Effects of splicing-regulatory polymorphisms in ABCC2, ABCG2, and ABCB1 on methotrexate exposure in Chinese children with acute lymphoblastic leukemia.

PURPOSE: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters play an important role in the response to methotrexate (MTX). In this study, we investigated the frequency distribution of three splicing-regulatory polymorphisms in ABC transporters (ABCC2 rs2273697 G>A, ABCG2 rs2231142 G>T, and ABCB1 rs1128503 A>G) and their effects on MTX concentrations and the clinical outcome in a Chinese pediatric population with acute lymphoblastic leukemia (ALL). METHODS: A fluorescence polarization immunoassay was used to measure the serum MTX concentrations in 24 h (C24h) and 42 h (C42h). The Sequenom MassARRAY system was used for single-nucleotide polymorphism (SNP) genotyping. RESULTS: The study population had significantly lower frequencies of ABCC2 rs2273697 A, ABCG2 rs2231142 G, and ABCB1 rs1128503 G than African and European samples (P < 0.05). The dose-normalized MTX concentrations after 24 h and the proportion of C42h > 0.5 µmol/L were significantly lower in patients with the ABCG2 rs2231142 GG genotype than in patients with the GT or TT genotype (P = 0.01 and 0.006, respectively). No significant effects on MTX pharmacokinetics were observed for ABCC2 rs2273697 and ABCB1 rs1128503 polymorphisms. Bioinformatics analysis suggested that the three SNPs overlapped with the putative binding sites of several splicing factors. CONCLUSION: In conclusion, our study confirmed the ethnicity-based differences in the distribution of the three investigated SNPs. The ABCG2 rs2231142 polymorphism exerted a significant effect on the level of MTX exposure. These findings may help explain the variability in MTX responses and optimize MTX treatment in pediatric patients with ALL.

Microevolutionary mechanism of high-altitude adaptation in Tibetan chicken populations from an elevation gradient.

As an indigenous breed, the Tibetan chicken is found in highland regions and shows physiological adaptations to high altitude; however, the genetic changes that determine these adaptations remain elusive. We assumed that the microevolution of the Tibetan chicken occurred from lowland to highland regions with a continuous elevation range. In this study, we analyzed the genome of 188 chickens from lowland areas to the high-altitude regions of the Tibetan plateau with four altitudinal levels. Phylogenetic analysis revealed that Tibetan chickens are significantly different from other altitude chicken populations. Reconstruction of the demographic history showed that the migration and admixture events of the Tibetan chicken occurred at different times. The genome of the Tibetan chicken was also used to analyze positive selection pressure that is associated with high-altitude adaptation, revealing the well-known candidate gene that participates in oxygen binding (HBAD), as well as other novel potential genes (e.g., HRG and ANK2) that are related to blood coagulation and cardiovascular efficiency. Our study provides novel insights regarding the evolutionary history and microevolution mechanisms of the high-altitude adaptation in the Tibetan chicken.

Analysis of the frequency distribution of five single-nucleotide polymorphisms of the MTRRgene in a Chinese pediatric population with acute lymphoblastic leukemia.

STUDY OBJECTIVE: The objective of the present study was to examine the frequency distribution of five single-nucleotide polymorphisms (SNPs; rs1801394 A>G, rs1532268 C>T, rs162036 A>G, rs10380 C>T, and rs9332 C>T) of the methionine synthase reductase (MTRR) gene, their effects on methotrexate (MTX) concentration, and the risk of relapse in a Chinese pediatric population with acute lymphoblastic leukemia (ALL). DESIGN: This was a retrospective single-center study, and all analyses were exploratory. SETTING: Pediatric Department of Beijing Shijitan Hospital, Capital Medical University, Beijing, China. PATIENTS: One hundred and forty pediatric patients with ALL. INTERVENTION: All patients were treated according to the Chinese Children's Leukemia Group (CCLG)-ALL 2008 protocol. MEASUREMENTS AND MAIN RESULTS: Serum MTX concentrations were measured using fluorescence polarization immunoassay. Genotyping of five SNPs was performed using the Sequenom MassARRAY iPLEX platform. Chinese children with ALL had a significantly lower frequency of rs1801394 G than European (EUR) and South Asian (SAS) populations; significantly lower frequency of rs1532268 T than American (AMR), EUR, and SAS populations; and significantly lower frequencies of rs162036 G, rs10380 T, and rs9332 T than African and AMR populations (p < 0.01). Seven haplotypes were observed, with the ACACC being the most common haplotype (49.9%) in our study. The median dose-normalized concentrations of MTX in serum at 24 h in children with rs1532268 CT and TT genotypes were significantly higher than those with CC genotype (p = 0.04). Compared with children with AA-CC-AA-CC-CC diplotype, a significantly higher risk of relapse was observed in children with AG-CC-AA-CC-CC and AG-CC-AG-CC-CC diplotypes (p = 0.03 and 0.003, respectively). CONCLUSIONS: The present study confirmed the ethnic differences in the distribution of MTRR rs1801394, rs1532268, rs162036, rs10380, and rs9332 polymorphisms. The rs1532268 polymorphism had greater effects on MTX disposition. The AG-CC-AA-CC-CC and AG-CC-AG-CC-CC diplotypes were significantly associated with higher risk of relapse of ALL.

Association of GGH Promoter Methylation Levels with Methotrexate Concentrations in Chinese Children with Acute Lymphoblastic Leukemia.

BACKGROUND: It is known that γ-glutamyl hydrolase (GGH) is involved in the disposition of methotrexate (MTX), and GGH activity is regulated by DNA methylation in acute lymphoblastic leukemia (ALL) cells. The present study explores the methylation status of the GGH promoter in peripheral blood and its association with MTX levels and toxicities in Chinese children with ALL. METHODS: Serum MTX concentrations were determined by fluorescence polarization immunoassay. Methylation quantification and genotyping for GGH rs3758149 and rs11545078 was performed by Sequenom MassARRAY in 50 pediatric patients with ALL. RESULTS: Overall, the investigated region of the GGH promoter was in hypomethylated status. The methylation levels of cytosine phosphate guanine (CpG)_7, CpG_12, CpG_17, and CpG_20 were significantly higher in patients with B-cell ALL than other immunotypes (p<0.05). The methylation levels of CpG_13.14, CpG_17, and CpG_19 showed a significant negative correlation with MTX C24 hr (p<0.05). The methylation level of CpG_8.9 correlated significantly with MTX C42 hrs (p<0.05). The methylation level of CpG_19 was significantly lower in patients with MTX toxicities (p<0.05). CONCLUSIONS: The methylation levels of the GGH promoter might affect MTX exposure and toxicities. These findings provided reasonable explanations for the variability of MTX responses in patients with childhood ALL.

Convergent genomic signatures of high-altitude adaptation among domestic mammals.

Abundant and diverse domestic mammals living on the Tibetan Plateau provide useful materials for investigating adaptive evolution and genetic convergence. Here, we used 327 genomes from horses, sheep, goats, cattle, pigs and dogs living at both high and low altitudes, including 73 genomes generated for this study, to disentangle the genetic mechanisms underlying local adaptation of domestic mammals. Although molecular convergence is comparatively rare at the DNA sequence level, we found convergent signature of positive selection at the gene level, particularly the EPAS1 gene in these Tibetan domestic mammals. We also reported a potential function in response to hypoxia for the gene C10orf67, which underwent positive selection in three of the domestic mammals. Our data provide an insight into adaptive evolution of high-altitude domestic mammals, and should facilitate the search for additional novel genes involved in the hypoxia response pathway.

[Preventive effects of Salvia miltiorrhiza on multiple organ edema in the rats of limb ischemia/reperfusion].

OBJECTIVE: To investigate the preventive effects of Salvia miltiorrhiza (SM) on multiple organ edema in the rats which suffered from hind limb ischemia/reperfusion( LI/R). METHODS: Twenty four Wistar rats were randomly divided into 3 groups (n = 8): control group (C group), ischemia/reperfusion group (I/R group ), Salvia miltiorrhiza group (SM group). Referring to Tourniquet method, the model rats which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made. Thirty minutes before reperfusion, SM was given to the rats in SM group by tail vein injection at the dose of 5 mL/kg. Accurately weighed one gram of heart, liver, kidney, lung, brain, intestine and skeletal muscle from every animals, weigh these specimens after baking (60 degrees C, 55 hours), calculated the ratio of wet and dry (Wet/Dry,W/D). The levels of interleukin-1 (IL-1) ,interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in plasma and the contents of Superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The morphologic changes of skeletal muscle were observed with microscope. RESULTS: It was found that after suffering from ischemia/reperfusion, the W/D of every specimens increased in different degree (P < 0.05, P < 0.01). In plasma, the values of SOD decreased but MDA increased obviously (P < 0.05, P < 0.01). The levels of IL-1, IL-6 ,TNF-alpha-a in plasma increased (P < 0.05, P <0.01). After LI/R, infiltration of inflammatory cells, broaden interstitial around muscle fiber and disordered arrangement of muscle fibers could be seen under microscope. However, Compared with LI/R group, W/D and levels of serum inflammatory factors in SM group were all lower, the values of SOD in plasma increased but MDA in plasma failed down. Pathological changes in skeletal muscle were improved. CONCLUSION: Limb ischemia/reperfusion can lead to multiple organ edema, Salvia miltiorrhiza can prevent the edema in some degree by anti-oxidation and anti-inflammation.

[Effects of taurine on hemorheology of rats with type 2 diabetes].

OBJECTIVE: To observe the effects of taurine on hemorheology and oxidative stress of diabetic rats. METHODS: 40 rats were divided into control group, diabetes group and treatment group at random. Diabetic model was reproduced by intraperitoneal injection of streptozotocin. After having been treated with taurine for 8 weeks, glycosylated hemoglobin (HbA1c) and the serum contents of glucose, superoxide dismutase(SOD), malondialdehyde (MDA) were measured. The changes of hemorheology in different groups were detected respectively. RESULTS: Compared with control group, the content of glucose, MDA and HbA1c in diabetic rats was increased, the activity of SOD was decreased, the levels of whole blood viscosity and the aggregation index of red blood cells and hematocrit were increased and RBC deformability index was decreased in diabetic rats. Moreover, taurine was able to apparently reduce high blood glucose and HbA1c (P < 0.05), markedly elevated the activity of SOD, lowered the content of MDA (P < 0.01); and taurine also could significantly reduce the levels of whole blood viscosity and the aggregation index of red blood cells and hematocrit in the meanwhile, and increase RBC deformability index (P < 0.05 or P < 0.01). CONCLUSION: Taurine could enhance the ability of oxidation resistance, improve blood rheology property in diabetic rats, at the same time it could be beneficial to prevent and cure the development of diabetic blood vessel complication.

[Experimental research of prevention and therapy effect of anisodamine on acute respiratory distress syndrome (ARDS)].

AIM: To study preventive and therapeutic effect of anisodamine on acute respiratory distress syndrome(ARDS) induced by oleic acid and their mechanism of action. METHODS: Model of ARDS was made in rabbits by oleic acid (OA). The effect of anisodamine on the malondialdehyde (MDA), fibronectin (FN), lactate dehydrogenase (LDH) and acid phosphatase (ACP) in plasma, and superoxide dismutase (SOD) in erythrocyte and MDA, SOD and pulmonary surfactant (PS) in lung tissues homogenate and pathological examination of lung were observed. RESULTS: The administration of anisodamine before and after 30 minutes of injection OA decreased MDA, LDH and ACP, prevented the reduction of SOD, FN and PS. Compared with ARDS group, there was marked difference between the two, and alleviated lung injury. CONCLUSION: Anisodamine possesses preventive and therapeutic effects on ARDS by inhibiting lipid peroxidation and stabilizing membranes.

[Effects of taurine on TNF-alpha and NF-kappaB expression of liver injury after limbs ischemia/reperfusion in rats].

AIM: To investigate the effect of pretreatment with taurine on liver injury changes and the change of tumor necrosis factor alpha and NFkappaB expression following rats limbs ischemia/reperfusion. METHODS: The model of limbs ischemia/reperfusion injury on rats was adopted in the experiment. Wistar rats were randomized into 4 groups (n = 10): Control group, T group, I/R group and TR group. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and MDA in the plasma, MDA, MPO, calcium in liver tissues were measured by colorimetric method. The content of TNF-alpha in plasma and liver tissues was determined by radioimmunoassay. The morphologic changes were observed with HE staining. The expressions of NF-kappaBp65 in liver tissues were tested by immuno-histochemistry method. RESULTS: It was found that against the control group, the test values of ALT, AST, et al. and expressions of TNF-alpha, NF-kappaB increased in I/R group and TR group, but values of those in TR group were lower than in I/R group. CONCLUSION: Taurine can decrease the levels of TNF-alpha and NF-kappaB. It can mitigate the liver injury after limb ischemia/reperfusion injury in rats.

[Effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts].

AIM: To study the effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts. METHODS: Cultured L-6TG cells were divided into 3 groups: control group (C), ischemia/reperfusion group (I/R), PMA + ischemia/ reperfusion group (PMA), SOD, XOD and free calcium and mitochondrial respiration in L-6TG cell were evaluated in each group. Apoptosis was detected by flow cytometer with PI staining method and agarose gel electrophoresis, the immunohistochemical method was used to determine the expression of caspase-3. RESULTS: Compared with I/R group, in PMA group, XOD , free calcium in L-6TG cell and apoptotic percentage all decreased significantly, while SOD and mitochondrial respiration in L-6TG cell increased. DNA fragmentation analysis of L-6TG cell showed no laddering pattern. The expression of caspase-3 was down regulated significantly. CONCLUSION: Activation of PKC can lessen ischemia/reperfusion injury and apoptosis through lessening oxidative injury and mitochondrial injury, adjusting calcium dyshomeostasis and down expression of caspase-3.