Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Bioreactors ; Catalysis ; Escherichia coli ; genetics ; Glucose ; Glucosyltransferases ; Industrial Microbiology ; Organisms, Genetically Modified ; Trehalose ; biosynthesis

Objective To explore the effect of fat on 1,2-dimethylhydrazine (DMH)-induced colon tumors.Methods A total of 50 7-week-old male Wistar rats were further divided into four groups:standard diet feed control group (n =10),standard diet feed plus DMH-induced tumor group (SDT,n =15),high-fat diet feed control group (n =10) and high-fat diet feed plus DMH-induced tumor group (HFDT,n =15).Rats were killed 18 weeks later,and enzyme-linked immunosorbent assay was used to detect serum triglyeeride,tumor necrosis factor (TNF-α),and colonic TNF-α,interleukin-6.After the intestinal tracts were removed,the location,amount,and size of the tumors were observed.The pathological changes of the tissue sections were observed,and the distributions of TNF-α and Ki-67 in the normal tissues and tumors were detected by immunohistochemistry.Results Upon the completion of the study,the mortality rate of rats was 20.00％ in the SDT group and 26.67％ in the HFDT group,the tumor formation rate was 75.00％ in the SDT group and 81.82％ in the HFDT group,and the tumor-bearing rate was 117％ in the SDT group and 191％ in the HFDT group.No statistical significance difference between the two groups in mortality rate,tumor formation rate (P =0.545) and tumor bearing rate (x2 =1.343,P =0.247).The average tumor volume was significantly different between the standard diet feed control group and high-fat diet feed control group (28.57％ vs 66.67％,P =0.030).Also,the serum triglyceride and TNF-α levels significantly differed between the SDT group and HFDT group [TG (1.39 ± 0.31) mmol/L and TNF-α (124.80 ± 21.69) ng/L in the HFDT group and TG (0.46 ±0.20) mmol/L and TNF-α (85.83 ± 17.45) ng/L in the SDT group] (P =0.000).The expressions of TNF-α,IL-6,and Ki-67 in colonic mucosa were significantly higher in the high-fat diet feed control group than in the standard diet feed control group [TNF-α:(6.22 ± 0.63) ng/g vs (2.33 ± 0.44) ng/g,P=0.020; IL-6:(13.50±0.67) ng/gvs (7.31 ±0.41) ng/g,P=0.000; and Ki-67:40％ vs 10％,P =0.028].The Ki-67 expression rate was 90.48％ in the HFDT group,compared to 50％ in the SDT group (P =0.015).Conclusions High-fat diet can increase the serum triglyceride and TNF-α levels in rats,upregulate the expressions of TNF-α,IL-6 and Ki-67,and thus promote inflammation and cell proliferation,and ultimately affect the tumor formation and development.However,the effect of fat on DMH-induced colon tumors warrants further studies.

Aim To determine the function of nuclear factor-κB ( NF-κB ) in immunological liver injury of rat model and its effect on CYP2E1 expression, content and metabolic activity. Methods The immunological liver injury rat model was prepared by injection of Ba-cillus Calmette Guérin ( BCG,125 mg · kg-1 ) for 14 days. The hepatic tissue injury was revealed by hema-toxylin and eosin ( HE ) method and serum concentra-tion of alanine aminotransferase( ALT) , aspartate ami-notransferase ( AST ) respectively. CYP450 total con-tent in hepatic homogenate was determined by spectro-photography. The expression of CYP2E1 protein was detected by Western blot analysis. The enzyme kinetics of CYP2 E1 probe drug chlorzoxazone was evaluated by high-performance liquid chromatography ( HPLC ) as-say. Results The results showed that BCG-pretreat-ment ( 125 mg · kg-1 ) significantly increased the weight of liver and spleen, serum levels of ALT and AST(P<0. 01) , and decreased CYP2E1 expression, content and metabolic activity ( P <0. 05 ) . Adminis-tration of ammonium pyrrolidine dithiocarbamate (PDTC) (50, 100, 200 mg·kg-1) reversed the a-bove hepatic injury stimulated by BCG in vivo. Moreo-ver, PDTC dose-dependently inhibited the down regu-lation of CYP2 E1 ( P<0. 05 ) . Conclusion Passiva-tion of NF-κB can inhibit the down regulation of CYP2 E1 in liver tissue of immunological liver injury rats;NF-κB may be involved in CYP2 E1 down-regula-tion.

This study was aimed to verify the effects of staged sequential therapy on expressions of matrix metalloproteinase-9 (MMP-9) and its inhibitor TIMP-1 within lung tissues in asthmatic rats with the airway remodeling, by applying a series of tests such as the immunohistochemistry, western blot and real-time fluorescent quantitative PCR. SD rats were randomly divided into 7 groups, which were the asthmatic group (Group X), the normal group (Group Z), the No. 1 sequential therapy group (Group A1), the No. 2 sequential therapy group (Group A2), the No. 3 sequential therapy group (Group A3), the montelukast group (Group M), and the budesonide group (Group B). The asthmatic model was established in each group except Group Z, by sensitization with both ovalbumin (OVA) and aluminium hydroxide via injection at the 1st, 8th and 15th day in a 22-day duration, followed by OVA aerosol inhalation every other day for 8 weeks for asthma activation. At the 8th day after the asthmatic model was established, Group A1 was orally given Ma-Xing Er-Chen Tang (MXECT) during acute phase while given normal saline (NS) during catabasis and stable phase; Group A2 was given MXECT during acute phase, and given modified Jin-Shui Liu-Jun Jian (JSLJJ) during catabasis as well as given NS during stable phase; Group A3 was given MXECT during acute phase, and given modified JSLJJ during catabasis as well as given Liu-Wei Di-Huang (LWDH) Powders during stable phase;Group M was given salbutamol via aerosol inhalation after stimulation, while orally given montelukast during catabasis and stable phase; Group B was given salbutamol via aerosol inhalation after stimulation, while given inhaled budesonide during catabasis and stable phase; Group X was given NS. After the 7-week intervention, the immunohistochemistry, western blot and real-time quantitative PCR were applied to analyze the location and quantitative expression of MMP-9 and TIMP-1, so as to find out the biological mechanism on expressions of MMP-9 and TIMP-1 in lung tissues of asthmatic rats from molecular levels to gene levels. The results of immunohistochemistry, western blot and real-time fluorescent quantitative PCR showed as follows. Compared with Group Z, the contents of MMP-9 and TIMP-1 increased significantly within lung tissues in Group X. Compared with Group X, the contents of MMP-9 and TIMP-1 decreased within lung tissues of asthmatic rats in each treatment group. It was concluded that the expression of MMP-9 and TIMP-1 elevated during asthma. TCM staged sequential therapy can regulate the ratio between MMP-9 and its inhibitor so as to block the airway remodeling, by decreasing the expression of MMP-9 and its inhibitor within lung tissues in asthmatic rats. This is one of the important action mechanisms.

Object To study the effects of active fraction L.F04 from the ground part of Lycopus lucidus Turcz. var. hirtus Reg. on platelet aggregation and thrombosis formation and investigate its mechanisms of promoting blood circulation and removing blood stasis. Methods The effects of L.F04 on platelet aggregation induced by ADP in vivo, thrombosis of artery vein side road and thrombus formed in rotary loop in vitro were examined, the rat model of blood stasis made by injecting high molecular weight dextran (HMWD) was used. Results L.F04 0.408 and 0.204 g/kg evidently inhibited the ADP induced increase of platelet maximum aggregation rate in vivo in HMWD model, with a concentration dependent manner. Compared with the control group, the thrombus weight in rat model of blood stasis was increased significantly and the length of thrombus was shown an increasing trendency. L.F04 0.408 and 0.204 g/kg both shown the anti thrombosis effect. L.F04 0.408 g/kg showed better effects of lessening the thrombus dry weight and wet weight. Both L.F04 0.408 and 0.204 g/kg could inhibit the thrombosis of artery vein side road, the inhibition rates are separately 27.41% and 27.14%. Conclusion L. lucidus var. hirtus F04 could significantly inhibit platelet aggregation and thrombosis formation.

With the rapid development of modern science and technology,the post-genomics period has come with the complement of the sequence of body genome including several tens of human genome,the emphasis of life science transfer from instruction genome to the post-genomics period,functional genome.Proteomics is the most important part of it.The technology of proteomics is advanced day by day.Except the classical two dimensional gel electrophoresis,the technology of multi-dimensional liquid chromatography and the technology of isotope coded-affinity tags have been successively developed,as well as protein chip and phage display have been applied extensively.This article simply summarizes the key technologies of proteomics.

Objectives To explore the expression of the receptor for advanced glycation end products (RAGE) in pancreas and the serum concentration of RAGE in rats with acute necrotizing pancreatitis (ANP).Methods Sixty four male Spraque-Dawley rats were randomly divided into control group,ANP 6,18,24,36,48,72,96 h group with 8 rats in each group.The rat model of ANP was established by injecting 20％ L-arginine intraperitoneally at the dose of 250mg/100g body weight for twice at the interval of 1 h.Rats in control group were injected intraperitoneally with the same amount of saline.The pancreas samples were histologically examined by light microscope and scored.The amount of ascites,levels of serum amylase and RAGE was determined; Real-time PCR was performed to detect the expression of RAGE mRNA in pancreatic tissue.Results Pancreatic injuries aggravated with time.The amount of ascites increased from ( 1.98 ± 0.64) ml at 6h to (8.69 ±0.62)ml at 96 h.Serum amylase level began to increase at 6h after L-arginine intraperitoneal injection and reached the peak value at 18 h[(5069.88 ± 603.25 ) U/L],and returned to normal at 36 h.The serum concentration of RAGE and RAGE mRNA expression in pancreatic tissue were ( 18.33 ± 2.99) ng/ml and 0.41 ±0.13 in the control group.The corresponding values increased at 6 h in ANP group,which were (30.31 ±5.03) ng/ml and 1.57 ±0.19,and they were significantly higher than those in the control group (P ＜0.05) ; they reached the peak value at 24 h[( 105.41 ±21.31 ) ng/ml and 4.23 ±0.73],which were significantly higher than those in other ANP groups ( P ＜ 0.05 ) ; at 96 h they decreased to the lowest point [( 33.54 ± 6.96) ng/ml and 1.19 ± 0.19],which were still significantly higher than those in the control group (P ＜ 0.05 ).Conclusions The expression levels of RAGE in pancreatic tissues and serum level of RAGE increase within 36 h of ANP onset,then decrease gradually,but they are always higher than normal values.

Objective To investigate the effects of acute reserpine-induced pain and depression co-morbidity model on behavior and related inflammatory cytokines in rats. Methods Twelve male 8-week-old SD rats were randomly divided into control group and model group,6 in each group. Rats in the model group were intraperitoneally injected with reserpine(1 mg/kg/d)for 3 days to establish the model. Rats in the con-trol group were injected with the same amount of distilled water. The pain threshold of rats was measured by Von Frey Hairs and Hot Plate Analgesia Test. The depression behavior of rats was evaluated by open-field test,Forced Swimming Test and Sucrose Consumption Test. Serum IL-1β, IL-6, IL-18 and TNF-α content were detected by ELISA. Results Compared with the control group(1d (15. 00±0. 00) g;3d ( 13. 20±4. 03)g),the PWTs of the model group (1d (6. 20±0. 45) g;3d (4. 20±1. 64) g) decreased significantly(t=44. 00,4. 63,both P<0. 05). Compared with the control group (( 8. 82 ± 1. 08) s),the PWTL (( 3. 16 ± 0. 24)s) was significantly reduced on the first day in model group,and the difference was statistically signifi-cant (t=11. 48,P<0. 05). Compared with the control group (( 1 815. 18± 541. 40) cm,(98. 20± 26. 25) s, (87. 78±9. 38)g),the total distance of the open-field test ((948. 91±494. 35)cm)significantly shortened (t=2. 64,P<0. 05),swimming time ((143. 60±21. 45)s) was significantly prolonged (t=-2. 65,P<0. 05), and the sucrose consumption (( 22. 23 ± 6. 97) g) significantly decreased (t=12. 55,P<0. 05) in model group. Compared with the control group (( 285. 80 ± 11. 93) ng/ml, ( 233. 07 ± 8. 47) ng/ml,( 280. 41 ± 14. 31) ng/ml,( 213. 10 ± 33. 87) ng/ml), serum levels of IL-1β, IL-6, IL-18, TNF-α in model group ((471. 23±24. 15) ng/ml,(364. 82±17. 16) ng/ml,(471. 81± 28. 98) ng/ml,(821. 19±93. 16) ng/ml) increased significantly(F=-15. 39,-15. 39,-13. 24,-13. 72,all P<0. 05). Conclusion A large number of intraperitoneal injections of acute reserpine can cause hyperalgesia,depressive behavior and serum inflamma-tory factors in rats,effectively simulating the symptoms of pain and depression,and can be used as a model of pain and depression comorbidity for biological mechanisms and treatment research.

Objective To investigate the antidepressant effects of Chaihu Shugan Powder(CSP) on depressive rats induced by reserpine and its influences on the kynurenine (KYN),indoleamine 2,3-dioxyge-nase(IDO),interleukin-6(IL-6) and tumor necrosis factor-α( TNF-α). Methods Forty rats with similar behavior results were divide into 4 groups randomly,including Control group(Con),Model group(Res),Flu- oxetine group(Res+Flu) and Chaihu Shugan Powder group(CSP). The depressive rat model was established by intraperitoneal injection reserpine. The rats in Res+Flu group were administered with fluoxetine by intrap-eritoneal and rats in Res+CSP group were administered with CSP by intraperitoneal. After 14 days,the be-havior of rats was measured and then the rats were executed and sampled. The content of tryptophan and kynurenine in raphe nuclei tissue were detected. The mRNA expression level of IDO,IL-6,TNF-α in raphe nuclei tissue were detected. Results ( 1) Compared with Con group (( 81. 81 ± 36. 13) s, ( 83. 51 ± 5. 34)%), the swimming immobility time((150. 50±31. 45)s) in Res group increased(t=68. 7, P<0. 05) and the sucrose perference (59. 73±11. 30)%) in Res group decreased(t=23. 8,P<0. 05). Compared with Res group, the swimming immobility time in Res+Flu group((114. 90± 14. 29) s) and Res+CSP group ((111. 7±11. 34)s) decreased(t=35. 6,35. 8,both P<0. 05). Compared with Res group, the sucrose pref-erence in Res+Flu group((78. 21±10. 07)%) increased(t=18. 3, P<0. 05). (2)Compared with Con group (KYN/TRP:(0. 023±0. 016),IDO mRNA:(1. 00±0. 05),IL-6 mRNA:(1. 00±0. 58),TNF-α mRNA:(1. 00±0. 32)), the activity of IDO(KYN/TRP(0. 039±0. 003)) and the mRNA levels of IDO mRNA(3. 63± 0. 31),IL-6 mRNA(2. 36±0. 23),TNF-α mRNA( 3. 56± 0. 14) of Raphe Nuclei tissue in Res group were significantly increased (t=21. 2,12. 9,38. 3,19. 7,all P<0. 05). Compared with Res group, the activity of IDO(KYN/TRP(0. 030±0. 013)),the mRNA expression levels of IDO mRNA( 1. 56±0. 36),IL-6 mRNA (1. 62±0. 16),TNF-α mRNA(2. 64±0. 20)of Raphe Nuclei tissue in Res+Flu group were significantly de-creased(t=38. 8,15. 8,12. 8,26. 4,all P<0. 05). And compared with Res group,the activity of IDO( KYN/TRP(0. 028±0. 021)) ,the mRNA expression level of IDO mRNA( 1. 33± 0. 29),IL-6 mRNA(1. 36± 0. 34),TNF-α mRNA(1. 93±0. 21)of raphe nuclei tissue in Res+CSP group were also significantly decreased (t=23. 21,17. 3,19. 8,29. 8,all P<0. 05). Compared with Res+Flu group,the level of IDO mRNA and in-flammatory factors' mRNA in Res+CSP group were significantly decreased(t=18. 3,20. 8,31. 5,all P<0. 05). Conclusion Chaihu Shugan Powder has antidepressant effect,and the mechanism is related with de-creasing the inflammatory factors,inhibiting IDO activation and decreasing the IDO mRNA.

Objective:To investigate the clinical effect of minimally invasive plate osteosynthesis (MIPO) through anterolateral approach in treatment of middle-distal humeral shaft fracture.Methods:A retrospective case series study was conducted to analyze the clinical data of 21 patients with middle-distal humeral shaft fracture admitted to 7th Medical Center of Chinese PLA General Hospital from August 2015 to March 2018, including 12 males and 9 females, aged 18-68 years [(31.3±3.6)years]. All were closed fracture. According to AO classification, the fracture were classified as type A in 6 patients, type B in 10 and type C in 5. All patients were treated with anterolateral minimally invasive plate fixation. The operation time, intraoperative blood loss and hospital stay were recorded. The fracture healing was observed after operation. The visual analogue scale (VAS), University of California at Los Angeles (UCLA) shoulder rating scale, and Mayo elbow performance score (MEPS) were used to evaluate the effectiveness before operation and at 2 weeks, 3 months and 12 months after operation.Results:All patients were followed up for 12-26 months [(18.2±2.4)months]. The operation time was 50-82 minutes [(68.2±19.4)minutes], with intraoperative blood loss of 40-95 ml [(60.2±21.3)ml]. The hospital stay was 6-16 days [(6.8±1.2)days]. There was no iatrogenic vascular or nerve injury during operation. The patients with radial nerve injury before operation were all adventitia contusion. The nerve function returned to normal within 3 months after operation. All fractures were healed within 5-10 months [(5.3±1.2)months]. At 2 weeks, 3 months and 12 months after operation, the VAS [(3.6±0.8)points, (2.1±0.4)points, (1.8±0.3)points] was lower than that before operation [(8.3±1.6)points] ( P<0.05); UCLA shoulder rating scale [(31.2±1.5)points, (33.6±0.8)points, (34.6±0.5)points] was higher than that before operation [(28.4±2.3)points] ( P<0.05); and MEPS [(80.2±3.4)points, (93.4±2.2)points, (96.4±3.5)points] was higher than that before operation [(60.5±4.5)points] ( P<0.05). At the last follow-up, the UCLA shoulder rating scale and MEPS showed excellent results. Conclusion:For middle and lower humeral shaft fracture especially for the fracture line relatively distal to the shaft, MIPO technique through anterolateral approach can attain satisfactory results in terms of pain, range of motion of shoulder and elbow joint, and joint function.