Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.
Adenine/metabolism* ; Adenine Nucleotides ; Adenine Phosphoribosyltransferase/genetics* ; Adenosine Monophosphate ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Recombinant Proteins/genetics*

Objective To determine the purine adenylate [adenosine triphosphate （ATP）, adenosine diphosphate （ADP）, adenosine monophosphate （AMP）] content in the muscles of both hind limbs of rats at different postmortem interval （PMI）, calculate the changes in the total adenine nucleotide （TAN） content and the adenylic-acid energy charge （AEC）, and explore their relationship with PMI. Methods Healthy rats were sacrificed by cervical dislocation and kept at 20 ℃. The muscles of their hind limbs were extracted at 0, 24, 48, 72, 96, 120, 144, and 168 h after death. Reversed-phase high performance liquid chromatography was used to determine the content of purine adenylates, the TAN and AEC of the muscles of the both hind limbs were calculated, and the related regression equations of their relationship with PMI were established. Results Within 168 h of death of rats, the trend of ATP change was different from ADP, and the content of AMP continuously increased. The TAN value gradually increased with the extension of PMI, and the AEC showed a downward trend within 168 h after death. Among them, the patterns of AEC changes with PMI were obvious, the correlation coefficient was high （ R²=0.903）, and the curve fitting relationship was good; the fitting relationship between ATP, ADP, AMP, TAN and PMI was poor （ R²=0.198-0.754）. Conclusion The postmortem change patterns of AEC provide new research ideas for PMI estimation in the forensic field.
Adenine Nucleotides ; Adenosine Monophosphate ; Animals ; Forensic Pathology ; Muscles ; Rats ; Rats, Sprague-Dawley ; Time Factors

We cultured the rabbit inner medullary collecting duct (IMCD) cells as monolayers on collagen-coated membrane filters, and investigated distribution of the P2Y receptors by analyzing nucleotide-induced short circuit current (Isc) responses. Exposure to different nucleotides of either the apical or basolateral surface of cell monolayers stimulated Isc. Dose-response relationship and cross-desensitization studies suggested that at least 3 distinct P2Y receptors are expressed asymmetrically on the apical and basolateral membranes. A P2Y2-like receptor, which responds to UTP and ATP, is expressed on both the apical and basolateral membranes. In addition, a uracil nucleotide receptor, which responds to UDP and UTP, but not ATP, is expressed predominantly on the apical membrane. In contrast, a P2Y1-like receptor, which responds to ADP and 2-methylthio-ATP, is expressed predominantly on the basolateral membrane. These nucleotides stimulated intracellular cAMP production with an asymmetrical profile, which was comparable to that in the stimulation of Isc. Our results suggest that the adenine and uracil nucleotides can interact with different P2Y nucleotide receptors that are expressed asymmetrically on the apical and basolateral membranes of the rabbit IMCD cells, and that both cAMP- and Ca2+-dependent signaling mechanisms underlie the stimulation of Isc.
Adenine ; Adenosine Diphosphate ; Adenosine Triphosphate ; Membranes ; Nucleotides ; Uracil ; Uracil Nucleotides ; Uridine Diphosphate ; Uridine Triphosphate

BACKGROUND: Adenosine 5'-tetraphosphate(ATPP), an endogenous nucleotide, is stored in cells and released into the extracellular space upon stimulation. Some of the biological responses to ATPP were reported, but characteristics of its receptor were not well known. Present study was conducted to investigate the effects of ATPP on mechanical contractility, resting membrane potential and action potential of rat left atrium. METHODS: Left atrium was isolated from Sprague-Dawley rat. Mechanical contraction induced by electrical field stimulation(EFS) was recorded on polygraph using force transducer. With glass microelectrodes(10 MOmega), potential difference across the membrane was measured and recorded on an oscilloscope and a polygraph. RESULTS: ATPP reduced the left atrial contractility with concentration-dependent manner. ATPP also hyperpolarized the resting membrane potential and decreased the action potential duration of the left atrial cell. Nucleotides other than ATPP, such as ATP, ADP, AMP and adenosine, have the same effect as ATPP. However, there is no difference among the nucleotides. Prior treatment of DPCPX, a P1-purinoceptor blocker, inhibited the ATPP-induced negative inotropism and changes of the membrane potential. But suramin, a nonselective P2-purinoceptor blocker, did not alter the effects of ATPP. alpha, beta methylene ADP and adenosine deaminase, which attenuates hydrolysis of adenine nucleotides and inactivates adenosine respectively, did not influence the effects of adenine nucleotides except for adenosine. CONCLUSION: ATPP reduced the mechanical contractility, hyperpolarized the resting membrance potential and decreased duration of action potential of rat left atrium. These effects were induced by ATPP directly, not by adenosine from hydrolyzed ATPP.
Action Potentials ; Adenine Nucleotides ; Adenosine Deaminase ; Adenosine Diphosphate ; Adenosine Triphosphate ; Adenosine* ; Animals ; Extracellular Space ; Glass ; Heart Atria ; Hydrolysis ; Membrane Potentials ; Membranes ; Muscle Contraction ; Nucleotides ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic ; Suramin ; Transducers

After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB-treated group and 36.7% decrease in 1 mM 3-AB-treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells.
Animals ; Cell Death ; Epithelial Cells* ; Glucose ; Glucose-6-Phosphate Isomerase ; Glycolysis ; Hexokinase ; Kidney* ; LLC-PK1 Cells ; Oxidoreductases ; Phosphofructokinase-1 ; Phosphopyruvate Hydratase ; Poly Adenosine Diphosphate Ribose* ; Poly(ADP-ribose) Polymerases* ; Pyruvate Kinase ; Swine

Cyclic adenosine monophosphate (cAMP) is one of the significant and conserved second messengers in mammals, and it participates in regulating the developmental and physiological functions of various organs and tissues through transducting extracellular signals. Studies have shown that the process of meiosis in female mammalian oocytes is closely related to the level of cAMP and strictly regulated. In oocytes, cAMP is mainly synthesized by adenylate cyclase 3 (AC3) and degraded by phosphodiesterase 3A (PDE3A), both of which jointly regulate the level of cAMP in oocytes and play important roles in the follicular development and oogenesis of female ovaries. It has been well illuminated that high level of cAMP in the cytoplasm of oocytes in growing follicles could maintain the arrest of the first meiotic of oocytes for a long time. The oocytes will resume meiosis and mature either when the synthesis of cAMP is down-regulated, or when cAMP is degraded by PDE3A. In recent years, the novo physiological functions of cAMP in oogenesis have been reported. To better understand the regulatory role and mechanism of cAMP in mammalian gametogenesis, this paper reviews the relevant research regarding the relationship between cAMP and germ cell development.
Adenosine Monophosphate ; Animals ; Cyclic AMP ; Female ; Mammals ; Meiosis ; Oocytes ; Oogenesis

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na(+)-K(+)-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 micromol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 micromol/L), an inhibitor for reverse mode of Na(+)-Ca(2+) exchangers (NCX), but did not by L-type Ca2+ channel blocker nifedipine (1.0 micromol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 micromol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 micromol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 micromol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 micromol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.
8-Bromo Cyclic Adenosine Monophosphate ; Adenosine ; Cardiomegaly ; Colforsin ; Cyclic AMP-Dependent Protein Kinases ; Endothelin-1* ; Heart Diseases ; Nifedipine ; Ouabain* ; Phosphotransferases ; Protein Kinases

OBJECTIVES: This study was aimed to examine whether participants of a Korean candlelight rally had correct medical information about human mad cow disease and rational attitudes about imported U.S. beef in relation to human mad cow disease. METHODS: A total of 393 face-to-face interviews were conducted, and subjects completed questions about prevalence of senile dementia and human mad cow disease in U.S. and whether they will eat U.S. beef even if no cases of human mad cow disease occurred in the U.S. or if the chance of being affected with human mad cow disease was lower than dying in a plane crash. RESULTS: Correct answer rates to the questions about prevalence of senile dementia and human mad cow disease were 28.2% and 36.1%, respectively. A majority of respondents answered that they would not eat U.S. beef even if there were no reported cases of human mad cow disease in the U.S. or if their chance of being affected with human mad cow disease was lower than dying in a plane crash (75.6% and 86.0%, respectively). CONCLUSION: At least 64.4% of participants had incorrect medical information about human mad cow disease, and their attitudes about imported U.S. beef may be rooted in emotion rather than fact.
Adenine Nucleotides ; Alzheimer Disease ; Animals ; Cattle ; Surveys and Questionnaires ; Encephalopathy, Bovine Spongiform ; Humans ; Korea ; Mycophenolic Acid ; Prevalence

Glucose and lipid metabolism is the most fundamental metabolic activity of higher organisms. This process is affected by both genetic polymorphisms and environmental factors. Excessive uptake and accumulation of lipids lead to obesity and disorder of glucose metabolic homeostasis characterized by insulin resistance and hyperglycemia, suggesting that the cross-regulation between lipid and glucose metabolism happens precisely at organ, cellular and molecular levels by known mechanisms. Adenine nucleotides and their metabolites have emerged as mediators in the mutual regulation of glucose and lipid metabolism. This review summarizes the roles of purinergic signaling induced by fatty acids in glucose metabolism and the development of type 2 diabetes.
Adenine Nucleotides ; Diabetes Mellitus, Type 2 ; Glucose ; Homeostasis ; Humans ; Insulin Resistance ; Lipid Metabolism

PURPOSE: The purpose of this study was to evaluate clinical results and accuracy of femoral cutting in the coronal plane in total knee arthroplasty (TKA) using a fixed length intramedullary guide. MATERIALS AND METHODS: From 2005 to 2008, 101 patients (154 knees) underwent TKA (NexGen LPS implant). The minimal follow-up period was 3 years (mean, 4.4 years). The patients were divided into two groups (group 1, 94< or =alpha angle<98; group 2, 94>alpha, 98< or =alpha). Mechanical axis deviation (MAD), patellar tilting angle (PTA), Knee Society Knee Score (KSKS), and Knee Society Function Score (KSFS) were measured in both groups. RESULTS: There were 120 knees in group 1 and 34 knees in group 2. There was no significant intergroup difference in the postoperative MAD (group 1, 1.59degrees; group 2, 1.91degrees). The number of outliers with > or =2degrees MAD was 65 in group 1 and 24 in group 2. The mean PTA, KSKS, and KSFS were 10.17degrees, 96.0, and 96.6, respectively, in group 1 and 11.58degrees, 84.5, and 85.5, respectively, in group 2. CONCLUSIONS: The percentage of coronal alignment outliers was relatively high (34 in 154 cases, 22%) after TKA using a fixed length intramedullary guide. However, there was no statistically significant intergroup difference in clinical results (KSKS, p=0.67; KSFS, p=0.56).
Adenine Nucleotides ; Arthroplasty ; Axis, Cervical Vertebra ; Follow-Up Studies ; Humans ; Knee ; Mycophenolic Acid