Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.

Objective To investigate the distribution characteristics of interleukin-27 gene rs17855750 G/T,rs40837 A/G polymorphism in Zhuang populations of Guangxi,and to compare the distribution differences of genotype and allele frequencies of interleukin-27 gene rs17855750 G/T,rs40837 A/G polymorphisms among different races.Methods The interleukin-27 gene rs17855750 G/T,rs40837 A/G polymorphisms were detected by SNaPshot SNP genotyping technique on 168 persons in Zhuang populations of Guangxi,frequencies of genotype and allele of interleukin-27 gene rs17855750 G/T,rs40837 A/G polymorphisms were analyzed in Zhuang populations,and was compared with the other four populations (HapMap-HCB,HapMap-JPT,HapMap-YRI,HapMap-TSI) from HapMap database.Results The most common genotype and allele of interleukin-27 gene rs17855750 G/T polymorphysms were TT(70.2％) and G(50.3％) in Zhuang populations of Guangxi,and the most common genotype and allele of interleukin-27 gene rs40837 G/T polymorphysms were AC(35.7 ％) and C(52.1 ％).There were no significant differences in the genotype and allele frequencies of interleukin-27 gene rs17855750 G/T,rs40837 A/G polymorphysms between male and female gender in Zhuang populations of Guangxi(P＞0.05).The frequencies of allele and genotype distribution of IL-27 gene rs17855750 G/T polymorphisms were not significantly different when compared with HapMap-HCB(P＞0.05),but were significantly different when compared with HapMap-JPT,HapMap-TSI and HapMap-YRI(P＜0.01);The frequencies of allele and genotype distribution of intetleukin-27 gene rs40837 A/G polymorphisms were significantly different when compared with HapMap-HCB(P＜ 0.05),and were significantly different when compared with HapMap-JPT,HapMap-YRI and HapMap-TSI(P＜0.01).Conclusion There are significant differences in the frequencies of allele and genotype distribution of interleukin-27 gene rs17855750 G/T,rs40837 A/G between Zhuang populations and other ethnic populations,and this variation may lead to a variety of clinical manifestation and morbidity of some diseases.

Objective To explore the effect of reconstructing unilateral cleft lip by changing the arc-shaped incision, combined with the 3D reconstruction of upper lip muscles.Methods Twenty unilateral cleft lip patients were treated by using a new surgical operation, the 3D reconstruction of upper lip muscle, to restore normal anatomy and stress of the mucous membrane, muscle and skin.Operation scar was designed for straight line, located on the philtral ridges of the contour line;phitrum and philtral ridges were rebuilt, and postoperative scar reduced.Results A lot of 20 patients had no local infection, hemorrhage, complex crack, and were stage I incision healing.Followed up for 1-8 months postoperatively, the patient's lip bow line continuity was good, with symmetrical shape and good phitrum and philtral ridges;scar was hidden on the philtral ridges of the contour line, and no obvious upper lip scar contracture found through the follow-up period.Conclusions This improved method is simple in the incision design, and less scar hidden on the philtral ridges of the contour line after operation, which can maximize the recovery of the appearance of nose and upper lip with satisfactory effect.It is a feasible improvement method of repairing unilateral cleft lip.

Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.
Cloning, Molecular ; DNA, Complementary ; Datura ; enzymology ; genetics ; Escherichia coli ; Hyoscyamine ; chemistry ; Mixed Function Oxygenases ; genetics ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Recombinant Proteins ; genetics ; Scopolamine Hydrobromide ; chemistry

Referring to the rules for agricultural seed testing (GB /T 3543-1995) issued by China, the test of sampling, seed purity, weight per 1 000 seeds, seed moisture, seed viability and germination rate had been studied for screening seed quality test methods of Pesudostellaria heterophylla. The seed quality from different collection areas was measured. The results showed that at least 6.5 g seeds should be sampled and passed through 10-mesh sieve for purity analysis. The weight of 1 000 seeds was determined by using the 500-seed method. The phenotypic observation and size measurement were used for authenticity testing. The seed moisture was determined under the higher temperature (130 ± 2) degrees C for 5 hours. The seeds were dipped into 0.2% TTC sustaining 1 hour at 40 degrees C, then the viability could be determined. The break dormancy seeds were cultured on sand at 10 degrees C. K cluster analysis was applied for the data analysis, the seed quality from different collection areas grading of P. Heterophylla was described as three grades. The seed quality of each grade should reach following requirements: for first grade seeds, germination rate ≥ 86%, 1 000-grain weight ≥ 2.59 g, purity ≥ 87%, moisture ≤ 13.1%; for second grade seeds, germination rate ≥ 70%, 1 000-grain weight ≥ 2.40 g, purity ≥ 77%, moisture ≤ 14.3%; for third grade seeds, germination rate ≥ 41%, 1 000-grain weight ≥ 2.29 g, purity ≥ 76%, moisture ≤ 15.8%. The seed testing methods for quality items of P. heterophylla had been initially established, as well as the primary P. heterophylla seed quality classification standard.
Caryophyllaceae ; chemistry ; growth & development ; Germination ; Quality Control ; Seeds ; chemistry ; growth & development ; Water ; analysis

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

OBJECTIVETo explore the role of the amino acids between 551 and 565 in the cytoplasmic domain of glycoprotein (GP) I b alpha in the VWF binding to GP I b alpha.

METHODSThe VWF binding to GP I b alpha induced by ristocetin was analyzed by flow cytometry, in three GP I b-IX-expressing Chinese hamster ovary (CHO) cell lines 1b9, delta 565 and delta 551, adhesion of above cells on VWF by flow chamber analysis at shear rate of 200 s(-1). The spread of GP I b-IX-expressing cells were stimulated with botrocetin on VWF-coated coverslips by confocal microscope.

RESULTSThe VWF binding to GP I b alpha was higher in delta 565 cells stimulated by ristocetin than in delta 551 or 1b9 cells. The number of delta 565 cells adhered on the VWF-coated-chamber was more than that of controls at shear rate of 200 s(-1). Moreover, the surface spreading areas of delta 565 cells were greater than that of the controls on VWF-coated coverslips.

CONCLUSIONSThe amino acids between 551 and 565 in the cytoplasmic domain of GP I b alpha regulates the VWF binding to GP I b alpha.

Amino Acid Sequence ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Platelet Adhesiveness ; Platelet Glycoprotein GPIb-IX Complex ; genetics ; metabolism ; von Willebrand Factor ; metabolism

ObjectiveTo probe the indication,treatment algorithm,anesthesia method,safety and efficacy ofextracorporealshockwavelithotripsy(ESWL)incombinationwithendoscopicretrograde cholangiopancreatography (ERCP) for pancreatic duct stones.MethodsThe patients with chronic pancreatitis and large pancreatic duct stones ( ＞ 5 mm diameter) and receiving ESWL and ERCP between March and July 2011 in Changhai Hospital were prospective studied.The third generation of extracorporeal shockwave lithotripsy was applied in ESWL,and the localization of stone was determined by X-ray.No more than 5000 shocks were given per session,and ESWL was performed continuously till the calculi were fragmented,and then was cleared by ERCP.ResultsA total of 100 patients underwent ESWL during the 5 months,among whom 84 patients received ERCP treatment and 41 cases failed to deep cannulation (41/84,48.8％ ).Multiple stones were seen in 83 patients.Ninety five patients had radio-opaque stones,two patients had radiolucent calculi,while three patients had both radio-opaque and radiolucent stones.Seventy five percent,14％ and 11％ stones were located in pancreatic head,pancreatic head and body,pancreatic body and tail,respectively.A total of 175 ESWL procedures were performed,43 patients needed 2 or more sessions for successful fragmentation.Anesthesia method was mainly intravenous sedation,accounting for 96％ (168/175).ERCP was successful in 96 patients after ESWL,only 4 patients failed after ESWL. Forty one cases which failed ERCP procedures before ESWL underwent ERCP,and 37 patients (90.2％) achieved successful cannulation.Successful fragmentation ratewas 100％.Complete clearance was achieved in 78 patients,and complication rate of post-ERCP pancreatitis,fever was 1.71％ (n =3 ),0.57％ (n =1 ),and the overall complication rate was 2.28％.Conclusions ESWL is an effective,safe and necessary modality for fragmentation of large PD stones in the management of minimal invasive treatment of chronic pancreatitis.

Objective To investigate the effect of silment information regulator factor related enzymes 1 (SIRT1) on the apoptosis of retinal ganglion cells (RGCs) in rats with diabetic retinopathy and its downstream molecular mechanisms.Methods Together 60 healthy male Sprague-Dawley rats were collected and randomly divided into normal group,diabetic group,SIRTI activator-resveratrol treatment group (treatment group),and diabetic rat model was induced by intraperitoneal injection of streptozotocin at 60 mg · kg-1 in the latter two group rats,while the normal group was injected with sodium citrate buffer at 60 mg · kg-1.Then,after 72 h,rats with blood glucose ＞ 16.7 mmol · L-1 were designated as diabetic rats by blood glucose test.Then each rat in the treatment group was treated with SIRT1 activator-resveratrol at 20 g · kg-1 once a day at the 2nd day after the success of the model,and the normal group and diabetic group were given methylene chloride.Finally,after immunohistochemical staining for retina,TUNEL assay was used to evaluate the apoptosis of RGCs,while the expression of SIRTI,p38 MAPK and Caspase-3 protein was detected by Western blot.Results The apoptotic index of RGCs in the normal group,diabetic group and treatment group was (0.848+0.131)％,(19.038 + 1.327)％,(10.461 + 1.089)％ respectively at 8 weeks,and the difference among the three groups was statistically significant (F =670.497,P =0.000),while the differences between each two groups were also statistically significant (all P =0.000).Furthermore,when compared with the normal group (0.132 ± 0.043),the expression of SIRT1 protein in the diabetic group (0.060 ± 0.028) and the treatment group (0.073 ± 0.026) was significantly decreased,and the overall difference among the three groups was statistically significant (F =1 310.663,P =0.000),while the differences between each two groups were also statistically significant (all P =0.000).The expression levels of p38 MAPK and Caspase-3 were increased in diabetic group (1.121 ± 0.082,0.266 ± 0.005) and treatment group (0.574 ± 0.012,0.190 ±0.060) respectively,and the overall difference and pairwise comparison in the three groups approached statistically significance (all P =0.000,0.000).Conelusion Up-regulation of SIRT1,can inhibit the apoptosis of RGCs,and protect RGCs against apoptosis in rat model of diabetic retinopathy,which may be correlated with the downregulation of p38 MAPK signal pathway.

OBJECTIVETo provide new germplasm materials for breeding new varieties of Pseudostellaria heterophylla.

METHODThe method of single plant selection was adopted, with the comparative experiments being carried out under the same conditions in Shibing county. The 8 plants of Shibing SB-4 were compared respectively with factor analysis for 27 phenotypic traits and 8 yield traits, and single factor variance analysis for the contents of polysaccharides.

RESULTUsing factor analysis, 27 phenotypic traits were classified into 7 principal divisors and 8 yield traits were simplified into 3 principal divisors. The 4 strains of P. heterophylla, ZT-01, ZT-02, ZT-06 and ZT-07, performed better than others in the phenotypic traits, and ZT-01, ZT-02, ZT-03 and ZT-07 in the yield traits. The contents of polysaccharides of ZT-01, ZT-02, ZT-05 and ZT-08 showed significantly higher value.

CONCLUSIONThere is significant difference among the 8 strains of P. heterophylla in phenotypic traits, yield traits and quality traits, making it possible to select certain strains for different purposes. ZT-01 and ZT-02 can be breaded further. ZT-06 and ZT-07 were used as ornamental cultivars for its great phenotypic traits. ZT-03 with good resistance and high yield was taken as resistant variety, and ZT-05 would face next selection on the basis of its high content of polysaccharide.

Breeding ; Caryophyllaceae ; chemistry ; growth & development ; China ; Phenotype ; Polysaccharides ; analysis