ObjectiveTo investigate the effects of different doses of obestatin on amylase secretion of pancreatic acinar and lobules of rats in vitro.MethodsPancreatic acinar cells of rats were separated in vitro and incubated with different doses of obestatin (0,0.1,1,10,30 nmol/L) for 1h,another group of pancreatic acinar ceils was incubated with obestatin for 30 min,then was incubated with 100 pmol/L CCK-8 for another 30 min.Pancreatic lobules,which containing intrapancreatic nerve terminals and islets,were prepared and were incubated with different concentrations of obestatin for 30 min at 37℃ with or without 75 mmol/L KCl.Amylase levels in the supernatants,acinar cells and pancreatic lobules were calculated as a percentage of total amylase content.ResultsObestatin (0,0.1,1,10,30 nmol/L) produced no significant change in basal amylase release of acinar cells [ (3.48 ± 1.44) ％,(3.70 ±- 1.39) ％,(3.36 ± 1.24) ％,(3.86 ± 1.41 ) ％,(4.54 ± 2.01 ) ％ ].CCK-8 significantly increased amylase release of acinar cells [ ( 13.84 ± 2.63 ) ％ vs (3.48 ± 1.44)％,P ＜0.05],but obestatin (0.1,1,10 nmol/L) has no effect on the amylase release inducedby CCK-8 [(14.55 ± 1.7)％,(13.79 ± 1.81)％,(14.39 ± 1.12)％].Obestatin (0.1,1,10,30 nmol/L) did not affect the amylase release of pancreatic lobuole.KCl significantly increased anylase release,which was ( 1.84 ± 0.29 ) folds higher than that of control group ( P ＜ 0.05 ),but obestatin has no effect on the amylase release induced by KCl,which were (2.01 ± 0.30 ),( 1.89 ± 0.41 ),( 1.74 ± 0.14 ),( 1.88± 0.33) folds higher than those of control group.ConclusionsExogenous obestatin has no effects on pancreatic exocrine of acinar cells and lobules of rats in vitro and cannot block or assist the increased amylase release induced by CCK-8 or KCl.

Objective:To study the inhibition effects of four dihydropyridine calcium antagonists felodipine, nicardipine, lercani-dipine and nifedipine on CYP3A4 enzyme to provide the theoretical basis for the understanding of the drug interactions between dihydro-pyridine calcium antagonists and other drugs. Methods:Using the probe drugs method, the SD female rats induced by 80 mg·kg-1 · d-1 dexamethasone for three days were divided into the negative control group, positive control group, four DHPs groups with six ones in each. Dapsone was used as the probe substrate, and the concentration was determined by HPLC. Data analysis software WinNonLin was used in the pharmacokinetic model fitting process and the paired t-test was used in the statistical analysis. Results: AUC0-24 and CL/F of dapsone in the negative control group showed statistically significant differences when compared with those in the four DHPs groups and the positive group (P<0. 05). Although the inhibition effect of the four DHPs was in the order of nifedipine inhibition >nicardipine > lercanidipine > felodipine, the difference was not statistically significant (P>0. 05). Cmax of dapsone in the DHPs groups and the positive group had no statistically significant difference when compared with that in the negative control group ( P>0. 05). Conclusion:Although there are different inhibition effects on CYP3A4 among the four DHPs, the differences are not significant in vivo, and there is no influence on the combination drugs which is not mainly metabolized by CYP3A4.

Objective To investigate the role of bone marrow mesenchymal stem cells (MSCs) transplantation in repairing injured intestinal mucosa of acute pancreatitis.Methods MSCs were harvested and cultured from femurs of male SD rats.Twenty female SD rats were divided into three groups,and serve acute pancratitis (SAP) model was induced by intraperitoneal injection of L-arginine (2 g/kg) twice.Twelve hours after SAP model established,MSC transplantation group (n=8) were injected MSCs (5 × 106 cell/rat) through tail vein for three days,and SAP group (n=6) were injected the same volume of saline through tail vein as control.Control group (n=6) were only injected the same volume of saline without any treatment.All the rats were sacrificed at 72 hours after model established.The small intestinal tissues were taken for HE staining and pathological score,the TNF-α mRNA and IL-1β mRNA expression level in small intestine and pancreas were tested by RT-PCR.Y chromo-some (Sry) gene in pancreatic and intestinal tissue was examined by polymerase chain reaction (PCR).Results The relative expression quantity of TNF-a mRNA and IL-1β mRNA in pancreas was significant higher in SAP group and MSC transplantation group than in control group (7.22 ± 1.99,3.46± 1.75 vs 1.32 ± 1.04 ; 2.71 ± 0.56,1.92 ± 0.28 vs 0.61 ± 0.45 ),the difference was statistically significant (F=18.375,F=22.701; P＜0.05).Compared with SAP group,the expression quantity of TNF-α mRNA and IL-1β mRNA in pancreas was significantly decreased in MSC transplantation group,the difference was statistically significant (P＜0.05).The relative expression quantity of TNF-α mRNA and IL-1β mRNA in small intestine was significantly higher in SAP group and MSC transplantation group than in control group (3.93 ± 1.08,2.13 ± 0.53 vs 0.68 ± 0.42 ; 2.44 ± 1.54,1.02±0.44 vs 0.60±0.14),the difference was statistically significant (F=21.772,F=6.132; P＜0.05).The expression of TNF-αmRNA and IL-1β mRNA in MSC transplantation group was lower than that in SAP group,the difference was statistically significant (P＜0.05).Compared with SAP group,pathological score indicated that small intestine injure was slighter in MSC transplantation group (3.83±0.28 vs 2.83±0.56),the difference was statistically significant (F=12.013,P＜0.05).Sry gene could be detected in the pancreatic and intestinal tissue of MSC transplantation group.Conclusion Allogeneic MSC transplantation group can inhibit Pro-inflammatory cytokines expression in acute pancreatitis,relieve the intestinal mucosa injury and may involve in the intestinal tissue repair.

Objective To estimate the performance of confocal laser scanning microscopy (CLSM) in the diagnosis of superficial cutaneous fungal infections. Methods This study recruited 59 patients with clinically suspected superficial cutaneous fungal infections.Three typical lesions were selected in each patient for CLSM and microscopic examination.Results CLSM revealed hyphae in stratum corneum in 56％ (14/25) of tinea manus or pedis and 79.17％ (19/24) of tinea cruris lesions,7 out of 8 tinea manus or pedis and 94.12％( 16/17 ) of tinea cruris early lesions (＜ 3 weeks ),and 41％ (7/17) of tinea manus or pedis and 3 out of 7 tinea croris old lesions (＞ 3 weeks).All the CLSM-positive specimens were positive for microscopic examination,and among the CLSM-negative specimens,fungal elements were observed by microscopic examination in 8 out of 11 tinea manus or pedis specimens,4 out of 5 tinea cruris specimens,1 tinea manus or pedis and 1 tinea cruris specimen of early lesions,and 7 out of 10 tinea manus or pedis and 3 out of 4 tinea cruris specimens of old lesions.No hypha was found by CLSM in any of the 10 tinea versicolor specimens,while microscopic examination revealed fungal elements in 8 of them.Neither CLSM nor microscopy revealed fungal elements in lesions from 5 patients with tinea manus or pedis and 5 patients with tinea cruris after treatment with topical bifonazole cream for 2 weeks.Conclusions CLSM shows a good consistency with light microscopy in the examination of early lesions of tinea marnus and pedis as well as tinea croris,and may serve as a valuable tool for clinical diagnosis.

Objective To investigate quercetin's protective effect against oxidative stress in and impact on the biological activity of mouse B10BR melanocytes. Methods B10BR cells were cultured and treated with different concentrations of quercetin followed by additional culture. Then, cell viability was measured by using MTT assay, hydrogen peroxide-induced cell apoptosis by flow cytometry, and cell morphological changes by microscopy. The tyrosinase activity in and melanin synthesis by B10BR cells were measured by dopa oxidation assay and sodium hydroxide (NaOH)-lysis method, respectively. Results After treatment with quercetin of 33.33 μmol/L for 24 hours, the survival rate of B10BR cells reached (94.22 ± 3.36)%, tyrosinase activity (107.15 ± 10.96)%, and melanin content (111.85 ± 9.49)%. A significant difference was observed in tyrosinase activity and melanin content between hydrogen peroxide-induced and 33.33 μmol/L quercetin-treated B10BR cells and those only induced by hydrogen peroxide (both P < 0.01). Flow cytometry revealed that quercetin inhibited hydrogen peroxide-induced apoptosis in melanocytes. Conclusion The protective effect of quercetin against hydrogen peroxide-induced apoptosis in melanocytes may provide a new idea for the treatment of vitiligo.

Objective To investigate the effect of gavaged yAGA2-sCT, a recombinant Saccharomyces cerevisiae which expressed salmon calcitonin on its surface, on serum calcium concentration of rats. Methods The sCT gene was artificially synthesized in advance and cloned into the pYD1 vector, a kind of surface displaying plasmid, to yield pYD1-sCT. The recombinant plasmid pYD1-sCT was transformed into S. cerevisiae EBY100 by LiAC method and achieved yAGA2-sCT. The sCT protein, which was expressed on the surface of recombinant S. cerevisiae yAGA2-sCT, was indirectly labeled with FITC. Labeled yeast was immediately detected under the fluorescent microscope. Before being gavaged to rats, recombinant yeast was frozen and vacuum-dried. Lyophilized yeast yAGA2-sCT was administered to rats in dosage of 0.1g/kg, 0.5g/kg and 5.0g/kg, respectively, with 6 rats in each group. Positive drug group (200mU/kg calcitonin via hypodermic injection) and two conditional control groups (being gavaged 5g/kg yAGA2-sCT and 30?g/kg calcitonin, respectively) were acted as controls. The serum was collected from canthus venous plexus and the calcium concentration was immediately measured by automatic biochemical analyzer. Results The sCT protein was successfully expressed on the surface of recombinant yAGA2-sCT, and FITC-labeled yAGA2-sCT was observed under the fluorescent microscope. The recombinant S. cerevisiae yAGA2-sCT was able to reduce the serum calcium concentration of rats in a dose-dependent manner. In the three subgroups, the hypocalcemic effect of high-dosage subgroup, with 5g/kg lyophilized yAGA2-sCT, was the highest (P

Objective To investigate the expression of melatonin MT1 receptor in rats with acute necrotizing pancreatitis (ANP) and the protective effects of melatonin (MT) pre-intervention for the pancreas. Methods Fifty-four male Sprague-Dawley (SD) rats were randomly divided into three groups:sham-operation group, ANP group and MT-pretreated group. The models of ANP were induced by retrograde injection sodium taurocholate into the bili-pancreatic duct. MT group undergoing intraperitoneal injection 50 mg/kg 30 minutes before the establishment of ANP models. Four, 8 and 12 hours after the onset of operation, the levels of serum amylase and pathological changes of the pancreas were observed. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) and tumor necrosis factor-alpha (TNFα) in the pancreas were measured. The expression of MT1 protein and MT1 mRNA in pancreas were separately analyzed by immunohistochemistry and real-time PCR. Results (1) Pancreatic pathological damage in ANP groups was progressive exacerbated. It was obviously ameliorated in MT group as compared with ANP group ( P ＜ 0.05 ); (2) Compared with SO group, the levels of serum amylase, MDA and TNFα in the pancreas were significantly increased in ANP group (P ＜0.05 or P ＜0.01 ). They were markedly decreased in MT group as compared with ANP group [ 12 h, (2348.00 ±278.90)U/L vs (3194. 83 ±538.10)U/L,(2.255 ± 0.472 ) μmol/L vs ( 2.960 ± 0.722 ) μ mol/L, ( 102.929 ± 29.399 ) ng/L vs ( 378. 544 ±183.454)ng/L, P ＜ 0.05 ]. The level of SOD was decreased in ANP group compared with SO group (P ＜0.05) and increased in MT group[ 12h, (11.448 ± 1.594)U/L vs (8.427 ± 1.950)U/L, P＜0.05] ;(3)Compared with SO group, the expression of MT1 protein and MT1 mRNA in ANP group were down-regulated as the severity of the disease increased ( P ＜ 0.05 ). They were significantly higher in MT group than ANP group. Conclusions Melatonin pre-intervention is able to increase SOD level and decrease MDA, TNFα levels, thereby reducing pancreatic injury. The MT1 might play an important role in the pathogenesis of ANP. MT might exert protective effects for the pancreas in ANP rats through increase the expression of MT1.

Objective To investigate the expression of Smac/DIABLO, XIAP mRNA in acute pancreatitis (AP) and the relationship with the severity in rats.Methods Fifty-four SD rats were randomly divided into three groups:sham-operation (SO) group, acute edematous pancreatitis (AEP) group and acute necrotizing pancreatitis (ANP) group.The models of AEP and ANP were induced by retrograde injection of 1％ and 3.5％ sodium deoxycholate into the pancreaticobiliary duct respectively.The specimens of pancreatic tissue at 3 h, 6 h, 12 h were collected, pathological changes of the pancreas were observed, apeptosis in pancreas were detected by TUNEL method and the expression of Smac/DIABLO, XIAP mRNA were analyzed by real-time PCR.Results Pathological changes of the pancreas confirmed the establishment of AEP and ANP.Apeptosis indexes in SO group, AEP group and ANP group were 0.67±0.82, 6.62 ±0.78 and 4.70 ±0.82, and the differences were significant (P< 0.05).The expression of Smac/DIABLO mRNA of AEP group increased with time, while the expression of ANP group decreased with time.Compared with SO group, Smac/DIABLO mRNA expressions at 6 h in AEP and ANP group were 2.41 ± 0.92 and 1.47± 0.53, and the differences were significant (P<0.05).By contrast, the expressions of XIAP mRNA in AEP group decreased with time,while the expressions in ANP group increased with time.The expressionsof XIAP mRNA at 6 h in AEP and ANP group were 5.51 ± 1.07 and 6.99 ± 1.00, and the differences were significant (P<0.05).Conclusions In acute pancreatitis, the expression of Smac/DIABLO mRNA was consistent with the apoptosis of pancreatic acinar cells, but not consistent with the severity of pancreatitis.The expression of XIAP mRNA was consistent with the severity of pancreatitis.Smac/DIABLO, XIAP mRNA is associated with regulation of apoptosis.

In the present study, through a functional strategy, a metagenome library of the marine microbes from the surface water of the South China Sea was screened for beta-glucosidase and six positive clones were obtained. One of these clones, pSB47B2, was subcloned and further analysed in sequence. The result showed that there was an open reading frame for a novel beta-glucosidase, which was nominated as bgl1B. Using pET22b(+) as vector and Escherichia coli BL21(DE3) as host, Bgl1B was overexpressed recombinantly with high yield obtained and substantial enzymatic activity detected. The recombinant protein (rBgllB) was purified by Ni-NTA affinity chromatography and further biochemically characterized. The results indicated that, with pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgl1B were about 6.5 and 40 degrees C respectively. Under the optimum conditions, rBgl1B hydrolyzed pNPG with an activity up to 39.7 U/mg, Km and Vmax being 0.288 mmol/L and 36.9 micromol/min respectively. In addition, rBgl1B could also hydrolyze cellobiose, with a Km of 0.173 mmol/L and a Vmax of 35 micromol/min. However, we did not detect evident hydrolytic activity of rBgl1B to lactose, maltose, sucrose, and CMC. The enzymatic activity of rBgl1B to pNPG was stimulated to certain degrees by low concentration of Ca2+ or Mn2+, whereas it exhibited significant tolerance against high Na+. Distinguished from most of the beta-glucosidases derived from fungi, which display the highest activities under acidic conditions, rBgl1B exhibited relatively higher activity and stability at pH between 7.0 and 9.0.
Amino Acid Sequence ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Metagenome ; genetics ; Metagenomics ; methods ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Seawater ; microbiology ; beta-Glucosidase ; biosynthesis ; genetics

Objective To study the effect of artesunate on inflammatory responses to severe pneumonia by regulating macrophage migration inhibitory factor (MIF) in rats.Methods Total of 100 SD by random (random number) assigned,20 rats were control group,80 SD rats with severe pneumonia were caused by Klebsiella pneumoniae,60 SD rats were treated with different concentrations (20,40,80 mk/kg) of artesunate after modeling.The pathological changes of lung tissue,the level of MIF myeloperoxidase activity and inflammatory cell infiltration in lung tissue of rats were evaluated.Results After treatment with artesunate,the severity of inflammation was significantly alleviated in rats with severe pneumonia evidenced by decrease in myeloperoxidase activity [severe pneumonia:(17.5 ± 1.5) vs.treatment group:(7.5 ±2.0)] and reduction in inflammatory cell infiltration (severe pneumonia:27 × 106 vs.treatment group:12.5 × 106).Similarly,the artesunate also reduced the production of inflammatory cytokines significantly in bronchoalveolar lavage fluid (IL-1 in severe pneumonia group:(1 100 ± 50) pg/ml vs.treatment group:(400 ± 60) pg/ml;IL-6 in severe pneumonia group:(700-± 30) pg/ml vs.treatment group:(200 ±40) pg/ml;IL-10 in severe pneumonia group:(500 ± 70) pg/ml vs.treatment group:(200 ± 40) pg/ml;TNF-αin severe pneumonia group:(500 ± 80) pg/ml vs.treatment group:(150 ± 50) pg/ml.In addition,artesunate inhibited the level and production of MIF,thus inhibiting the inflammatory responses mediated by MIF.Conclusions Artesunate had a protective effect on pneumonia caused by Klebsiella pneumoniae in rats via inhibiting the inflammation responses mediated by MIF.This study provided a molecular basis for newly developed drugs applied to the treatment of pneumonia caused by Klebsiella pneumoniae in rats.