Guanine Phosphoribosyltransferase Research Articles

Impact of denervation-induced muscle atrophy on housekeeping gene expression in mice.

Immobilization induced by experimental denervation leads to rapid and progressive alterations in structural and biochemical properties of skeletal muscle. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a popular method of elucidating the molecular mechanisms involved in muscle atrophy. Identification of suitable reference genes that are not affected by experimental conditions is a critical step in accurate normalization of real-time RT-PCR. We investigated the impact of denervation-induced muscle atrophy for 2 weeks on the expression of common housekeeping genes. Denervation differentially affected the expression levels of these genes. RefFinder software identified TATA box binding protein (Tbp) as the most stable gene and showed that the stability of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and hypoxanthine guanine phosphoribosyl transferase (Hprt) genes was low, even though they are widely used for normalization. The appropriate reference gene for normalization of genes of interest in denervated muscle is Tbp.

Hyperuricemia and gout due to deficiency of hypoxanthine–guanine phosphoribosyltransferase in female carriers: New insight to differential diagnosis

BackgroundX-linked hypoxanthine–guanine phosphoribosyltransferase (HPRT) deficiency in an inherited disorder of purine metabolism is usually associated with the clinical manifestations of hyperuricemia. A variable spectrum of neurological involvement occurs predominantly in males. Females are usually asymptomatic. Carrier status cannot be confirmed by biochemical and enzymatic methods reliably. MethodsWe studied clinical, biochemical and molecular genetic characteristics of Czech families with hyperuricemia and HPRT deficiency. We analyzed age at diagnosis, clinical symptoms, uricemia, urinary hypoxanthine and xanthine, HPRT activity in erythrocytes, mutation in the HPRT1 gene, X-inactivation, and major urate transporters. ResultsA mutation in the HPRT1 gene in family A was confirmed in one boy and four females. Three females with hyperuricemia had normal excretion of purine. One female was normouricemic. An 8-month-old boy with neurological symptoms showed hyperuricemia, increased excretion of urinary hypoxanthine and xanthine and a very low HPRT activity in erythrocytes. We have found three other unrelated female carriers with hyperuricemia and normal excretion of hypoxanthine and xanthine among other families with HPRT deficiency. ConclusionsHPRT deficiency needs to be considered in females with hyperuricemia with normal excretion of purine metabolites. Familiar hyperuricemia and/or nonfamiliar gout should always be further investigated, especially in children.

Hydrophilic-interaction liquid chromatography-tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine-guanine phosphoribosyltransferase deficiency.

Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC-MS/MS system. All measurement results showed good precision with RSD <6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV.

Clinical severity in Lesch–Nyhan disease: The role of residual enzyme and compensatory pathways

Mutations in the HPRT1 gene, which encodes the purine salvage enzyme hypoxanthine–guanine phosphoribosyltransferase (HGprt), cause Lesch–Nyhan disease (LND) and more mildly affected Lesch–Nyhan variants. Prior studies have suggested a strong correlation between residual hypoxanthine recycling activity and disease severity. However, the relevance of guanine recycling and compensatory changes in the de novo synthesis of purines has received little attention. In the current studies, fibroblast cultures were established for 21 healthy controls and 36 patients with a broad spectrum of disease severity related to HGprt deficiency. We assessed hypoxanthine recycling, guanine recycling, steady-state purine pools, and de novo purine synthesis. There was a strong correlation between disease severity and either hypoxanthine or guanine recycling. Intracellular purines were normal in the HGprt-deficient fibroblasts, but purine wasting was evident as increased purine metabolites excreted from the cells. The normal intracellular purines in the HGprt-deficient fibroblasts were likely due in part to a compensatory increase in purine synthesis, as demonstrated by a significant increase in purinosomes. However, the increase in purine synthesis did not appear to correlate with disease severity. These results refine our understanding of the potential sources of phenotypic heterogeneity in LND and its variants.

Validation of housekeeping genes in the brains of rats submitted to chronic intermittent hypoxia, a sleep apnea model.

Obstructive sleep apnea (OSA) is a syndrome characterized by intermittent nocturnal hypoxia, sleep fragmentation, hypercapnia and respiratory effort, and it has been associated with several complications, such as diabetes, hypertension and obesity. Quantitative real-time PCR has been performed in previous OSA-related studies; however, these studies were not validated using proper reference genes. We have examined the effects of chronic intermittent hypoxia (CIH), which is an experimental model mainly of cardiovascular consequences of OSA, on reference genes, including beta-actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase and eukaryotic 18S rRNA, in different areas of the brain. All stability analyses were performed using the geNorm, Normfinder and BestKeeper software programs. With exception of the 18S rRNA, all of the evaluated genes were shown to be stable following CIH exposure. However, gene stability rankings were dependent on the area of the brain that was analyzed and varied according to the software that was used. This study demonstrated that CIH affects various brain structures differently. With the exception of the 18S rRNA, all of the tested genes are suitable for use as housekeeping genes in expression analyses.

Hypoxanthine Guanine Phosphoribosyl Transferase Is the Most Stable Reference Gene for Gene Expression Analysis by Quantitative PCR in Peripheral Blood Mononuclear Cells from Women with the Polycystic Ovary Syndrome

Summary Background: The polycystic ovary syndrome (PCOS) is a frequent endocrine disorder that affects women of reproductive age. As the syndrome is strongly associated with obesity, it is of interest to examine the gene expression diffe rences that accompany its development and the associ a ted metabolic disturbances. Real-time RT PCR is a standard method for studying changes in gene expression. However, to obtain accurate and reliable results, validation of reference genes is obligatory. The aim of this study was to identify a suitable reference for the normalization of gene expression in peripheral blood mononuclear cells (PBMCs) from obese and normal-weight women with PCOS. Methods: The expression stability of four potential reference genes: hypoxanthine guanine phosphoribosyl trans-ferase 1 (HPRT), β-actin (BA), β2-microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was assessed in PBMCs from healthy women, and from normal-weight and obese women with PCOS. The variability in the expression of potential reference genes was analyzed by the TaqMan real-time RT PCR method, using GeNorm and NormFinder software packages. Results: Direct comparison of cycle threshold (Ct) values showed inter-individual variations for all validated genes, the Ct values of HPRT being less variable than those of BA, GAPDH and B2M. Both software packages pointed to HPRT as the most steadily expressed gene in the PBMCs of women with PCOS and healthy controls. Conclusions: Cross-validation of the expression stability of four potential reference genes identified HPRT as the most stable reference, suitable for further investigations of gene expression in PBMCs from women with PCOS.

Investigating Genetic Damage in Peripheral Lymphocytes of Radiation Workers at Al-Tuwaitha Site in Iraq Using Four Genetic End-Points

The present study aims to use the biological techniques in a genotoxicity assessment of DNA damage in peripheral lymphocytes of radiation workers at Al-Tuwaitha site due to decommissioning to radioactive contamination as a result of work during January 2010 to December 2011. The subjects were divided into two groups: (i) 85 workers from radiation workers at Al-Tuwaitha site; (ii) 50 controls were matched non-smoking and no alcohol drink. Fresh blood samples were collected from the workers and controls. Four genetic parameter were studied using the micronucleus (MN) test, nuclear division index (NDI) test, the comet assay and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation assay. The results of the MN test showed that the average of MN per cell (Mean ± SE) in workers were 0.025 ± 0.0016 MN/ cells, which were significantly higher than those 0.010 ± 0.0006 MN/ cells in controls P&lt; 0.01. While, the results of NDI test the average of NDI (Mean ± SE) in workers were 1.154 ± 0.0089 when compared with the control 1.322 ± 0.0117, which were significant increase p&lt;0.01. It was found in the comet assay that the mean tail length (Mean ± SE) of radiation workers and controls were 17.69 ± 0.23 µm and 14.05 ± 0.13 µm, respectively. There was a significant difference between radiation workers and controls for mean tail length P &lt; 0.01, but the difference between the mean tail moment (Mean ± SE) 14.22 ±0.21 of workers and mean tail moment 12.96± 0.15 of controls was not significant P&gt; 0.01. Mean while, the results of the average of mutation frequency for HPRT were no significant differences rate for radiation workers compared with the control group P&gt; 0.01. In conclusion, the results of our experiment suggest that the accumulation of genetic damage is detectable in peripheral lymphocytes of radiation workers at Al-Tuwaitha site. Also, the current results of frequency MN and NDI within of normal values according of the technical report of International Atomic Energy Agency (IAEA) No. 405, 2001.

Epigenetic regulation in amyloid precursor protein and the Lesch-Nyhan syndrome

Lesch-Nyhan syndrome (LNS) is a neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine–guanine phosphoribosyltransferase (HPRT) is defective. A major unsolved question is how the loss of HPRT enzyme function affects the brain to cause the neurobehavioural syndrome in LNS and its attenuated variants (LNVs). To address this issue, a search for a link between LNS and the amyloid precursor protein (APP) is developed. Here, I identified, for the first time in fibroblasts from normal subjects as well as from LNS and LNV patients: (a) several APP-mRNA isoforms encoding divers APP protein isoforms ranging from 120 to 770 amino acids (with or without mutations and/or deletions) accounted for epigenetic mechanisms in the regulation of alternative APP pre-mRNA splicing and (b) five novel independent polymorphisms in the APP promoter: −956A>G, −1023T>C, −1161A>G, −2224G>A, −2335C>T relative to the transcription start site. A role for epistasis between mutated HPRT and APP genes affecting the regulation of alternative APP pre-mRNA splicing in LNS is suggested. An accurate quantification of various APP isoforms in brain tissues for detection of initial pathological changes or pathology development is needed. My findings may provide new directions not only for investigating the role of APP in neuropathology associated with HPRT-deficiency in LNS but also for the research in neurodevelopmental and neurodegenerative disorders by which various APP isoforms involved in the pathogenesis of the diseases such as Alzheimer’s disease.

A modified intraoral resin mouthguard to prevent self-mutilations in lesch-nyhan patients.

Lesch-Nyhan syndrome, described in 1964 by Lesch and Nyhan, is a X-linked recessive disorder, occurring in 1 : 100000 to 1 : 380000 live births. LNS is characterized by a decrease in activity of hypoxanthine guanine phosphoribosyl transferase, an enzyme involved in purine metabolism, resulting in overproduction of uric acid. Hyperuricemia and neurological features including choreoathetoid spasticity, self-mutilation, and mental retardation clinically characterize this syndrome. In LNS patients the typical feature is loss of tissue from biting themselves with partial or complete amputation of fingers, lips, and tongue. The self-mutilation compares with the eruption of the deciduous teeth. Several drugs trials have been administered to improve self-destructive behavior and invasive treatment approaches, such as extractions of teeth and orthognathic surgery, have been suggested with variable effectiveness. Nowadays prevention is, therefore, the standard of care. The role of dentistry is essential in the management of the self-mutilating behavior, because the teeth represent the main self-injury instrument. This report presents a revision of various therapeutic approaches to manage self-destruction, highlighting the effectiveness of a preventive treatment. It describes a new technique: a resin mouthguard, realized at Gaslini Hospital, to obtain immediate healing of the oral lesions, confirmed in the follow-up period.

Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV).

The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.

A Global Increase in 5‐Hydroxymethylcytosine Levels Marks Osteoarthritic Chondrocytes

To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA). Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation. A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment. This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.

HIV-1 TAT-mediated protein transduction of human HPRT into deficient cells

Lesch–Nyhan disease (LND) is a severe and incurable X-linked genetic syndrome caused by the deficiency of hypoxanthine–guanine phosphoribosyltransferase (HPRT), resulting in severe alterations of central nervous system, hyperuricemia and subsequent impaired renal functions. Therapeutic options consist in supportive care and treatments of complications, but the disease remains largely untreatable. Enzyme replacement of the malfunctioning cytosolic protein might represent a possible therapeutic approach for the LND treatment. Protein transduction domains, such as the TAT peptide derived from HIV TAT protein, have been used to transduce macromolecules into cells in vitro and in vivo. The present study was aimed to the generation of TAT peptide fused to human HPRT for cell transduction in enzyme deficient cells. Here we document the construction, expression and delivery of a functional HPRT enzyme into deficient cells by TAT transduction domain and by liposome mediated protein transfer. With this approach we demonstrate the correction of the enzymatic defect in HPRT deficient cells.Our data show for the first time the feasibility of the enzyme replacement therapy for the treatment of LND.

The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease.

Phenotypic variation among seven members of one family with deficiency of hypoxanthine–guanine phosphoribosyltransferase

We describe a family of seven boys affected by Lesch–Nyhan disease with various phenotypes. Further investigations revealed a mutation c.203T>C in the gene encoding HGprt of all members, with substitution of leucine to proline at residue 68 (p.Leu68Pro). Thus patients from this family display a wide variety of symptoms although sharing the same mutation. Mutant HGprt enzyme was prepared by site-directed mutagenesis and the kinetics of the enzyme revealed that the catalytic activity of the mutant was reduced, in association with marked reductions in the affinity towards phosphoribosylpyrophosphate (PRPP). Its Km for PRPP was increased 215-fold with hypoxanthine as substrate and 40-fold with guanine as substrate with associated reduced catalytic potential. Molecular modeling confirmed that the most prominent defect was the dramatically reduced affinity towards PRPP. Our studies suggest that the p.Leu68Pro mutation has a strong impact on PRPP binding and on stability of the active conformation. This suggests that factors other than HGprt activity per se may influence the phenotype of Lesch–Nyhan patients.

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

D-glucose- and 3-O-methyl-D-glucose-induced upregulation of selected genes in rat hepatocytes and INS1E cells: Re-evaluation of the possible role of hexose phosphorylation

The biochemical events involved in the upregulation of selected glucose‑responsive genes by 3‑O‑methyl‑D‑glucose (3‑MG) remain to be elucidated. The present study mainly aimed to re‑evaluate the possible role of 3‑MG phosphorylation in the upregulation of the thioredoxin interacting protein (TXNIP) and liver pyruvate kinase (LPK) genes in rat hepatocytes and INS1E cells. TXNIP and LPK transcription was assessed in rat liver and INS1E cells exposed to a rise in D‑glucose concentration, 2‑deoxy‑D‑glucose (2‑DG), 3‑MG and, when required, D‑mannoheptulose. The phosphorylation of D‑[U‑14C]glucose and 3‑O‑[14C]methyl‑D‑glucose (14C-labeled 3-MG) was measured in rat liver, INS1E cell and rat pancreatic islet homogenates. The utilization of D‑[5‑3H]glucose by intact INS1E cells was also measured. In rat hepatocytes, a rise in the D‑glucose concentration increased the TXNIP/hypoxanthine‑guanine phosphoribosyl transferase (HPRT) and LPK/HPRT ratios, while 2‑DG and 3‑MG also increased the TXNIP/HPRT ratio, but not the LPK/HPRT ratio. In INS1E cells, the TXNIP/HPRT and LPK/HPRT ratios were increased in response to the addition of D‑glucose, 2‑DG and 3‑MG. Furthermore, D‑mannoheptulose abolished the response to D‑glucose and 2‑DG, but not to 3‑MG, in these cells. Liver cell homogenates catalyzed the phosphorylation of 3‑MG to a modest extent, whilst INS1E and rat pancreatic islet cell homogenates did not. Moreover, 3‑MG marginally decreased D‑glucose phosphorylation in INS1E cell homogenates but not in liver cell homogenates. D‑[5‑3H]glucose utilization by intact INS1E cells was decreased by 2‑DG, but not by 3‑MG. These findings reinforce the view that the upregulation of the TXNIP and LPK genes induced by 3‑MG is not attributable to its phosphorylation or any favorable effect on D‑glucose metabolism.

International residency for the development of the emergency department clinical nurse specialist role

Brown adipocytes oxidize fatty acids to produce heat in response to cold or caloric overfeeding. The motivation and function of the development of brown fat may thus counteract obesity, though this remains uncertain. We investigated the brown adipocyte proteome by two-dimensional gel electrophoresis followed by mass spectrometry. Comparative analyses of proteins focused on total protein spots to filter differentially expressed proteins during the differentiation of mouse primary brown preadipocytes. A Western blot analysis was performed to verify the target proteins. The results indicated that 10 protein spots were differentially expressed with significant changes, including the three up-regulated proteins of prohibitin, hypoxanthine–guanine phosphoribosyltransferase, and enoyl-CoA hydratase protein; the 5 down-regulated proteins of triosephosphate isomerase, elongation factor 2, α-tropomyosin slow, endophilin-B1, and cofilin-1 (CFL1); and the two unequivocally expressed proteins of peroxiredoxin-1 and collagen α-1(i) chain precursor. We found that during brown adipogenesis, CFL1 has an inhibitory effect on brown adipocyte differentiation. The overexpression of CFL1 inhibited the brown fat deposition and repressed the brown marker genes UCP1, PRDM16, PGC-1α and PPARγ via actin dynamics and polymerization. These observations may be novel findings that bring new insight into the detailed mechanisms of brown adipogenesis and identify possible therapeutic targets for anti-obesity.We use 2-DE to compare the proteomes of adipocytes during the brown adipogenesis of primary mouse preadipocytes. We identified 10 proteins that are differentially expressed. Among these, we found that the actin binding protein CFL1 inhibits the differentiation of brown preadipocytes. CFL1 overexpressing cells showed lower deposition of brown fat droplets, and the brown marker genes of UCP1, PRDM16, PGC-1α and PPARγ were decreased through actin dynamics and polymerization.

Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract

There is accumulating evidence for the involvement of pro-inflammatory cytokines associated with a T helper 17 response in intestinal disorders such as inflammatory bowel disease (IBD) in humans. The involvement of interleukin (IL)-17 or IL-23 in equine IBD has not been studied and most gene expression studies in the equine intestine have been limited to the use of a single non-validated reference gene. In this study, expression of the reference gene candidates β2 microglobulin (β2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H2A type 1, hypoxanthine–guanine phosphoribosyltransferase (HPRT), 60S ribosomal protein L32 (RPL32), succinate dehydrogenase complex subunit A (SDHA) and transferrin receptor 1 protein coding (TFRC)in the equine intestine was evaluated by quantitative PCR. Three to four reference genes were adequate for normalisation of gene expression in the healthy duodenum, mid-jejunum, colon and rectum, although each segment required a unique combination of reference genes. No combination of the evaluated genes was optimal for the caecum and ileum. Another combination of reference genes (GAPDH, HPRT, RPL32 and SDHA) was optimal for normalisation of rectal samples from healthy and IBD-affected horses, indicating that reference genes should be re-evaluated if material from diseased specimens is analysed. Basal expression of IL-12p40, IL-17A and IL-23p19 was detected in each segment, which will enable gene expression studies of these cytokines by relative quantification.

HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression: Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

Lesch-Nyhan Disease (LND) is the result of mutations in the X-linked gene encoding the purine metabolic enzyme, hypoxanthine guanine phosphoribosyl transferase (HPRT). LND gives rise to severe neurological anomalies including mental retardation, dystonia, chorea, pyramidal signs and a compulsive and aggressive behavior to self injure. The neurological phenotype in LND has been shown to reflect aberrant dopaminergic signaling in the basal ganglia, however there are little data correlating the defect in purine metabolism to the neural-related abnormalities. In the present studies, we find that HPRT-deficient neuronal cell lines have reduced CREB (cAMP response element-binding protein) expression and intracellular cyclic AMP (cAMP), which correlates with attenuated CREB-dependent transcriptional activity and a reduced phosphorylation of protein kinase A (PKA) substrates such as synapsin (p-syn I). Of interest, we found increased expression of phosphodiesterase 10A (PDE10A) in HPRT-deficient cell lines and that the PDE10 inhibitor papaverine and PDE10A siRNA restored cAMP/PKA signaling. Furthermore, reconstitution of HPRT expression in mutant cells partly increased cAMP signaling synapsin phosphorylation. In conclusion, our data show that HPRT-deficiency alters cAMP/PKA signaling pathway, which is in part due to the increased of PDE10A expression and activity. These findings suggest a mechanistic insight into the possible causes of LND and highlight PDE10A as a possible therapeutic target for this intractable neurological disease.

Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (&amp;lt;italic&amp;gt;Anser anser domestica&amp;lt;/italic&amp;gt;) Gene Expression

Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), β-actin (ACTB), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese.