Effects of needling acupoints at different nerve segments on oxytocin neurons in rat's hypothalamic paraventricular nucleus and intragastric pressure / 针灸推拿医学（英文版）

Objective:To compare and explore the effects of needling acupoints at different nerve segmentson the oxytocin (OT) neurons in the paraventricular nucleus of hypothalamus (PVN) and the intragastric pressure, and discuss the possible mechanisms. Methods: Thirty-two healthy adult Sprague-Dawley (SD) rats were numbered and divided into 4 groups according to the random number table, a Zusanli (ST 36) group, a Neiguan (PC 6) group, a Weishu (BL 21) group and a control group, with 8 rats in each group. Except the control group, rats in the other three groups received acupuncture at the corresponding acupoints. To observe the differences in double-labeled OT neurons and c-fos neurons of the hypothalamic PVN and the intragastric pressure after acupuncture among the three groups of needling acupoints at different nerve segments. Results:Compared with the control group, the numbers of double-labeled cells in the PVN of the Zusanli (ST 36) group and the Neiguan (PC 6) group decreased significantly, while the intragastric pressure increased significantly (allP<0.05), and the inter-group differences were statistically significant (P<0.05). The intragastric pressure in the Weishu (BL 21) group decreased significantly, and the inter-group difference was statistically significant (P<0.05). Compared with the Weishu (BL 21) group, the numbers of OT/c-fos double-labeled cells in PVN of the Zusanli (ST 36) group and the Neiguan (PC 6) group decreased significantly, and the intragastric pressure increased significantly, the inter-group differences were statistically significant (allP<0.01). Conclusion:Acupoints at different nerve segments have different regulation effects on intragastric pressure. The difference may be related to the different nerve conduction pathways by acupoints at different nerve segments in regulating the intragastric pressure. The PVN may be one common integration center for the regulation of gastric function in the three acupoints [Zusanli (ST 36), Neiguan (PC 6) and Weishu (BL 21)] at different nerve segments.

Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture

Aims@#Hyaluronic acid (HA) is a high molecular weight polymer and a major component of mucoid capsule in bacteria and extracellular matrix (ECM) of vertebrate tissue. Due to its unique characteristics, HA is used extensively in medical and cosmetic field. However, because of the exotoxins production from animal tissues extraction and Streptococcus zooepidemicus, HA production by recombinant microorganisms has gained interest. The present study was aimed at cloning of hasA gene in Escherichia coli and optimization of the medium components for HA production. @*Methodology and results@#A fragment of an approximate size of 1.5 kb that encodes the hyaluronan synthase (hasA) gene from S. zooepidemicus ATCC 39920 was amplified by PCR using hasA-specific primers. The hasA gene was ligated into the bacterial expression vector pLbADH and transformed into the expression host, Escherichia coli BL21 strain. Then, genetically engineered E. coli strain BL21 was used for the production of HA by fermentation using different glucose concentration (10-50 g/L) and different IPTG concentration (0.1, 0.5 and 1.0 mM) in shake flask culture. Amongst varying glucose concentrations, results showed that 50 g/L glucose with nutrient rich media containing nitrogen source was able to produce the highest HA concentration (0.115 ± 0.002 g/L). With addition of 1.0 mM IPTG, HA production reached a peak 0.532 ± 0.026 g/L which is around fivefold higher compared to without IPTG. @*Conclusion, significance and impact of study@#The hasA gene was cloned from S. zooepidemicus and successfully expressed in recombinant E. coli BL21 cells. This low molecular weight HA is gaining more importance in medical and cosmetic application due to possess pronounced free radical scavenging and antioxidant activities.

Role of hippocampus NMDAR subunits in electroacupuncture at "Zhongwan" (CV 12) and "Weishu" (BL 21) for gastric motility / 中国针灸

OBJECTIVE@#To observe the effects of electroacupuncture (EA) on gastric motility, protooncogene c-fos and hippocampus N-methyl-D-aspartate (NMDA) receptor subunits in rats with functional dyspepsia (FD), and to discuss the molecular mechanism of hippocampal in EA at "Zhongwan" (CV 12) and "Weishu" (BL 21) for gastric motility.@*METHODS@#Eighty-four Sprague Dawley (SD) rats were randomly divided into a normal group, a model group, a Zhongwan group, a Weishu group, an acupoint combination group and a non-acupoint group, 14 rats in each one. Except for the normal group, FD model were established by moderate tail-clipping infuriation method and irregular feeding. The rats in the Zhongwan group, Weishu group, acupoint combination group and non-acupoint group were treated with EA at corresponding acupoints, 20 min per treatment, once a day for 7 days. The rats in the normal group and the model group received no treatment; grabbing and fixation were applied in the model group. The stress transducer was used to record gastric motion waveforms; immunohistochemistry method was used to detect the expression of c-fos in hippocampus; Western blot method was used to detect the expression of NMDA receptor subunits NR1, NR2A and NR2B in hippocampus.@*RESULTS@#Compared with the normal group, the gastric motility range was decreased (0.05). Compared with the model group, the gastric motility range was increased, the expression of hippocampus c-fos and expression of hippocampus NR2A was increased but expressions of NR1 and NR2B were reduced in the Weishu group, Zhongwan group and acupoint combination group (0.05). Compared with the Zhongwan group and the Weishu group, the gastric motility range was increased, the expression of hippocampus c-fos and NR2A was increased but the expression of NR1 and NR2B was reducedin the acupoint combination group (0.05).@*CONCLUSION@#EA at "Zhongwan" (CV 12) and "Weishu" (BL 21) could increase gastric motility of FD rats, which is likely to be related with activating hippocampal neurons, upregulating the level of NR2A and downregulating NR1 and NR2B.

The effects of electroacupuncture at and points of stomach on gastric motility, the NMDA of vagus nerve dorsal nucleus and serum NO expression in functional dyspepsia rats / 中国针灸

<p><b>OBJECTIVE</b>To research the central molecular mechanism of gastric motility in functional dyspepsia (FD) rats treated with electroacupuncture (EA) at and points of stomach.</p><p><b>METHODS</b>A total of 30 SD rats were randomized into a blank group, a model group, a Zhongwan+Weishu group, a Weishu group and a Zhongwan group, 6 rats in each group. FD rats were established by moderate clipping tail infuriation and irregular feeding except in the blank group. EA was used at "Zhongwan"(CV 12),"Weishu"(BL 21), and"Zhongwan"(CV 12) +"Weishu"(BL 21) in the corresponding groups for 7 days, once a day, and 20 min a time. No intervention was used in the blank and model groups. Grabbing and fixation were applied in the model group. Gastric antrum motion range and frequency were recorded by gastrointestinal pressure transducer. The expression of subunit NR1 of N-methyl-D-aspartate recepter (NMDAR) in dorsal motor nucleus of the vagus (DMV) was determined by Western blotting. The content of serum nitric oxide (NO) was measured by ELISA.</p><p><b>RESULTS</b>Compared with the blank group, the gastric antrum motion range and NR1 in the DMV decreased and the serum NO content increased in the model group (all <0.05). Compared with the model group, the gastric antrum motion range and NR1 in the DMV increased and the serum NO content decreased in the three EA groups (all <0.05). Compared with the Zhongwan and Weishu groups, the gastric antrum motion range and NR1 in the DMV increased in the Zhongwan + Weishu group (all <0.05). Compared with Zhongwan + Weishu and Zhongwan groups, the expression of NO in the Weishu group decreased (both <0.05). The gastric antrum motion frequency among the 5 groups had no statistical significance (all >0.05).</p><p><b>CONCLUSION</b>EA at the and points can regulate the gastric motility in FD rats which may be by modulating the activity of NMDAR in the central DMV region, thus regulating the serum NO content.</p>

Effects on the regional homogeneity of resting-state brain function in the healthy subjects of gastric distention treated with acupuncture at the front- and back- points of the stomach, Weishu (BL 21) and Zhongwan (CV 12) / 中国针灸

<p><b>OBJECTIVE</b>To observe the regional homogeneity (ReHo) of resting-state brain function in the healthy subjects of gastric distention treated with acupuncture at the back- and front- points of the stomach, Weishu (BL 21) and Zhongwan (CV 12) and the correlation with gastric motility so as to explore the mechanism on the central integration of the front- and back- points of the stomach.</p><p><b>METHODS</b>The crossover test design was adopted. Twenty-four healthy subjects were assigned to a Weishu group, a Zhongwan group and a combined-point group separately, 8 cases in each one in each of the three times. Totally, 24 subjects were included in each group. Under the water load condition, the subjects received acupuncture at Weishu (BL 21), Zhongwan (CV 12) and the combined Weishu (BL 21) and Zhongwan (CV 12). Before and after each acupuncture, the resting-state brain functional magnetic resonance imaging (fMRI) scan and electrogastrogram (EGG) test were applied. The ReHo value was calculated in the collected fMRI imaging data. The changes in ReHo values were analyzed and compared before and after acupuncture in each group, as well as among the groups. The gastric motility was analyzed before and after acupuncture. Additionally, the correlative analysis was conducted between the gastric motility and ReHo changes before and after acupuncture.</p><p><b>RESULTS</b>(1) After acupuncture, EGG amplitudes in the subjects of each group were lower remarkably as compared with those before acupuncture (all <0.01). The EGG frequencies were not different significantly as compared with those before acupuncture (all >0.05). The EGG amplitudes in the Weishu group and the Zhongwan group were higher than those in the combined-point group (both <0.05). (2) As compared with the conditions before acupuncture, acupuncture at the combined front- and the back- points as well as Weishu (BL 21) and Zhongwan (CV 12) separately all induced the changes in the brain ReHo. Acupuncture at the combined front- and the back- points significantly increased Reho values in the right inferior temporal gyrus, the left thalamus, the precuneus and the posterior cingulate gyrus (all <0.05) and remarkably reduced the ReHo values in the the middle temporal gyrus of the right temporal pole, sulcus calcarinus and precuneus (all <0.05). Compared with the single point groups, acupuncture at the combined front- and the back- points induced the increase of ReHo value in the posterior cingulate gyrus and the decrease of ReHo in the temporal pole (all <0.05). (3) The correlative analysis showed that the changes in the ReHo values in the posterior cingulate gyrus, the thalamus and the precuneus were positively correlated to the changes of the gastric motility amplitudes. The changes in the ReHo values in the temporal pole was negatively correlated to the changes of the gastric motility amplitudes.</p><p><b>CONCLUSION</b>Acupuncture at the combined back- and front- points of the stomach, as well as acupuncture at single Weishu (BL 21) and Zhongwan (CV 12) induce the ReHo changes in the different brain regions. Acupuncture at the combined back- and front- points of the stomach may induce the ReHo changes in some new brain regions as compared with the acupuncture at the single point. The thalamus, the posterior cingulate gyrus and the precuneus may be the the important integrated brain regions for acupuncture at the back- and the front- points in regulating the gastric motility. The effects of acupuncture at the back- and the front- points for the regulation of the gastric motility are closely related to the thalamus, the limbic system and the default network of the brain regions.</p>

Expresión y purificación de las proteínas estructurales del rotavirus VP5* y VP8* en bacterias E. coli BL21(DE3) / Expression and purification of rotavirus structural proteins VP5* and VP8* in bacteria E. coli BL21(DE3)

La caracterización de las proteínas estructurales del rotavirus y de las proteínas de la superficie de la célula hospedera implicadas en la unión y penetración del virion requiere de la disponibilidad de cantidades suficientes y de alto grado de pureza de estas proteínas. Por lo tanto, el objetivo de este trabajo fue expresar y purificar las proteínas estructurales del rotavirus de la cepa RRV, VP5* y VP8*, y producir anticuerpos policlonales dirigidos contra ellas. Se expresaron las proteínas recombinantes VP5* (rVP5*) y VP8* (rVP8*) en bacterias E. coli BL21(DE3) transfectadas con el plásmido pGEX-4T que contenía sus secuencias codificantes. Se consideraron como variables el medio de crecimiento, número de bacterias antes de inducir la expresión, concentración del inductor y tiempo de inducción. La mayor proporción de rVP8* se obtuvo cuando las bacterias transformadas se cultivaron en medio LB y la inducción se llevó a cabo con 1 mM de IPTG cuando el cultivo alcanzó una OD 600 nm de 0.5 y la inducción se mantuvo durante 6 h. rVP5* alcanzó la mayor proporción cuando células a una OD 600 nm de 0.2 fueron inducidas con 0.5 mM de IPTG durante 4 h en medio 2XYT en presencia de glucosa al 2 %. Las proteínas recombinantes obtenidas, acumuladas en la fracción insoluble, fueron solubilizadas con detergentes iónicos y no iónicos, seguido de purificación mediante cromatografía de afinidad antes de ser empleadas como antígenos para la producción de anticuerpos policlonales en conejos. Estos anticuerpos se caracterizaron mediante su capacidad de reconocimiento de los antígenos correspondientes en ELISA, "Western blotting", y en ensayos de inmunocitoquímica en células infectadas con rotavirus RRV. La cantidad y el grado de pureza de las proteínas recombinantes obtenidas, y los anticuerpos dirigidos contra ellas, anticipan su utilidad como herramientas en la caracterización de la interacción virus-célula.

The characterization of rotavirus structural proteins and the cell surface proteins involved in virion binding and penetration depends on the availability of substantial amounts of highly purified proteins. The aim of the present work was to express and purify the rotavirus structural proteins VP5* and VP8* in order to produce polyclonal antibodies against them. Recombinant proteins VP5* (rVP5*) and VP8* (rVP8*) were expressed in E. coli cells transfected with pGEX-4T or pET 28a containing their corresponding encoding sequences. Culture medium, bacterial concentration before induction, inductor concentration and induction time were used as variables. The greater proportion of rVP8* was obtained when transfected bacteria were grown in medium LB and induction was started by adding 1 mM IPTG to cells at OD 600 nm 0.5 followed by 6 h-induction. The highest proportion of rVP5* was reached when cells at OD 600 nm 0.2 were induced with 0.5 mM IPTG for 4 h in medium 2XYT containing 2% glucose. The recombinant proteins accumulated in the insoluble fraction were solubilized with ionic and non-ionic detergents, and purified by affinity chromatography before being used as antigens for production of rabbit polyclonal antibodies. The antibodies produced were characterized through their ability to recognize the corresponding antigens in ELISA, Western blotting, and immunochemistry assays in rotavirus infected cells. The amount and purity of recombinant proteins obtained in this work, and the antibodies against them, are expected to be useful for the characterization of the virus-cell interaction.

Cloning,expression and purification of fragile X mental retardation protein / 基础医学与临床

Objective Explore the conditions of the cloning,expression and purification of FMRP.Methods The plasmid pET22b(+)-FMR1,constructed by molecular cloning,was transformed into E.coli BL21(DE3) competent cells and induced to express FMRP by IPTG.Recombinant FMRP was purified by affinity chromatography,verified by Western-blot,and tested for its RNA binding ability.Results FMR1 cDNA was successfully cloned into pET22b(+) vector and expressed in E.coli BL21(DE3).A protein with Mr 79 000 was purified and confirmed to be FMRP.This protein retained the RNA binding ability of FMRP.Conclusion We successfully expressed recombinant hFMRP with high purity and activity in E.coli,which provided a reliable material to study the function of FMRP.

Application of Element and Metabolism Balancing in the Production Phase of Human-like Collagen / 中国生物工程杂志

On the base of element and metabolism balancing,the mathematic model of the human-like collagen expression phase with recombinant Escherichia coli BL21 was developed and the unknown parameters in the model were estimated with the method of nonlinear optimization.The model was in agreement with the growth kinetics and the metabolic kinetics,and the key calculated parameters of ?h,?p and mx were 1.173 mol?C-mol-1,293.814 mol?C-mol-1 and 17.878 mol?C-mol-1?h-1 respectively.This model could preferably predict the macroscopic reaction rates,and in the synthesis phase of human-like collagen,the specific growth rate should be controlled at 0.04 h-1 with controlling glucose feeding rate to gain the highest specific production rate of human-like collagen.

Mechanism of acupuncture-induced gastric motility / 全日本鍼灸学会雑誌

Effects of acupuncture on gastric motility in anesthetized rats<BR>Acupuncture has been used to treat gastrointestinal symptoms in. China for more than 3, 000 years. However, the mechanism of the beneficial effects of acupuncture remains mysterious. Strain gage transducers were implanted on the serosal surface of the stomach to record circular muscle contractions in thiobutabarbialanesthetized rats. Acupuncture on the right lower abdomen caused a transient relaxation of the stomach. Acupuncture-induced gastric relaxations were abolished by guanethidine, propranolol, splanchnic ganglionectomy, spinal cord transection, and spino-medullary transection. In contrast, NG-nitro-L-arginine, phentolamine, truncal vagotomy and ponto-medullary transection had no effect. Acupuncture increased the number of c-Fos immunopositive cells at the rostral ventrolateral medulla (RVLM). It is concluded that acupuncture-induced gastric relaxations are mediated via somato-sympathetic reflex. Its afferent limb is composed of abdominal cutaneous and muscle afferent nerves. Its efferent limb is the gastric sympathetic nerve and the reflex center is within the medulla. RVLM neurons may play an important role in mediating this reflex.<BR>Effects of acupuncture on gastric motility in conscious rats<BR>Of 35 rats studied, 60% showed no cyclic groupings of strong contractions (type A) and 10 rats (40%) showed the cyclic phase III-like contractions (type B) in conscious rats. In 73 % of type A rats, acupuncture on the hind limb (ST36), but not on the back, produced strong cyclic contractions lasting for more than 3 hrs. Acupuncture failed to produce phase III-like contractions in type A rats treated with atropine, hexamethonium and vagotomy. Pretreatment with naloxone significantly shortened the duration of enhanced phase III-like contractions. In 60 % of type B rats, acupuncture suppressed phase III-like contractions. These results suggest that the acupuncture on the ST36 induces dual effects, either stimulatory or inhibitory effects, on gastric motility. The stimulatory effects of acupuncture on the hind limb are mediated via wagal efferent pathway and opioid pathway.<BR>Effects of acupuncture on vasopressin-induced emesis in conscious dogs<BR>Arginine vasopressin (AVP) is a posterior pituitary hormone and recent evidence implies an involvement of AVP in vomiting and nausea of humans and animals. Vasopressin was intravenously infused at a dose of 0.1U/kg/min. Number of retching and vomiting were visually counted during the experiment. Intravenous administration of vasopressin induced retching and vomiting in all dogs tested. Retrograde peristaltic contractions (RPCs) occurred before the retching and vomiting.<BR>Electro-acupuncture (EA) of 10 HZ at PC6 decreased the number of retching and vomiting induced by vasopressin. EA also suppressed the RPCs. EA at the PC6 significantly reduced the number of episodes of retching and vomiting to 20-30 % compared to that without EA, while EA at BL21 had no significant effects on the number of episodes of retching and vomiting induced by vasopressin.<BR>To investigate whether opioid pathway is involved in EA-induced anti-nociceptive effects, naloxone was administered before EA and vasopressin infusion. EA at PC6 had no more inhibitory effects on vasopressininduced nausea and vomiting when naloxone was given. This suggests that anti-emetic effects of acupuncture is mediated via (central) opioid pathway.<BR>Effects of acupuncture on EGG in healthy volunteers<BR>The point of PC6 and ST36 are the common points for the treatment of gastric symptoms. We compared the effect of EA between PC6 and ST36 on gastric myoelectrical activity using surface electrogastrography (EGG). Eight healthy volunteers were fasted for 12 hours. On the separate days, four sessions of EA (1 Hz, 30 min) were randomly performed to each subject. The percentage of normal slow waves and tachygastria, peak dominant frequency (PDF) and peak dominant power