Guanine Phosphoribosyltransferase Research Articles

Elevated Expression of Hypoxanthine Guanine Phosphoribosyltransferase within Malignant Tissue

Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) is a housekeeping enzyme involved in the purine synthesis of guanine and inosine in the salvage pathway. While other salvage pathway enzymes, such as TK1, have been established as biomarkers for both cancer cell proliferation and cancer development, little research been done to evaluate whether HPRT has the same potential as a cancer biomarker. We designed this study to determine if HPRT has value as an identifier of malignancy within the most common types of cancer. We utilized histological samples from lung, colon, prostate, and breast cancer with additional normal tissue to evaluate whether there was any elevation of HPRT within malignant samples. In addition, we also assessed general HPRT expression within patient’s samples from The Cancer Genome Atlas (TCGA) to confirm clinical relevance. We found that within a subset of patients, there was significant elevation of HPRT when compared to normal tissue controls. This elevation was seen in 33-55% of the malignant samples and appears to have no dependence on cancer stage. There were slight differences in staining patterns among all the organ types, but overall each organ displayed the same pattern of ‘HPRT high’ and ‘HPRT low’ populations within malignant samples. We found that in our TCGA samples, there was a similar elevation of HPRT that was significant when compared to normal controls. Overall, as an upregulated enzyme that does not directly correlate with stage, HPRT could become a valuable marker in the early diagnosis of a variety of solid tumors.

Bioenergetic Failure in Rat Oligodendrocyte Progenitor Cells Treated with Cerebrospinal Fluid Derived from Multiple Sclerosis Patients

In relapsing-remitting multiple sclerosis (RRMS) subtype, the patient’s brain itself is capable of repairing the damage, remyelinating the axon and recovering the neurological function. Cerebrospinal fluid (CSF) is in close proximity with brain parenchyma and contains a host of proteins and other molecules, which influence the cellular physiology, that may balance damage and repair of neurons and glial cells. The purpose of this study was to determine the pathophysiological mechanisms underpinning myelin repair in distinct clinical forms of MS and neuromyelitis optica (NMO) patients by studying the effect of diseased CSF on glucose metabolism and ATP synthesis. A cellular model with primary cultures of oligodendrocyte progenitor cells (OPCs) from rat cerebrum was employed, and cells were treated with CSF from distinct clinical forms of MS, NMO patients and neurological controls. Prior to comprehending mechanisms underlying myelin repair, we determine the best stably expressed reference genes in our experimental condition to accurately normalize our target mRNA transcripts. The GeNorm and NormFinder algorithms showed that mitochondrial ribosomal protein (Mrpl19), hypoxanthine guanine phosphoribosyl transferase (Hprt), microglobulin β2 (B2m), and transferrin receptor (Tfrc) were identified as the best reference genes in OPCs treated with MS subjects and were used for normalizing gene transcripts. The main findings on microarray gene expression profiling analysis on CSF treated OPCs cells revealed a disturbed carbohydrate metabolism and ATP synthesis in MS and NMO derived CSF treated OPCs. In addition, using STRING program, we investigate whether gene–gene interaction affected the whole network in our experimental conditions. Our findings revealed downregulated expression of genes involved in carbohydrate metabolism, and that glucose metabolism impairment and reduced ATP availability for cellular damage repair clearly differentiate more benign forms from the most aggressive forms and worst prognosis in MS patients.

In Vitro Supplementation of Leucine Increases Murine HC11 Cell Proliferation

Mammary tissue undergoes multiples cycles of cell growth and proliferation during the lifetime of the female to meet the nutrient demand of the suckling neonate. On the basis of studies in rodents, pigs and humans demonstrating the anabolic effects of leucine (Leu) through the activation of mammalian target of rapamycin (mTOR) signaling pathway, there has been recent interest in the potential of supplementary Leu to improve skeletal muscle protein synthesis and overall nitrogen retention. To determine whether Leu supplementation also upregulates mTOR pathway in mammary tissue and increases mammary cell proliferation, a murine mammary cell line (HC11) was cultured in different concentrations of Leu (0, 1, and 2 mM). Cell proliferation was measured using flow cytometry at 0, 8, and 16 days. Reverse transcription quantitative PCR was performed to measure mRNA abundance of mammary Leu transporter LAT1 (SLC7A5), the intracellular Leu sensor for the mTOR signaling pathway leucyl‐tRNA synthetase (LARS2), mTOR and its downstream effector ribosomal protein S6 kinase (RPS6K1), and cyclin D (CCND1), a marker for cell proliferation. Guanine phosphoribosyl transferase (HPRT), RNA polymerase 3 (POL3), and ribosomal protein L3a (RPL3a) were used as reference genes. Statistical analysis was performed by ANOVA using a linear mixed model to account for treatment and replication effects. After 16 d cell proliferation increased in response to 1 mM Leu compared to 0 mM (P < 0.05), and increased further (P < 0.05) in cells supplemented with 2 mM Leu. Gene expression of SLC7A5, LARS2, mTOR, RPS6K1, and CCND1 did not differ between groups. Taken together, these results suggest that Leu supplementation increases mammary cell proliferation in vitro, but this effect is not associated with an increase in mRNA abundance of aforementioned genes. Further work is needed to elucidate the mechanisms by which dietary Leu supplementation increases mammary cell proliferation, as it may hold the potential to enhance mammary growth and subsequent milk production in animals.Support or Funding InformationARI 58982

Investigation of toxicity and mutagenicity of cold atmospheric argon plasma.

Cold atmospheric argon plasma is recognized as a new contact free approach for the decrease of bacterial load on chronic wounds in patients. So far very limited data are available on its toxicity and mutagenicity on eukaryotic cells. Thus, the toxic/mutagenic potential of cold atmospheric argon plasma using the MicroPlaSter β® , which has been used efficiently in humans treating chronic and acute wounds, was investigated using the XTT assay in keratinocytes and fibroblasts and the HGPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 Chinese hamster cells. The tested clinical parameter of a 2 min cold atmospheric argon plasma treatment revealed no relevant toxicity on keratinocytes (viability: 76% ± 0.17%) and on fibroblasts (viability: 81.8 ± 0.10) after 72 hr as compared to the untreated controls. No mutagenicity was detected in the HGPRT assay with V79 cells even after repetitive CAP treatments of 2-10 min every 24 hr for up to 5 days. In contrast, UV-C irradiation of V79 cells, used as a positive control in the HGPRT test, led to DNA damage and mutagenic effects. Our findings indicate that cold atmospheric plasma using the MicroPlaSter β® shows negligible effects on keratinocytes and fibroblasts but no mutagenic potential in the HGPRT assay, indicating a new contact free safe technology. Environ. Mol. Mutagen. 58:172-177, 2017. © 2017 Wiley Periodicals, Inc.

In Silico Design of New Inhibitors of Guanine Phosphoribosyltransferase (GPRT) from Giardia lamblia as Antiparasitic Drug Candidates.

Guanine phosphoribosyltransferase (GPRT) is a very attractive target for the development of new drugs against G. lamblia because of its critical role in the synthesis of DNA and RNA. Herein we report the use of in silico approaches to identify potential G. lamblia GPRT inhibitors. Analyses of the binding site of the enzyme accomplished through the use of several methods allowed the construction of a pharmacophore model, which was screened against a database of commercial substances. The resulting retrieved compounds were then screened against GPRT by consensus docking with two different methods, and the top 10% scored compounds had their poses visually inspected. Root Mean Square Deviation (RMSD) values ≤ 2.0 Å were used to define a consensual pose while RMSD values between 2 and 3 Å defined a partial consensus. Main toxicity endpoints were predicted through substructural analyses. From the 1,230 compounds retrieved in the pharmacophore-based screening, eleven had their binding modes consensually ascribed by the docking methods, suggesting a better selectivity for the parasite enzyme in comparison to the human counterpart by avoiding steric bumps with a flexible loop in the human enzyme binding site. One compound, ZINC38139588, was predicted to be totally devoid of toxicity, being perhaps the most promising of this series. Through rigorously validated docking protocols, we predicted the binding mode of these compounds in the GPRT binding site. The use of a consensus docking strategy yielded more reliable predictions of the binding modes to guide the future biological assays.

Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane.

In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines.

Oligomeric state of hypoxanthine−guanine phosphoribosyltransferase from Mycobacterium tuberculosis

Sedimentation equilibrium and size-exclusion chromatography experiments on Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT) have established the existence of this enzyme as a reversibly associating mixture of dimeric and tetrameric species in 0.1 M Tris-HCl−0.012 M MgCl2, pH 7.4. Displacement of the equilibrium position towards the larger oligomer by phosphate signifies the probable existence of MtHGPRT as a tetramer in the biological environment. These data thus add credibility to the relevance of considering enzyme function in the light of a published tetrameric structure deduced from X-ray crystallography. Failure of 5-phospho-α-d-ribosyl-1-pyrophosphate (PRib-PP) to perturb the dimer−tetramer equilibrium position indicates the equivalence and independence of binding for this substrate (the first to bind in an ordered sequential mechanism) to the two oligomers. By virtue of the displacement of the equilibrium position towards dimer that is affected by removing MgCl2 from the Tris-HCl buffer, it can be concluded that divalent metal ions, as well as phosphate, can affect the oligomerization. These characteristics of MtHGPRT in solution are correlated with published crystal structures of four enzyme−ligand complexes.

Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase

Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed.

Novel mutation in HPRT1 causing a splicing error with multiple variations

ABSTRACTLesch-Nyhan disease (LND) is a rare X-linked recessive disorder caused by deficiency of the purine salvage enzyme hypoxanthine–guanine phosphoribosyltransferase (HPRT), encoded by the HPRT1. To date, nearly all types of mutations have been reported in the whole gene; however, duplication mutations are rare. We here report the case of a 9-month-old boy with LND. He showed developmental delay, athetosis, and dystonic posture from early infancy, but no self-injurious behaviors. Hyperuricemia was detected, and his HPRT enzyme activity in erythrocytes was completely deficient. A novel duplication mutation (c.372dupT, c.372_374 TTT > c.372_375 TTTT) was identified in exon 4 of the HPRT1, which causes aberrant splicing. This is the third case of a duplication mutation in the HPRT1 that causes splicing error.

Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism

ObjectiveMercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated...

Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT.

Review article: recent advances in pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy ininflammatory bowel disease.

Azathioprine and mercaptopurine have a pivotal role in the treatment of inflammatory bowel disease (IBD). However, because of their complex metabolism and potential toxicities, optimal use of biomarkers to predict adverse effects and therapeutic response is paramount. To provide a comprehensive review focused on pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy in IBD. A literature search up to July 2015 was performed in PubMed using a combination of relevant MeSH terms. Pre-treatment thiopurine S-methyltransferase typing plus measurement of 6-tioguanine nucleotides and 6-methylmercaptopurine ribonucleotides levels during treatment have emerged with key roles in facilitating safe and effective thiopurine therapy. Optimal use of these tools has been shown to reduce the risk of adverse effects by 3-7%, and to improve efficacy by 15-30%. For the introduction of aldehyde oxidase (AOX) into clinical practice, the association between AOX activity and AZA dose requirements should be positively confirmed. Inosine triphosphatase assessment associated with adverse effects also shows promise. Nucleoside diphosphate-linked moiety X-type motif 15 variants have been shown to predict myelotoxicity on thiopurines in East Asian patients. However, the impact of assessments of xanthine oxidase, glutathione S-transferase, hypoxanthine guanine phosphoribosyltransferase and inosine monophosphate dehydrogenase appears too low to favour incorporation into clinical practice. Measurement of thiopurine-related enzymes and metabolites reduces the risk of adverse effects and improves efficacy, and should be considered part of standard management. However, this approach will not predict or avoid all adverse effects, and careful clinical and laboratory monitoring of patients receiving thiopurines remains essential.

Topiroxostat Showed Renoprotection via Various Mechanisms Including Suppression of Xanthine Oxidoreductase-Induced-Oxidative Stress and Inflammation in db/db Mice

Background: Xanthine oxidoreductase (XOR) produces superoxide along with uric acid. This reactive oxygen species induces organ damage. Therefore, we aimed to investigate whether XOR-induced superoxide is associated with renal abnormality in diabetic mice. Methods: Db/db mice (9-week-old) were fed standard diets with or without topiroxostat (1, 3, and 10 mg/kg/day) or febuxostat (3, 10, 30, and 45 mg/kg/day) for 4 weeks. Furthermore, topiroxostat (1 and 3 mg/kg/day) and febuxostat (3 mg/kg/day) were administered for 7 days or 8 weeks. The levels of urinary albumin excretion (UAE), monocyte chemoattractant protein-1 (MCP-1), and angiotensin II were determined. For renal pathological or mechanistic analysis, periodic acid-Schiff (PAS), nitrotyrosine, and MCP-1 staining or renal gene expression, XOR activity, and purine body levels were evaluated. Results: Both topiroxostat and febuxostat suppressed UAE : topiroxostat 3 mg/kg/day, 11.5 ± 33 μg/day versus vehicle 253.7 ± 69 μg/day, p<0.05; febuxostat 3 mg/kg/day, 255 ± 92 μg/day versus vehicle 634 ± 101 μg/day, p=0.05, for 4 weeks, respectively, and reduced immunohistological staining of enhanced oxidative stress and inflammation markers without affecting glycated hemoglobin. Topiroxostat but not febuxostat decreased urinary MCP-1 and angiotensin II levels and attenuated morphological hypertrophy in the glomerulus and epithelial cell enlargement (diameter and height) in the proximal tubules. In the early stage, topiroxostat and febuxostat decreased renal XOR activity in response to increased purine body levels (topiroxostat 3 mg/kg/day, 2.25 ± 0.19 mg/g tissue, p=0.07; febuxostat 3 mg/kg/day, 2.34 ± 0.09 mg/g tissue, p<0.05 to vehicle 2.01 ± 0.12 mg/g tissue, for 7 days) relative to the vehicle. Moreover, febuxostat upregulated the gene expression of renin 1 (Ren1) and downregulated that of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HPRT) compared with that of the diabetic control. Conclusion: Topiroxostat and febuxostat exhibited renoprotective effects by suppressing oxidative stress and inflammation, and enhancing the salvage pathway via XOR inhibition.

Evaluation of Sister Chromatid Exchanges and Hypoxanthine guanine phosphoribosyl transferase Gene Mutation Assay in Peripheral Lymphocytes of Workers Exposed to Radiation at Al-Tuwaitha Site

The present study aims at using the biological techniques in a genotoxicity assessment of low doses of radiation in samples of workers in Al-Tuwaitha site due to decommissioning to radioactive contamination as a result of work. This study included 50 male blood samples, aged 35 - 63 years as well as 35 blood samples from non-smokers and non-alcoholic as control group which included 25 male and 10 female aged 25 -57 years. The endpoints used were sister chromatid exchange (SCE) and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation assay. The sister chromatid exchange in the human lymphocyte for radiation worker was significantly (P&lt;0.05) higher than in the control group. While the results of mutation for HPRT were non-significant when compared with the control group. In conclusion, the results indicated the possibility of using the changes in sister chromatid exchange as useful biomarkers for the detection of human exposure to ionizing radiation. In conclusion, the increase frequencies of sister chromatid exchange in radiation workers indicate the cumulative effect of low-level chronic exposure to ionizing radiation.

Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus)

In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD+), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise.

Effect of carbon monoxide on gene expression in cerebrocortical astrocytes: Validation of reference genes for quantitative real-time PCR

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as cytoprotection or inflammation. The accurate assessment of changes in gene expression depends on the selection of adequate internal reference gene(s). Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to this gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of CO on gene expression in cultured cerebrocortical astrocytes. The cells were exposed to CO by treatment with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) together with Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-A1 for up to 60 min. For validation, we analyzed the effect of CO on the expression of Bdnf and bcl-2. Different results were obtained, depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. These findings highlight the need for a proper and accurate selection of the reference genes used in the quantification of qRT-PCR results in studies on the effect of CO in gene expression.

Validation of astrocytic reference genes for qRT-PCR in CO treatment studies

Quantitative real-time reverse transcription-polymerase (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as the cytoprotection or inflammation. The selection of an adequate internal reference gene for accurate and consistent analysis of gene expression is of major importance. Carbon monoxide (CO) affects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to the gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the effect of the CO on gene expression in cultured cortical astrocytes. The cells were exposed to CO by incubation with CORM-A1 (CO releasing molecule A1) and four different algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to better evaluate the stability of eight putative reference genes. Our results indicate that Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Ppia (peptidylpropyl isomerase A) is the most suitable gene pair for normalization of qRT-PCR results under the experimental conditions used. Pgk1 (phosphoglycerate kinase 1), Hprt1 (hypoxanthine guanine phosphoribosyl transferase I), Sdha (Succinate Dehydrogenase Complex, Subunit A), Tbp (TATA box binding protein), Actg1 (actin gamma 1) and Rn18s (18S rRNA) genes presented less stable expression profiles in cultured cortical astrocytes exposed to CORM-1 for up to 60 min. Analysis of the effects of CO on the expression of Bdnf and bcl-2 gave different results depending on the reference genes used. A significant increase in the expression of both genes was found when the results were normalized with Gapdh and Ppia, in contrast with the results obtained when the other genes were used as reference. This study highlights the need for proper and accurate usage of reference genes in quantification of qRT-PCR results in studies on the effect of CO in gene expression.

Deciphering Differential Distortion of Purine Substrates by Human and Plasmodium falciparum HGPRT

Enzymes of the salvage pathway are essential to understanding the biochemistry of parasitic protozoa. These parasites lack de novo pathway for nucleotide synthesis and depend solely on purines salvage from their host. Hypoxanthine guanine phosphoribosyltransferase (HGPRT) from Plasmodium falciparum has long been regarded as a potential therapeutic target. Although structurally homologous to human enzyme, PfHGPRT differs from human (h) HGPRT in its expanded substrate specificity and activity towards common substrates. We used substrate analogues, 9‐deazapurines, in combination with UV resonance Raman spectroscopy to capture non‐covalent interactions in enzyme‐substrate complexes. Raman spectral band positions are sensitive to sub‐angstrom changes in bond length. We observed perturbations in Raman spectra that provide quantitative information about hydrogen bonding and stacking‐interactions with the enzyme active‐site. The bound ligands are extensively distorted towards the transition‐state of phosphoribosyltransferase reaction. Differential loss in Raman crosssection of the ligands bound to two enzymes suggests that active‐site is more compact in hHGPRT than in PfHGPRT. The rigidity of active‐site in hHGPRT is attributed to tighter stacking interaction between phenylalanine residue of the active‐site and the nucleobase.Research was supported by IISER, India and Council for Scientific and Industrial Research, India.

Alzheimer’s disease shares gene expression aberrations with purinergic dysregulation of HPRT deficiency (Lesch–Nyhan disease)

Transcriptomic studies of murine D3 embryonic stem (ES) cells deficient in the purinergic biosynthetic function hypoxanthine guanine phosphoribosyltransferase (HPRT) and undergoing dopaminergic neuronal differentiation has demonstrated a marked shift from neuronal to glial gene expression and aberrant expression of multiple genes also known to be aberrantly expressed in Alzheimer’s and other CNS disorders. Such genetic dysregulations may indicate some shared pathogenic metabolic mechanisms in diverse CNS diseases.

Mode of action of recombinant hypoxanthine–guanine phosphoribosyltransferase from Mycobacterium tuberculosis

HomodimericMycobacterium tuberculosisHGPRT follows a sequential compulsory ordered enzyme mechanism.