinoma NCCIT cells were evaluated.NCCIT cells were exposed to RA (10 µM) for 14 days to induce differentiation. RT-qPCR for candidate references genes was performed and normalization between untreated and RA-treated cells was performed using identical sample input amounts. Expression of OCT4, SOX2, NANOG during RA-induced differentiation was assessed by quantitative real-time PCR. RT-qPCR results indicated significant difference in expression level of GAPDH between untreated (Ct mean: 19.36667± 0.28) and RA-treated (Ct mean: 28.94± 0.18) NCCIT cells. However, transcriptional level of ACTB, HPRT and β2M remained unchanged after RA treatment. qRT-PCR analysis using ACTB, HPRT and β2M showed treatment of NCCIT cells with RA lead to significant down regulation of OCT4 (79%), NANOG (71%) and SOX2 (96%) transcript. ACTB, HPRT and β2M were recognized as valid reference genes for analysis of gene expression during RA-induced differentiation of NCCIT cells, while GAPDH was not suitable.

制备人脐静脉内皮细胞和人肺腺癌细胞融合细胞的新方法

Purpose:Human umbilical endothelial cells (HUVECs) have been proved to be an effective whole-cell vaccine inhibiting tumor angiogenesis. In this study, we fused HUVECs with human lung adenocarcinoma cells A549 s, aiming at preparing lung cancer vaccine to achieve dual effects of anti-tumor angiogenesis and specific immunity to tumor cells.Methods:A549 cells were induced by ethyl methane sulfonate (EMS) and 8-azaguanine (8-AG) to get hypoxanthine guanine phosphoribosyl transferase (HGPRT) auxotrophic A549 cells. Then Fused HGPRT auxotrophic A549 cells with primary HUVEC cells by combining electrofusion with polyethylene glycol (PEG). Afterward the fusion cells were screened by HAT and HT selective medium and sorted by flow cell sorter to obtain high-purity HUVEC-A549 cells. Finally, HUVEC-A549 cells were identified by karyotype analysis and western blotting.Results:The fusion efficiency of HUVEC-A549 cells prepared by combining electrofusion with polyethylene glycol (PEG) was significantly higher than that of electrofusion and PEG (43.0% vs 17.60% vs 2.71%, P < 0.05). After screened by HAT and HT selective medium and sorted by flow cell sorter, the proportion of HUVEC-A549 cells can count for 71.2% ± 3.2%. The mode of chromosomes in HUVEC-A549 cells was 68, and the chromosome was triploid. VE-cadherin and platelet endothelial cell adhesion molecule-1 (CD31) were highly expressed in HUVECs and HUVEC-A549 cells, but not in A549 cells.Conclusions:These results indicate that HUVEC-A549 cells retain the biological characteristics of human umbilical vein endothelial cells and A549 cells. It can be used in the experimental study of lung cancer cell vaccine.

A proteomic glimpse into the effect of antimalarial drugs on Plasmodium falciparum proteome towards highlighting possible therapeutic targets.

There is no effective vaccine against malaria; therefore, chemotherapy is to date the only choice to fight against this infectious disease. However, there is growing evidences of drug-resistance mechanisms in malaria treatments. Therefore, the identification of new drug targets is an urgent need for the clinical management of the disease. Proteomic approaches offer the chance of determining the effects of antimalarial drugs on the proteome of Plasmodium parasites. Accordingly, we reviewed the effects of antimalarial drugs on the Plasmodium falciparum proteome pointing out the relevance of several proteins as possible drug targets in malaria treatment. In addition, some of the P. falciparum stage-specific altered proteins and parasite-host interactions might play important roles in pathogenicity, survival, invasion and metabolic pathways and thus serve as potential sources of drug targets. In this review, we have identified several proteins, including thioredoxin reductase, helicases, peptidyl-prolyl cis-trans isomerase, endoplasmic reticulum-resident calcium-binding protein, choline/ethanolamine phosphotransferase, purine nucleoside phosphorylase, apical membrane antigen 1, glutamate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase, heat shock protein 70x, knob-associated histidine-rich protein and erythrocyte membrane protein 1, as promising antimalarial drugs targets. Overall, proteomic approaches are able to partially facilitate finding possible drug targets. However, the integration of other 'omics' and specific pharmaceutical techniques with proteomics may increase the therapeutic properties of the critical proteins identified in the P. falciparum proteome.