OBJECTIVETo establish the hair roots culture system of Datura metel and study the hair roots growth and biosynthesis of scopolamine and hyoscyamine in hair roots culturing system.

METHODDirect degermed cotyledon of wild D. metel was infected by Agrobacterium tumefaciens strain C58C1 to obtain hair roots. Growth curves and scopolamine and hyoscyamine biosynthesis curves were determined. The scopolamine and hyoscyamine from different hair roots lines were examined by HPLC.

RESULTHair roots induction rate reached 70%. After 25 days cultured in 1/2 MS liquid nutrient medium, the hair roots weight, content of scopolamine and hyoscyamine reached maximum, tow high efficient accumulation hyoscyamine and scopolamine hair roots lines M1 and M2 were obtained. The medial accumulation coefficient of hyoscyamine and scopolamine were 2.53 times and 5.37 times compared with the leaves of wild D. metel respectively.

CONCLUSIONThe established hair roots induction and culture system of D. metel provided a foundation for further obtaining scopolamine and hyoscyamine.

Atropine ; analysis ; biosynthesis ; Datura metel ; chemistry ; growth & development ; metabolism ; Plant Roots ; chemistry ; growth & development ; metabolism ; Scopolamine Hydrobromide ; analysis ; metabolism

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.
Atropa belladonna ; genetics ; metabolism ; Atropine ; metabolism ; Gene Expression ; Mixed Function Oxygenases ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Solanaceae ; enzymology ; genetics

OBJECTIVETo observe the effect of sodium bicarbonate combined with ulinastatin on cholinesterase activity for patients with acute phoxim pesticide poisoning.

METHODSA total of 67 eligible patients with acute phoxim pesticide poisoning, Who were admitted to the emeryency department of hospital from March 2011 to February 2014, Acording to different treatments au patients were randomly divided into the conventional treatment group (n=34) and the sodium bicarbonate+ulinastatin group (n=35) . The conventional treatment group were given thorough gastric lavage with water, the sodium bicarbonate + ulinastatin group were given gastric lavage with 2% sodium bicarbonate solution. Both groups were given such treatments as catharsis, administration of oxygen, fluid infusion, diuresis, and antidotes such as atropine and pralidoxime methylchloride. On the basis of comprehensive treatment, people in the sodium bicarbonate+ulinastatin group were given 5% sodium bicarbonate injection and ulinastatin. The clinical effect of the two groups were compared.

RESULTSThe serum cholinesterase activity of the sodium bicarbonate+ulinastatin group was significantly higher than the conventional treatment group from the 5th day, and the difference was statistically significant (P<0.05) . The total atropine dosage, total pralidoxime methylchloride dosage and hospitalization days were better than the conventional treatment group, and the differences were statistically significant (P<0.05) . The difference in the time of atropinization between the two groups was not statistically significant (P>0.05) . The results of arterial blood pH, HCO3- of the sodium bicarbonate + ulinastatin group were higher than the conventional treatment group, and the difference of HCO3- at the 10th day was statistically significant (P<0.05) .

CONCLUSIONSSodium bicarbonate combined with ulinastatin can improve the therapeutic effect and reduce complications in the treatment of acute phoxim pesticide poisoning, and have beneficial effects on the recovery of cholinesterase activity.

Atropine ; therapeutic use ; Cholinesterases ; metabolism ; Glycoproteins ; therapeutic use ; Humans ; Organophosphate Poisoning ; drug therapy ; Organothiophosphorus Compounds ; poisoning ; Pesticides ; poisoning ; Pralidoxime Compounds ; therapeutic use ; Sodium Bicarbonate ; therapeutic use

OBJECTIVE: To investigate the regulation of atropine to the expression and secretion of TGF-beta2 in retinal pigment epithelium (RPE) cells by observing the changes of those under different treatments of atropine and carbachol. METHODS: D407 cells were cultured conventionally and divided into 4 groups as follows: (1) An experimental group (Group A), cells were pretreated with 10(-4)-10(-8) mol/L atropine for 30 min, and then treated with 10(-5) mol/L carbachol; (2) a negative control group (Group B), cells were treated with 10(-4)-10(-8) mol/L atropine; (3) a positive control group (Group C), cells were treated with 10(-5) mol/L carbachol; (4) a blank control group (Group D). The concentration of TGF-beta2 in the supernate, and the level of TGF-beta2 mRNA and protein were measured by ELISA, RT-PCR, and Western blot after the 24-hour treatment. The data were analyzed by analysis of variance. RESULTS: The levels of TGF-beta2 mRNA and protein in the cytoplasm and the concentration of TGF-beta2 in the supernate in the experimental groups were lower than those of the positive control group. Atropine at 10-4 mol/L could completely inhibit the effect of carbachol at 10-5 mol/L. The effect of atropine was concentration-dependent (F=1,056.897,1,320.170, and 475.657; P<0.001). There was no change of TGF-beta2 level in the cytoplasm and supernate with the treatment of atropine alone (P>0.05). CONCLUSION Carbachol can promote the expression and secretion of TGF-beta2 in human RPE cells and atropine could reverse it effectively, suggesting that M receptor may be involved.
Adult ; Atropine ; pharmacology ; Carbachol ; pharmacology ; Cell Line ; Female ; Humans ; Male ; Muscarinic Antagonists ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Retinal Pigment Epithelium ; cytology ; metabolism ; Transforming Growth Factor beta2 ; genetics ; metabolism ; Young Adult

This study was undertaken to investigate the effects of halothane anesthesia alone on blood glucose metabolism in ten surgical patients by determining plasma growth hormone ,(HGH), insulin and glucose, and to compare these with the effects of halothane anesthesia plus surgery. Each patient was premedicated with diazepam, 5 mg and atropine sulfate, 0. 5mg intramuscu Jarly one hour before the induction of halothane anesthesia. After endotracheal intubatien had been carried out following SCC 30-40mg, anesthesia was mairrtained with halothane and oxygen. The muscle relaxant (gallamine) in divided doses was injected during surgery when needed and ventilation was controlled or assisted intermittently throughout the procedure. A moderate depth of anesthesia was maintained according to clinical judgement, based on signs including blood pressure, pulse rate and somatie reflex responses to the surgical xtimulation. Eight blood samples were obtained from each patient:( I ) at 09.00 a.m. immediately before :induction of anesthesia (this sample served as acoatrol value) (2) the next three were after 15. 30 and 45 minutes of halothane anesthesia but before the start of the operation; (3) further three, 15,30 and 45 minutes, after the start of the operation;(4) in the recovery room after fully awaking. HGH RIAKIT was utilized as a kit for determination of human growth hormoae by radioimmunoassay and also lNSULIN-RIAKlT for insulin levels in the blood, For the determination of blood glucose level, OTB method was utilized. The ratio between male and female pgtients was 5:5, mean age 39. 2+/-12. 1 and average body weights 55. 5+/-8 .5 kg. The mean control plasma growth hormone level(HGH) in 10 surgical patients after premedication, and immediately before induction of anesthesia was 2. 03+/-0. 28ng/ml. The mean yre-surgical concentration of HGH in plasma after 15, 30 and 45 minutes of halothane anesthesia rose to 2.63+/-0.41 ng/ml, 2.76+/-0.35 ng/ml, and 2.95+/-0.42 ng/ml, respectively but these values were not significantly elevated above the control value. The plasma levols significantly increased to 3. 42+/-0. 5 mg/ml (P <0. 05), 6. 64+/-0. 6 mg/ml (p <0.001) and 7.83+/-0,75 ng/ml. (p<0.001) 15 minutes, 30 minutes and 45 minutes respectively after the start of the operation. It decreased to 3. 58+/-0. 68 ng/ml. (p<0. 05) in the recovery room when the patient awoke fully. The mean pre-anesthetic control insulin level in plasma. in 10 surgical patients was 12. 6+/-2. 0 ng/ml which was within the normal range. This level. did not vary appreciably during halothane anesthesia alone, during operation or in the post-operative period. The mean control pre-anesthetic blood glucose level was 78. .7+/-3.9 mg/100ml. It rose to 80. 6+/-2. 9 mg/100ml. 83. 2+/-3. 6mg/100 ml 30minutes, 45minutes and of halothane anesthesia alone and 15 minutes of the start of the operation but these changes were not significant. It increased significanty to 98. 2+/-4. 4 mg/100ml(p<0. 05) and 103. 9+/-4. 0 mg/100ml (p<0. 001) 30 minutes and 45 minutes respectively after the start of the surgery but it rose to 95.7+/-3.1mg/ 100ml (p <0. 01) in the post-operative period. Consequently the plasma HGH level during halothane anesthesia alone for 45 minutes was slightly, increased and rose significantly during operation and in the post-operative period. The peak level was achieved 45 minutes after the start of surgery. Plasma insulin levels did not change appreciably during anesthesia alone or during surgery. Blood glucose levels increased slightly during anesthesia alone but rose significantly during operation and in the post-operative period.
Anesthesia* ; Atropine ; Blood Glucose ; Blood Pressure ; Body Weight ; Diazepam ; Female ; Glucose* ; Growth Hormone ; Halothane* ; Heart Rate ; Human Growth Hormone* ; Humans* ; Insulin ; Male ; Metabolism ; Methods ; Oxygen ; Plasma* ; Premedication ; Radioimmunoassay ; Recovery Room ; Reference Values ; Reflex ; Ventilation

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.
Acetic Acids/administration & dosage ; Acetic Acids/metabolism ; Acetic Acids/pharmacology* ; Adrenergic Antagonists/metabolism ; Adrenergic alpha-Antagonists/metabolism ; Analgesics/administration & dosage ; Analgesics/metabolism ; Analgesics/pharmacology* ; Animals ; Atropine/metabolism ; Dihydroergocristine/metabolism ; Enzyme Inhibitors/metabolism ; Excitatory Amino Acid Agonists/metabolism ; GABA Antagonists/metabolism ; Injections, Spinal ; Leucine/metabolism ; Male ; Mecamylamine/metabolism ; Muscarinic Antagonists/metabolism ; N-Methylaspartate/metabolism ; Naloxone/metabolism ; Narcotic Antagonists/metabolism ; Nicotinic Antagonists/metabolism ; Pain Measurement ; Quinazolines/metabolism ; Rats ; Rats, Sprague-Dawley ; Serine/metabolism ; Spinal Cord/drug effects* ; Thapsigargin/metabolism ; Triazoles/metabolism ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/metabolism

AIMTo study the vasodilation effect of atropine and its mechanism.

METHODSIsometric tension was recorded in isolated rat super mesenteric arteries precontracted by noradrenaline (NE) to study the vasodilation effect of atropine, and to investigate the role of endothelial cell and vascular smooth muscle cell on vasodilation.

RESULTSAtropine was shown to significantly dilate the endothelium-intact and endothelium-denuded arteries precontracted by NE. Nomega-Nitro-L-arginine methyl ester (L-NAME, nitric oxide synthase inhabitor), indomethacin (cyclooxygenase inhibitor), propranolol (general beta adrenoceptor antagonist) and glibenclamide (ATP sensitive potassium channel inhibitor) showed no effect on vasodilation of atropine. Atropine did not affect the concentration-contraction curve of K+. However, atropine suppressed the contraction induced by NE and CaCl2, but not that by caffeine in the Ca+ -free Krebs solution.

CONCLUSIONAtropine showed significant vasodilation effect which may derive, in part, from endothelium. Besides, atropine could inhibit the receptor-mediated Ca2+ -influx and Ca2+ -release, which was inferred to the mechanism of atropine on vasodilation.

Animals ; Atropine ; pharmacology ; Calcium ; metabolism ; Calcium Chloride ; antagonists & inhibitors ; Cyclooxygenase Inhibitors ; pharmacology ; Endothelial Cells ; physiology ; Female ; In Vitro Techniques ; Indomethacin ; antagonists & inhibitors ; Male ; Mesenteric Artery, Superior ; drug effects ; NG-Nitroarginine Methyl Ester ; antagonists & inhibitors ; Nitric Oxide Synthase ; antagonists & inhibitors ; Norepinephrine ; antagonists & inhibitors ; Potassium Chloride ; antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Vasodilation ; drug effects ; Vasodilator Agents ; pharmacology