Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var. yunnanensis / 中国中药杂志

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.

Crystal structures of plant uridine diphosphate-dependent glycosyltransferases / 生物工程学报

Glycosyltransferases (GTs) catalyze the transfer of a sugar residue of an activated sugar donor to an acceptor molecule. Many families 1 GTs utilize an uridine diphosphate (UDP) activated sugar as donor in the glycosylation reaction, and most of these belong to a group of GTs referred to as the UGTs. The relationship between the degree of amino acid sequence identity and substrate specificity of the plant UGTs is highly complicated, and the prediction of substrate specificity based on phylogenetic analyses need to be improved by more biochemical characterization. This review summarizes the three dimensional structures of plant UGTs published in the Protein Data Bank (PDB), including the detailed substrate interactions with the sugar and receptor binding pockets and mutational analyses of some critical amino acids. It will be helpful for biochemical characterization the substrate specificity of the individual UGT, and lay the foundation for the enzymatic and genetic manipulation of plant UGTs in the future.

Is it Possible to Perform Chest Compression in Various Alternative Positions in a Confined Space?: A Manikin and Simulation Study

PURPOSE: Standard chest compression is useful for cardiopulmonary resuscitation of victims but may be difficult to perform in a confined space if the victim is lying on his side. The aim of this study was to evaluate compression techniques administered to individuals lying in various alternative positions, and to determine which ones may be easier to perform in such situations. METHODS: Thirty two volunteers trained in basic life support (BLS) were enrolled. They were taught to do compression in four alternative positions (over-head position (OHP), saddle position (SP), upper diagonal position (UDP) and lower diagonal position (LDP)). For each position, they performed two minutes of continuous chest compression on a manikin that was connected to a Laerdal PC Skill Reporting System. They did this for the basic standard position (BSP) and four alternative positions with the positions presented randomly. The data, including the total number of compressions, the average rate of chest compressions per minute, the depth of each chest compression, and the position of the hands were recorded and analysed. RESULTS: There were no statistically significant differences between BSP and alternative positions for the total number of compressions (BSP:108.8 min-1; OHP:109.5 min-1; SP:107.8 min-1; UDP:108.5 min-1; LDP:107.7 min-1) of chest compressions. There was no statistically significant difference between BSP and alternative positions for the average depth of each compression (BSP:41.9 mm; OHP:44.4 mm; SP:41.8 mm; UDP:42.9 mm; LDP:41.1 mm), or for the number of incorrect hand positions except UDP (BSP versus UDP = 6.4 versus 32.5). p<0.054 is not normally considered significant. The p value has to be 0.050 or smaller. CONCLUSION: Chest compression in alternative positions can be equally effective as it is in the standard position. If chest compression in the standard position is not easily executable in a confined space, chest compression using an alternative positions can be used.

Is it Possible to Perform Chest Compression in Various Alternative Positions in a Confined Space?: A Manikin and Simulation Study

PURPOSE: Standard chest compression is useful for cardiopulmonary resuscitation of victims but may be difficult to perform in a confined space if the victim is lying on his side. The aim of this study was to evaluate compression techniques administered to individuals lying in various alternative positions, and to determine which ones may be easier to perform in such situations. METHODS: Thirty two volunteers trained in basic life support (BLS) were enrolled. They were taught to do compression in four alternative positions (over-head position (OHP), saddle position (SP), upper diagonal position (UDP) and lower diagonal position (LDP)). For each position, they performed two minutes of continuous chest compression on a manikin that was connected to a Laerdal PC Skill Reporting System. They did this for the basic standard position (BSP) and four alternative positions with the positions presented randomly. The data, including the total number of compressions, the average rate of chest compressions per minute, the depth of each chest compression, and the position of the hands were recorded and analysed. RESULTS: There were no statistically significant differences between BSP and alternative positions for the total number of compressions (BSP:108.8 min-1; OHP:109.5 min-1; SP:107.8 min-1; UDP:108.5 min-1; LDP:107.7 min-1) of chest compressions. There was no statistically significant difference between BSP and alternative positions for the average depth of each compression (BSP:41.9 mm; OHP:44.4 mm; SP:41.8 mm; UDP:42.9 mm; LDP:41.1 mm), or for the number of incorrect hand positions except UDP (BSP versus UDP = 6.4 versus 32.5). p<0.054 is not normally considered significant. The p value has to be 0.050 or smaller. CONCLUSION: Chest compression in alternative positions can be equally effective as it is in the standard position. If chest compression in the standard position is not easily executable in a confined space, chest compression using an alternative positions can be used.

A Case of Infantile Hepatic Hemangioendothelioma Incidentally Detected during the Evaluation of Galactosemia

Galactosemia is a group of inherited enzyme deficiencies characterized by increase in the blood galactose levels. This condition may be associated with deficiencies of galactose-1-phosphate uridyl transferase, galactokinase, or uridine diphosphate galactose-4-epimerase. However, the elevated galactose identified by neonatal screening tests has several other possible etiologies, including hepatic hemangioendothelioma, hepatic hemangioma, and patent ductus venosus with hypoplasia of the portal vein. We report a 13-day-old Korean male with hepatic hemangioendothelioma, which was incidentally detected during the evaluation for suspected galactosemia. Laboratory studies revealed that mildly elevated levels of galactose, galactose-1-phosphate and alpha-fetoprotein, at the time of admission, were gradually decreased to the normal range over the 6 months of observation. Ultrasonography showed a well-defined heterogeneous hypoechoic mass in the liver, and magnetic resonance imaging study showed multiple enhanced mass lesions, which was compatible with the diagnosis of a hepatic hemangioendothelioma. Thus, hepatic imaging, especially ultrasonography, should be performed if neonatal screening suggests galactosemia.

A Case of Gilbert's Syndrome with Severe Neonatal Hyperbilirubinemia

Gilbert's syndrome is caused by a reduction in the activity of uridine diphosphate glucuronosyltransferase (UGT) and induces chronic, non-hemolytic unconjugated hyperbilirubinemia. It has been suggested that 3-10% of the population has Gilbert's syndrome. Commonly, Gilbert's syndrome causes mild symptoms. However, a case of Gilbert's syndrome with severe neonatal hyperbilirubinemia is presented here. The patient developed jaundice three days after birth. Five days after birth, the patient's total serum bilirubin level was 34 mg/dL. The patient received intensive phototherapy and was given oral phenobarbital. Hemolytic hyperbilirubinemia was excluded on the basis of laboratory tests. Heterozygote polymorphisms of the promoter region (-3279T>G) and exon 1 (211G>A) were found in UGT1A1 gene. After discharge, the patient did not require any further treatment. This is the first case of proven Gilbert's syndrome with severe neonatal hyperbilirubinemia in Korea.

A Case of Gilbert's Syndrome with Severe Neonatal Hyperbilirubinemia

Gilbert's syndrome is caused by a reduction in the activity of uridine diphosphate glucuronosyltransferase (UGT) and induces chronic, non-hemolytic unconjugated hyperbilirubinemia. It has been suggested that 3-10% of the population has Gilbert's syndrome. Commonly, Gilbert's syndrome causes mild symptoms. However, a case of Gilbert's syndrome with severe neonatal hyperbilirubinemia is presented here. The patient developed jaundice three days after birth. Five days after birth, the patient's total serum bilirubin level was 34 mg/dL. The patient received intensive phototherapy and was given oral phenobarbital. Hemolytic hyperbilirubinemia was excluded on the basis of laboratory tests. Heterozygote polymorphisms of the promoter region (-3279T>G) and exon 1 (211G>A) were found in UGT1A1 gene. After discharge, the patient did not require any further treatment. This is the first case of proven Gilbert's syndrome with severe neonatal hyperbilirubinemia in Korea.

The relationship between Gly71Arg and TATA box polymorphism of UGT1A1 gene and prolonged hyperbilirubinemia of breast milk feeding infant in Korean / 소아과

PURPOSE: It has been known that breast milk cause prolonged unconjugated hyperbilirubinemia. UGT1A1 is a important gene of uridine diphosphate glucuronosyltransferase (UGT) which has a major role of bilirubin metabolism. These findings suggest that there is a relationship between UGT1A1 gene mutation and prolonged jaundice of breast feeding infant. The aim of study was to investigate whether a polymorphism of the UGT1A1 gene exist in prolonged hyperbilirubinemia of breast milk feeding Korean infant. METHODS: The genomic DNA was isolated from 50 full term Korean neonates, who had greater than a 10 mg/dL of serem bilirubin after 2 weeks of birth with no significant cause, and the other genomic DNA was isolated from 162 full term Korean neonates of the control population. Both group fed breast milk. We performed direct sequencing of TATA box and Gly71Arg polymorphism of the UGT1A1 gene. RESULTS: Two of the 50 neonates with hyperbilirubinemia had AA polymorphism, and 40 had GA polymorphism. Five of the 129 neonates of the control group had AA polymorphism, and 4 had GA polymorphism. The allele frequency of G>A polymorphism in the hyperbilirubinemia group was 44.0%; it was significantly higher than 5.4% of the control group. TATA box polymorpism was not different both group significantly. CONCLUSION: Our result indicated that Gly71Arg polymorphism is associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean, while TATA box polymorphism is not associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean.

The Efficacy of UGT1A1 Polymorphism in Chemoradiation Therapy Using Irinotecan in Patients with Locally Advanced Rectal Cancer

PURPOSE: Irinotecan (CPT-11) is hydrolyzed to an active SN-38, which is further detoxicated to SN-38G through conjugation by uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) enzymes. There are many reports that UGT1A1 polymorphisms are associated with irinotecan related dose-limiting toxicity. The aim of the present study is to determine whether UGT1A1 polymorphisms affect individual variations of the toxicity due to and the tumor response to irinotecan via the alteration of bioavailability of SN-38 in Korean patients with locally advanced rectal cancer. METHODS: Twenty patients with locally advanced rectal cancer, who had received surgery after irinotecan-containing chemoradiation from 2003 to 2006, were enrolled. We analyzed the association of UGT1A1 genotypes with toxicity and tumor response to chemoradiation therapy. A tumor response was assumed when a tumor regression grade I or II was obtained. Toxicity was graded in accordance with the NCI common toxicity criteria. RESULTS: The frequence of square53(TA)6>7 (UGT1A1*28), 211G>A (UGT1A1*6), 686C>A (UGT1A1*27), square3279T>G (UGT1A1*60), and square3156G>A were 25% (5/20), 25% (5/20), 0% (0/20), 55% (11/20), and 20% (4/20), respectively. There were five grade III neutropenia and one severe diarrhea. Patients with UGT1A1*28 and square3156G>A showed higher complete tumor response rates (40% vs. 6.7%, P=0.07; 50% vs. 6.3%, P=0.08), but there was no differences in toxicity and tumor response between responders and non-responders. Patients with square3279T>G (UGT1A1*60) showed a tendency for lower tumor response in tumor responders, but there was no statistically significant difference (P=0.07). CONCLUSIONS: This study suggested that square3279T>G (UGT1A1*60) may be useful in predicting tumor response of irinotecan. In the future, further study is warranted using large numbers of cases to reach statistical significance.

Intrahepatic Arterio-Venous Shunts in a Patient with Hypergalactosemia Detected by Neonatal Screening

We report a case of 15 days old newborn presenting with hypergalactosemia detected by newborn screening who had intrahepatic arterio-venous shunts with multiple pin-head sized cutaneous hemangiomas. Plasma level of galactose was elevated to 11.3 mg/dL at age of 7 days, but the activity of galactose-metabolizing enzymes including galactose-1- phosphate uridyltransferase, galactokinase, and uridine diphosphate galactose-4-epimerase were all normal. Intrahepatic arterio-venous shunts were diagnosed by abdominal ultrasonography with color doppler ultrasonography and abdominal computed tomography. At age of 3 months, the plasma level of galactose further elevated to 14.73 mg/dL, at which time lactose-free cows milk formula was started. At age of 6 months, the plasma level of galactose decreased to within normal range with disappearance of previously noted multiple cutaneous hemangiomas. In hypergalactosemia of the newborn, the intrahepatic shunts should be considered as a possible cause, once hereditary enzyme deficiencies have been ruled out.

Design of Mobile Emergency Telemedicine System Based on CDMA2000 1X-EVDO / 대한의료정보학회지

Using a high bandwidth cellular communication network is appropriate to implement a high-quality mobile emergency telemedicine system. In Korea, the commercial service of CDMA2000 1X-EVDO has been providing since 2002. In this paper, we designed the system that transfers the biological signal and the video information of a patient simultaneously based on this CDMA2000 1X-EVDO network environment. In CDMA2000 1X-EVDO, the maximum speed of its reverse link can be observed within 153.6Kbps. Before the system design, several field tests had been performed using commercial CDMA2000 1X-EVDO reverse link with the UDP data segments. The test had been taken under several velocity and tunnel areas of Seoul. With the test result we implemented an efficient emergency telemedicine system fitted to the features of CDMA2000 1X-EVDO reverse link using UDP packets. Additional header information is added to the UDP packet data. With the header information the emergency system can transmits the ECG signal prior to the video data and controls the transmission error. The designed system has the ability to transmit both the biological signals and MPEG4 video of 640x480 spatial resolution at the same time. We set up the ambulance with this system and test it on the road.

Asymmetrical distribution of P2Y nucleotide receptors in rabbit inner medullary collecting duct cells

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.

Effects of Uridine 5'-Triphosphate on the Vascular Tone of Rat Thoracic Aorta

BACKGROUND: Uracil nucleotides are stored in platelets and all other cells, and are released into the extracellular space upon stimulation. They show various biological responses but their actions and mechanism are not well understood. This study was conducted to investigate the effects of uridine 5'-triphosphate(UTP) on vascular tone and to identify the characteristics of their receptors. METHODS: Aortic ring preparation were made from the rat descending thoracic aorta. Endo-thelial cells were preserved or removed by gentle rubbing, The basal tension of aortic ring was lgm and isometric contraction were recorded on polygraph using force transducer. RESULTS: In aortic ring Precontracted by 100nM norepinephrine, UTP induced dual effect with various concentrations. UTP elicited endothelium-dependent relaxation at low concentrations(100nM-10microM), and endothelium-independent contraction at high concentrations(more than 30microM). Among uracil nucleotides, UDP was as much effective as UTP in vascular tone, but UMP and uridine were not. UTP(pA50 6.15) was more potent than ATP(5.17), ITP(4.75) and other nucleotides(TTP, GTP, CTP). At basal tension, UTP induced relaxation at low concentrations and contraction at hige concentrations in endothelium-intact ring. But in endothelium-removed ring, UTP elicited only contraction. Prior treatment of aortic ring with suramin, a non-selective P2-purinoceptor blocker, inhibited UTP-Induced relaxation and contraction. Reactive blue-2, a P2gamma purinoceptor blocker, inhibited relaxation only, but alpha, beta-methylene ATP, a P2x Purinoceptor blocker, enhanced contractile response. ATP inhibited the UPT-induced relaxation, but 2-methylthio ATP did not alter the effects of UTP. It means that UTP and ATP act at the same receptor but 2-methylthio ATP does not. CONCLUSION: These results suggest that UTP-induced relaxation is mediated by nucleotide receptors on endothelium and the contraction is mediated by pyrimidinoceptors on vascular smooth muscle.

Activation of the Cardiac ATP-Sensitive K+Channel by KR-30816,Newly Synthesized Potassium Channel Opener

BACKGROUND: The effects of a newly synthesized potassium channel opener, KR-30816((-)(nitro-2-hydroxymethyl-2-methy-2H-1-benzopyran-4-y1)pyridine oxide) on the action potential of papillary muscles of guinea pigs and the ATP-sensitive potassium channel current(IKATP) of single ventricular muscle cells of rats were examined to make clear its action mechanism of the KATPchannel. METHODS: We used the conventional microelectrode and the excised inside-out patch configuration. RESULTS: KR-30816 caused a shortening of the action potential duration in dose-dependent manner, which was inhibited by glibenclamide(3microM). Before run-down of the K+channel, KR-30816 activated the cardiac ATP-sensitive K+ channel only in the presence of ATP and shifted the dose-response relation curve between [ATP]i and the channel activity to the right in parallel. After run-down of the KATP channel, KR-30816 did not after the channel opening either in the absence or in the presence of UDP. CONCLUSION: These results suggest that KR-30816 antagonizes the inhibitory effect of ATP on the KATPchannel in a competitive manner, thereby enhancing the channel openings.

Reduçäo do efeito hipnótico da ketamina pelo uso prévio de um ativador metabólico / Reduction of the ketamin hypnotic effect by previous use of a metabolic activator

A análise quantal pelo método dos próbitos mostrou que a UDPG (20 mg/kg i.p.) foi capaz de diminuir 1/3 do efeito da ketamina em produzir a perda do reflexo de endireitamento em ratos, onde a DE50% de 54,0 mg passou para 69,8 mg (p=0,05). As retas de regressäo da Ketamina sozinha e Ketamina UDPG sem desvios de linearidade (p=0,05) mostram que a UDPG diminui a potência hipnótica da ketamina e näo foi capaz de interferir com seu mecanismo básico de açäo no sistema nervoso central. Sugere-se que a UDPG bloqueia os efeitos da Ketamina pelo aumento da glicuroconjugaçäo da norketamina e do metabólito II da Ketamina no fígado e provavelmente aumentando suas eliminaçöes renais. Contudo a açäo direta da UDPG no sistema nervoso central e sua interaçäo com os enantiomorfos da Ketamina e seus metabólitos fica para serem elucidados