The DNA fragment sized 2 139bp, the same Sequence with AtKup1 gene from Arabidopsis thalianna was used as the templates for DNA family shuffling. The shuffeld AtKup1 gene library was expressed in the mutant of 5. cerevisae in which potassium transporter gene TRK1 and TRK2 were knocked out by homologous recombination. Then the screening was carried out in the low potassium media containing 5. 0 mmol/L KC1 and no histidine in it. it was found that both of diverse and wild AtKup1 gene can rescues the trk1△trk2△yeast mutant strain in low [ K + ] medium. The growth of 2 clones yeast containing diverse AtKup1 were beter than that of AtKup1 wild gene transformant. The sequencig results of the shuffeld AtKup1 showed that there were 2 nucleotide changed, which resulted in 2 amino acid variations in it compared with the original AtKup1. The potassium uptaking capacity of shuffled AtKup1 gene increased significantly when it was transformed into tobacco.

Objective To investigate the influences of mutation at precore and basic core promoter(BCP) region in hepatitis B virus(HBV) on the immune response of specific cytotoxic T lymphocytes(CTL) in patients with chronic hepatitis B(CHB).Methods The number of specific CTL in peripheral blood mononuclear(PBMC) of CHB patients were tested by cytokine flow cytome- try(CFC) and HBV core18-27 peptide.HBV precore and BCP fragments were directly sequenced. Results Twenty-one(38.9%) samples were HBV precore G1896A mutation.Twenty-six(48.1%) samples were BCP region 1762/1764 combined mutation.Thirteen(24.1%) stains were three sites mutated simultaneously.Stimulated with HBV core 18-27 in vitro,the specific CTL level was signifi- cantly higher in the patients with G1896A mutation and BCP region mutation [(0.41?0.09)%, (0.36?0.08)%,(0.48?0.08)%,respectively]than those without mutation[(0.11?0.06)%, P

Objective To study the presence of CD4+CD25+regulatory T cells and hepatitis B virus(HBV)specific cytotoxie T lymphocyte(CTL)in peripheral blood and liver tissues of patients with chronic hepititis B(CHB)and its clincial significance.Methods One hundred and fifty-seven HBV-infected patients,including 20 cases of acute hepatitis B,115 cases of chronic hepatitis B,and 22cases of HBV-related liver cirrhosis,and 20 healthy controls were enrolled in this study.Peripheral blood was collected and liver tissues were obtained from some of the enrolled subjects.The CD4+CD25+regulatory T cells and HBV specific CTL were analyzed using flow cytometry and cytokine flow cytometry(CFC).The comparison between groups was done by t test.Results The percentages of CD4+CD25+ regulatory T cells in the peripheral blood of patients with acute hepatitis B and CHB of mild,moderate and severe degree were(2.87±0.94)%,(3.53±1.56)%,(4.59±2.98)%and(3.65±1.73)%,respectively,which were higher than that of controls(2.36±0.60)%(t=2.04,5.97,3.30 and 3.17,respectively,P<0.01).The percentages of HBV specific CTL in the peripheral blood of patients with mild,moderate and severe degree of CHB and HBV-related liver cirrhosis were (0.189±0.152)%,(0.103±0.110)%,(0.118±0.120)%and(0.098±0.101)%,respectively,which were significantly lower than that of acute hepatitis patients [(0.815±0.360)%](t=10.09,11.87,9.17 and 8.96,respectively,P<0.01).CD4+CD25+ regulatory T cells and HBV specific CTL in liver tissues were both higher than those in the peripheral blood.Conclusion CD4+CD25+regulatory T cells may play an important role in anti-HBV immune response through inhibiting CD8+T eell function.

Objective To explore the mechanism of opioid-psychic dependence involving the aspects of pre-receptor and receptor by observing the changeable expressions of glutamate neurotransmitter and NR2B of N-methyl-D-aspartic acid (NMDA) receptor in the neuroanatomical circuit of ventral temental area, nucleus accumbens and prefrontal cortex (VTA-Nac-PFC) of rats subjected to morphine-induced conditioned place preference (CPP). Methods The models of CPP were validated by escalating doses of morphine in rats (n=16). The colorimetry and immunohistochemistry ways were applied to detect the contents of glutamic acid and the expression level of NR2B in VTA, Nac and PFC. Results As compared with those in the control group physiological saline), the prolonged detention time of white compartment in the model group was notably observed (P<0.05), and increased content of glutamic acid and expression level of NR2B in fields of VTA, Nac and PFC in the model group were significantly detected (P<0.05). Conclusion Increased level of giutamic acid and expression level of NR2B in nuroanatomieal circuit of VTA, Nac and PFC could play key roles in inducing morphine-psychic dependent rats.

Objective To understand iodine and iron nutritional status of lactating women in Aksu Prefecture of Xinjing Autonomous Region, and to provide proper intervention in control of iodine deficiency disorders (IDD) combined with iron deficiency. Methods Four townships as survey points were selected in Baicheng County of Aksu, where severe iodine deficiency was confirmed, 30 to 40 lactating women were investigated for this study in each townships. Samples of urine and drinking water from their family were collected, and then the iodine concentrations were determined. Whole blood was collected by venipuncture for determinations on serum ferritin (SF), serum iron (SI), total-iron-binding capacity(TIBC), and their thyroid function, including FT3, FT4, TSH. Results Median urinary iodine concentration in 137 lactating women was 134.99 μg/L, however, median urinary iodine in lactating women in Daqiao (99.73 μg/L), Tuokexun(44.17 μg/L) of 4 townships was below 100 μg/L. The proportion of urinary iodine below 50 μg/L was higher than 20% in Chaerqi [21.1% (8/38)], Daqiao [21.4% (6/28)], Tuokexun [47.8% (11/25)]. The medium of iodine concentration in drinking water, that was collected from 78 resident families, was 2.15 μg/L. Lactating women of serum SF, SI, FT4, that lower than the normal value, was accounted for 47.6% (59/124), 16.9%(21/124), 11.8% (15/127)respectively. Lactating women of serum TIBC, TSH, that higher than the normal value, was accounted for 20.2% (25/124),10.2% (13/127). Conclusion There is existence in of the combination severe iodine and iron deficiency in a historical serious IDD endemic area in Aksu Prefecture of Xinjiang Autonomous Region, and iron deficiency may be another important environmental factor for the deterioration of IDD prevalence.

Objective To investigate the iodine nutritional status of school children, lactating women and infants in iodine deficient areas of Baicheng and Wushi Counties in Xinjiang Autonomous Region. Methods According to the geographic location of east, south, west and north of county, 10 primary schools of 8 townships were selected. In each school, 10 children among each gender and age group from 8 to 10 years old were selected. A total of 300 school children were chosen. One hundred and four infants aged 0-2 years old and their mother were selected in 8 townships. Iodine content in edible salt at household level, the urinary iodine in school children and lactating women, total goiter rate(TGR) and the status of the intelligence quotient (1Q) of school children, the status of psychological development of infants were observed. Direct titration assay for testing the salt iodine, colorimetric ceric-arsenic assay and vitriolic ammonium assimilation were used for testing urinary iodine. The size of thyroid gland was measured by palpation. The Combined Raven Test for Chinese Rural was used to test the IQ. The psychological development of infants was tested by Danver Development Screening Test (DDST). Results The coverage rate of iodized salt at household level was 73.1% (123/182), however, the proportion of households using adequately iodized salt was 64.1% (118/182). The medium of urinary iodine in school children was 103.7 μ/L, with 47.8%(75/157) less than 100 μg/L and 28.0% (44/157) less than 50 μg/L; it was 123.0 μg/L in Baieheng County, with 44.4%(28/63) less than 100 μg/L and 33.3%(21/63) less than 50 μg/L; it was 100.3 μg/L in Wushi County, 50.0%(47/94) less than 100 μg/L and 24.5%(23/94) less than 50 μg/L. The medium of urinary iodine in locating women was 143.3 μg/L, it was 119.7 μg/L and 184.6 μg/L in Baicheng and Wushi Counties, respectively. The total rate of goiter in school children was 14.3%(43/300), it was 10.8%(13/120) and 16.6%(30/180) in Baicheng and Wushi Counties, respectively. The average IQ in school children was 80.6±11.6, it was 83.0±11.6 and 79.0±11.7 in Baicheng and Wushi Counties, respectively. The proportion of mental retardation in school children (IQ≤69) was 13.0% (39/300), it was 6.7% (8/120) and 17.2%(31/180) in Baicheng and Wushi Counties, respectively. In addition, the proportion of psychological development in infants being normal, suspicious and abnormal was 78.8%(82/104), 12.5% (13/104) and 8.7%(9/104), respectively. Conclusion This study confirms the fact that there is also existence of mental retardation in children and infants, caused by iodine deficiency.

OBJECTIVETo study the acting mechanism of anti-morphine conditioned place preference (CPP) between aqueous extract of Corydalis yanhusuo and L-THP and compare their effects.

METHODThe CPP model was established by injecting morphine in rats with a increasing dose for 10 days, with the initial dose of 10 g x kg(-1) and the final dose of 100 g x kg(-1), 10 mg x kg(-1) was increased each day, thus 100 mg x kg(-1) was injected by d 10. Having been treated with differential doses (2, 1 and 0.5 g x kg(-1)) of C. yanhusuo (containing L-THP: 0.153, 0.077 and 0.038 mg x kg(-1) respectively) and L-THP (3.76, 1.88 and 0.94 mg x kg(-1)) for six days, the CPP effect in rats was detected. Both colorimetry and immunohistochemistry methods were adopted to detect the content of glutamate neurotransmitter in each brain region and the expression of NR2B in VTA-NAc-PFC neuroanatomical circuit.

RESULTCompared with the physiological saline treatment group, C. yanhusuo (2, 1 g x kg(-1)) and L-THP (3.76 and 1.88 mg x kg(-1)) groups showed a notably shorter retention period of rats in white boxes (morphine-accompanied boxes) (P < 0.05 or P < 0.01) and remarkably lower glutamic acid content in VTA, NAc and PFC and NR2B expression.

CONCLUSIONBoth C. yanhusuo and L-THP can substantially inhibit the effect of morphine CPP, reduce the increasing glutamic acid content in VTA-NAc-PFC neuroanatomical circuit and down-regulated NR2B expression, which may be one of mechanisms on reducing the effect of morphine CPP. C. yanhusuo preparations containing L-THP (1 x ) showed 24-fold effect of L-THP monomer of single application in terms of the behaviouristics of inhibitory effect on CPP as well as the similarity in terms of transmitter glutamic acid of in VTA-NAc-PFC neuroanatomical circuit and pharmacological mechanism of NR2B.

Animals ; Berberine Alkaloids ; therapeutic use ; Conditioning, Operant ; drug effects ; Corydalis ; chemistry ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Male ; Morphine ; antagonists & inhibitors ; Morphine Dependence ; drug therapy ; psychology ; Rats ; Rats, Sprague-Dawley

ObjectiveTo observe the thyroid status of pregnant women during the 1st and 2nd trimester of gestation,and its role in brain and neural development of their offspring's.MethodsFrom 2008 to 2009,pregnant women from nine townships of two counties in Wushi and Baicheng in Aksu prefecture of Xinjiang were selected as research subjects according to the survey standard.After informed consent signed,their urinary iodine,serum thyroid stimulating hormone(TSH) and free thyroxin(FT4) were analyzed.The value of thyroid hormone of normal pregnant women was used in diagnosis of subclinical hypothyroidism and hypothyroxinemia in pregnant women.From 2010 to 2011, The brain and neural development status among offspring born by those pregnant women were evaluated with DDST.In accordance with the results of Denver Development Screen Test (DDST) screening,pregnant women were classified into survey and control groups,and the survey group was the suspicious and abnormal of the result of DDST screening(delay),the control group was normal of the result.According to gestational age,pregnant women were divided into 4 gestation groups:G1(6 to 13 weeks),G2(14 to 18 weeks),G3 ( 19 to 23 weeks) and G4(24 to 28 weeks).ResultsA total of 396 cases of pregnant women during the 1st and 2nd trimester of gestation were investigated(survey group 102 cases,control group 294 cases).The median value of urinary iodine concentration among pregnant women in survey group was 152.4 μg/L The prevalence of subclinical hypothyroidism and hypothyroxinemia among pregnant women was 10.78％(11/102) and 3.93％(4/102),respectively.In control group,the median value of urinary iodine concentration was 180.0 μg/L The prevalence of subclinical hypothyroidism and hypothyroxinemia among pregnant women was 7.48％ (22/294) and 4.42％ (13/294),respectively.During the pregnant period from G1 to G3,the median serum TSH of pregnant women in DDST survey group (2.24,3.49,2.85 mU/L) was higher than that of DDST control group( 1.59,2.70,2.28 mU/L).Especially,the difference of TSH between the two groups during the period of G3 was statistically significant (t =4.906,P ＜ 0.05 ).ConclusionsHypothyroidism tendency of pregnant women during the period from gestation week 19th to 23rd may be an important factor in the development of brain abnormalities of their offsprings.

Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ～ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ～ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ～ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ～-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ～-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.

OBJECTIVETo identify the relationship between coagulation factor V (FV) gene single nucleotide polymorphisms (SNPs) and venous thromboembolism (VTE).

METHODSThe FV clotting activity (FV: C) and FV antigen (FV: Ag) in plasma of VTE group (111 patients) and normal control (110 patients) were detected using one-stage clotting assay and ELISA, respectively. Five pairs of primers of the F V polymorphisms including Asp79His, Arg306The, Arg306Gly, Arg506Gln and Ile359The/His1299 Arg were synthesized and amplified by PCR. The PCR products were digested by restriction enzyme using PCR-RFLP. The detected polymorphisms were confirmed by direct sequencing. The samples containing the polymorphisms were screened for coding regions of all F V exons with direct sequencing.

RESULTSThe plasma levels of F V: C and F V: Ag of VTE group and normal control were (106.9 +/- 28.0)%, (110.4 +/- 33.3)% and (102.4 +/- 30.9)%, (102.1 +/- 24.1)%, respectively. The plasma level of FV: Ag was significantly different between VTE group and normal control. However, there was no difference in F V: C levels. Polymorphisms for the fore mentioned 5 primer pairs were not found in either patients or normal controls. Polymorphism of His1299Arg was identified in 5 patients with VTE and 3 normal controls. And these 5 cases also combined Met1736Val polymorphism, 3 of them combined another Asp2194Gly polymorphism.

CONCLUSIONThe higher plasma level of F V: Ag contribute to venous thromboembolism. There is no relationship between polymorphisms of Asp79His, Arg306The, Arg306Gly, Arg506Gln, Ile359The and venous thromboembolism in Chinese studied. Polymorphism His1299Arg is higher in VTE group than in normal control, but has no statistical difference. Polymorphisms of His1299Arg, Met1736Val and Asp2194Gly are linked disequilibrium in Chinese Han population.

Factor V ; genetics ; Female ; Gene Frequency ; Humans ; Linkage Disequilibrium ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Venous Thromboembolism ; genetics