[Cloning and expression of the key enzyme hyoscyamine 6 beta-hydroxylase gene (DaH6H) in scopolamine biosynthesis of Datura arborea].

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.

[Serumimmunological study of moxibustion on helicobacter pylori gastritis in rats].

OBJECTIVE: To explore the immune mechanism of moxibustion on protecting gastric mucosa injury. METHODS: Forty healthy SD rats were randomly divided into four groups: a blank group, a model group, a moxibustion acupoint group and a moxibustion non-acupoint group, 10 rats in each one. Eight days before model establishment, moxibustion at "Zusanli" (ST 36), "Zhongwan" (CV 12), "Guanyuan" (CV 4), "Pishu" (BL 20) and "Weishu" (BL 21) was applied in the moxibustion acupoint group while these acupoints' controlled points were selected in the moxibustion non-acupoint group, and no treatment was given in the model group, once a day in three groups for continuous 16 days. The helicobacter pylori (Hp) model was established by intragastric administration of Hp. HE staining microscopic examination was used to observe inflammation severity in gastric mucosa, and enzyme-linked immunosorbent assay (ELISA) was adapted to measure content of heat shock protein (HSP) 72, TNF-alpha and IL-1beta, and real-time quantitative PCR was used to measure the expression of TLR2 mRNA, TLR4 mRNA, CD14 mRNA and MyD88 mRNA in peripheral blood mononuclear cells, and western blot method was used to measure content of NFkappaB and IkappaBalpha in peripheral blood mononuclear cells. RESULTS: Compared with the blank group, the expression of HP could be seen in the smear of gastric mucosa by Gram's staining in the model group; the inflammation severity score was obviously increased as well as content of serum HSP 72 and TNF-alpha and IL-1beta in gastric tissue; and expression of TLR2, 4 mRNA, CD14 mRNA, MyD88 mRNA, NFkappaB was increased (P < 0.01), but the expression of IkappaBalpha was reduced (P < 0.05). After the moxibustion, the inflammation severity score was reduced in the moxibustion acupoint group, and the content of serum HSP 72 was increased, and the expression of TNF-alpha and IL-1beta in gastric tissue and expression of TLR2 mRNA, TLR4 mRNA, CD14 mRNA, MyD88 mRNA and NFkappaB were reduced (P < 0.01), but the expression of IkappaBalpha was increased (P < 0.05). The differences between the moxibustion non-acupoint group and the model group were not significant (P > 0.05). CONCLUSION: The pretreatment of moxibustion at acupoints could induce the over expression of serum HSP 72. By combining TLR 2 and 4 receptors to trigger receptor signal transduction pathways, the releases of downstream signal substances are regulated; as a result, the releases of related immune substances are regulated to relieve the gastric mucosa injury of rats with HP gastritis.

Design, synthesis, and fungicidal evaluation of a series of novel 5-methyl-1H-1,2,3-trizole-4-carboxyl amide and ester analogues.

Succinate dehydrogenase inhibitors (SDHIs) are efficient fungicides that are widely used to control plant diseases caused by phytopathogenic fungi, although their effectiveness is undermined by the development of resistance across a range of different fungi. One of the most common structural features of SDHIs is their amide bond. The introduction of greater structural diversity to SDHIs is a promising strategy to delay the onset of resistance. A series of novel SDHIs containing a bioactive 1,2,3-triazole moiety have been designed and synthesized and their fungicidal and insecticidal activities evaluated. The results of these analyses show that most of the newly synthesized 1,2,3-trizole-4-carboxyl amide (ester) analogues exhibit good fungicidal activities, especially towards Sclerotinia sclerotiorum, and a structure-activity relationship study confirmed that the replacement of the amide group with an ester group had little effect on fungicidal activity, which could be provideous in terms of issues and metabolism. 1,6-Dimethyl phenyl was confirmed as the most efficient substituent of the current study when it was placed on both the amide and ester compounds. Interestingly, some of the newly synthesized compounds displayed good insecticidal activities against Culex pipiens pallens. The results of the current study show that these 1,2,3-triazole-4-carboxyl amide and ester analogues represent a new type of SDHI that could be used for the development of novel pesticides.

[Effect of manual acupuncture stimulation of "Zusanli" (ST 36) on gastric motility, and SP and motilin activities in gastric antrum and nucleus raphe magnus in gastric hyperactivity and hypoactivity rats].

OBJECTIVE: To observe the changes of gastric motility and levels of substance P (SP) and motilin (MTL) in the gastric antrum and Nucleus Raphe Magnus (NRM) after manual acupuncture stimulation of "Zusanli" (ST 36) in gastric hyperactivity and hypoactivity rats, so as to analyze the role of NRM in acupuncture mediated adjustment of gastric motility. METHODS: Fifty SD rats were randomly and equally divided into control, gastric hyperactivity (G-Hypera) model, gastric hypoactivity (G-Hypoa) model, acupuncture + G-Hypera and acupuncture + G-Hypoa groups (10 rats/group). G-Hypera model was established by intravenous (tail vein) injection of Maxolon (0.5 mL/200 g) and G-Hypoa model established by intravenous injection of Atropin (0.5 mL/200 g), respectively. After insertion of acupuncture needles into bilateral "Zusanli" (ST 36), the needles were repeatedly manipulated at a frequency of about 2 Hz for 5 min. The intragastric pressure was recorded and analyzed using a physiological signal analysis system. The SP and MTL contents of gastric antrum were measured by ELISA, and SP and MTL immunoactivity of NRM was determined by immunohistochemistry. RESULTS: In gastric hyperactivity rats, compared with the control group, the intragastric pressure (not systolic frequency), SP and MTL contents in the gastric antrum and MTL immunoactivity of NRM were significantly increased (P < 0.05, P < 0.01), while SP immunoactivity of NRM was apparently decreased in the G-Hypera model group (P < 0.01). After acupuncture stimulation, the intragastric pressure, gastric SP and MTL contents, and MTL immunoactivity of NRM were all significantly down-regulated (P < 0.05, P < 0.01), and SP immunoactivity of NRM was markedly up-regulated (P < 0.01) in comparison with the model group. In gastric hypoactivity rats, compared with the control group, the intragastric pressure and gastric systolic frequency, gastric SP and MTL contents, and MTL immunoactivity of NRM were significantly decreased (P < 0.05, PF < 0.01), while SP immunoactivity of NRM was remarkably up-regulated in the G-Hypoa model group (P < 0.01). After acupuncture stimulation, the above-mentioned 6 indexes were reversed significantly (P < 0.05, P < 0.01) in comparison with those of the model group. CONCLUSION: Acupuncture stimulation of "Zusanli" (ST 36) has an obvious bi-directional adjustment effect on gastric motility, which is closely associated with its functions in regulating gastric SP and MTL level and the expression of MTL and SP in the NRM of brainstem.