D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The K_m and k_cat/K_m values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.
Recombinant Proteins/metabolism* ; Paenibacillus/metabolism* ; Mannose/metabolism* ; Escherichia coli/metabolism* ; Mannitol/metabolism*

BACKGROUNDLeakage of the intestinal mucosal barrier may cause translocation of bacteria, then leading to multiorgan failure. This study hypothesized that rhubarb monomers might protect the gut mucosal barrier in sepsis through junction proteins.

METHODSHealthy male Sprague-Dawley rats (weighing 230-250 g) under anesthesia and sedation were subjected to cecal ligation and perforation (CLP). After surgical preparation, rats were randomly assigned to eight groups (n = 6 or 8 each group): sham group (Group A: normal saline gavage); sepsis group (Group B: normal saline gavage); Group C (intraperitoneally, dexamethasone 0.5 mg/kg) immediately after CLP surgery; and rhubarb monomer (100 mg/kg in normal saline)-treated groups (Group D: rhein; Group E: emodin; Group F: 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid; Group G: 1-O-caffeoyl-2-(4-hydroxy-O-cinnamoyl)-D-glucose; and Group H: daucosterol linoleate). Animals were sacrificed after 24 h. Intestinal histology, lactulose, mannitol concentrations were measured, and zonula occludens (ZO)-1, occludin and claudin-5 transcription (polymerase chain reaction), translation (by Western blot analysis), and expression (by immunohistochemistry) were also measured.

RESULTSIntestinal histology revealed injury to intestinal mucosal villi induced by sepsis in Group B, compared with Group A. Compared with Group A (0.17 ± 0.41), the pathological scores in Groups B (2.83 ± 0.41, P < 0.001), C (1.83 ± 0.41, P < 0.001), D (2.00 ± 0.63, P < 0.001), E (1.83 ± 0.41, P < 0.001), F (1.83 ± 0.75, P < 0.001), G (2.17 ± 0.41, P < 0.001),and H (1.83 ± 0.41, P < 0.001) were significantly increased. Lactulose/mannitol (L/M) ratio in Group B (0.046 ± 0.003) was significantly higher than in Group A (0.013 ± 0.001, P< 0.001) while L/M ratios in Groups C (0.028 ± 0.002, P< 0.001), D (0.029 ± 0.003, P< 0.001), E (0.026 ± 0.003, P< 0.001), F (0.027 ± 0.003, P< 0.001), G (0.030 ± 0.005, P< 0.001), and H (0.026 ± 0.002, P< 0.001) were significantly lower than that in Group B. ZO-1, occludin and claudin-5 transcription, translation, and expression in Group B were significantly lower than that in Group A (P < 0.001), but they were significantly higher in Groups C, D, E, F, G, and H than those in Group B (P < 0.05).

CONCLUSIONRhubarb monomer treatment ameliorated mucosal damage in sepsis via enhanced transcription, translation, and expression of junction proteins.

Animals ; Claudin-5 ; metabolism ; Intestinal Mucosa ; drug effects ; metabolism ; Lactulose ; metabolism ; Male ; Mannitol ; metabolism ; Occludin ; metabolism ; Plant Extracts ; chemistry ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Rheum ; chemistry ; Sepsis ; drug therapy ; metabolism ; Zonula Occludens-1 Protein ; metabolism

BACKGROUND/AIMS: This animal study aimed to define the underlying cellular mechanisms of intestinal barrier dysfunction. METHODS: Rats were fed 4% with dextran sodium sulfate (DSS) to induce experimental colitis. We analyzed the sugars in 24-hour urine output by high pressure liquid chromatography. The expression of claudins, mannan-binding lectin (MBL), and MBL-associated serine proteases 2 (MASP-2) were detected in the colonic mucosa by immunohistochemistry; and apoptotic cells in the colonic epithelium were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method assay. RESULTS: The lactulose and sucralose excretion levels in the urine of rats with DSS-induced colitis were significantly higher than those in the control rats. Mannitol excretion was lower and lactulose/mannitol ratios and sucralose/mannitol ratios were significantly increased compared with those in the control group (p<0.05). Compared with the controls, the expression of sealing claudins (claudin 3, claudin 5, and claudin 8) was significantly decreased, but that of claudin 1 was increased. The expression of pore-forming claudin 2 was upregulated and claudin 7 was downregulated in DSS-induced colitis. The epithelial apoptotic ratio was 2.8%+/-1.2% in controls and was significantly increased to 7.2%+/-1.2% in DSS-induced colitis. The expression of MBL and MASP-2 in the intestinal mucosa showed intense staining in controls, whereas there was weak staining in the rats with colitis. CONCLUSIONS: There was increased intestinal permeability in DSS-induced colitis. Changes in the expression and distribution of claudins, increased epithelial apoptosis, and the MASP-2-induced immune response impaired the intestinal epithelium and contributed to high intestinal permeability.
Animals ; Apoptosis/*physiology ; Claudins/*metabolism ; Colitis/chemically induced/immunology/*physiopathology ; Colon/immunology/physiopathology ; Dextran Sulfate ; Intestinal Mucosa/*physiopathology ; Lactulose/metabolism ; Mannitol/metabolism ; Mannose-Binding Lectin/*immunology ; Permeability ; Rats ; Rats, Sprague-Dawley ; Sucrose/analogs & derivatives/metabolism ; Up-Regulation

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.
Animals ; *Gene Expression Regulation/drug effects ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Male ; Mannitol/pharmacology ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Reactive Oxygen Species/*metabolism ; Rotenone/pharmacology ; Sodium Chloride/pharmacology ; Toll-Like Receptors ; Transcription Factors/genetics/*metabolism

OBJECTIVEThe complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype.

METHODSThe crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl.

RESULTSIn this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon.

CONCLUSIONThe five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism.

Bacterial Proteins ; genetics ; Base Sequence ; Cyclic AMP ; metabolism ; Cyclic AMP Receptor Protein ; Gene Expression Regulation, Bacterial ; Mannitol ; Molecular Sequence Data ; Operon ; Phosphotransferases ; Vibrio cholerae

OBJECTIVETo study the protective effect of cold autologous blood cardioplegic solution on the heart of infants with cyanotic congenital heart disease (CCHD).

METHODSNinety-six infants with CCHD who underwent cardiopulmonary bypass (CPB) were randomly and equally divided into three groups: histidine-tryptophan-ketoglutarate (HTK) solution, cold non-autologous blood cardioplegic solution, and cold autologous blood cardioplegic solution. The right auricular tissues were taken before aortic cross-clamping and at 30 minutes after aortic declamping, and ATP level and energy charge (EC) in the myocardium were measured. Venous blood was collected before and immediately after CPB, and the serum levels of creatine kinase (CK)-MB and cardiac troponin I (cTnI) were measured. The clinical parameters, such as the re-beat time and re-beat rate during CPB, cardiac index, dependence on positive inotropic agents, and left ventricular ejection fraction (LVEF) at 2 hours after CPB, the incidence rate of arrhythmia within 24 hours after CPB, and postoperative complications and mortality, were recorded.

RESULTSAt 30 minutes after aortic declamping, the three groups showed significantly decreased ATP and EC levels (P<0.05), and the cold autologous blood group had significantly higher ATP and EC levels than the other two groups (P<0.05). Immediately after CPB, the three groups showed significantly increased serum levels of CK-MB and cTnI (P<0.05), and the cold autologous blood group had significantly lower serum levels of CK-MB and cTnI than the other two groups (P<0.05). The cold autologous blood group had significantly better outcomes than the other two groups in terms of the re-beat time during CPB and the dependence on positive inotropic agents and LVEF at 2 hours after CPB (P<0.05).

CONCLUSIONSCold autologous blood cardioplegic solution is superior to HTK and cold non-autologous blood cardioplegic solutions in preserving myocardial energy and reducing myocardial injury in infants with CCHD who undergo CPB, thus providing a better protective effect on the heart.

Cardioplegic Solutions ; pharmacology ; Cardiopulmonary Bypass ; Energy Metabolism ; Female ; Glucose ; pharmacology ; Heart Defects, Congenital ; metabolism ; surgery ; Humans ; Infant ; Infant, Newborn ; Male ; Mannitol ; pharmacology ; Myocardium ; metabolism ; Potassium Chloride ; pharmacology ; Procaine ; pharmacology ; Ventricular Function, Left

D-Mannitol has wide applications in food, pharmaceutical, and chemical industries. In this study, we constructed a genetically stable Escherichia coli strain for D-mannitol production by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC 12291 into chromosome of E. coli ATCC 8739 and inactivating other fermentation pathways (including pyruvate formate-lyase, lactate dehydrogenase, fumarate reductase, alcohol dehydrogenase, methylglyoxal synthase and pyruvate oxidase). Using mineral salts medium with glucose and fructose as carbon sources, the engineered strain could produce 1.2 mmol/L D-mannitol after anaerobic fermentation for 6 days. Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for 80 generations, D-mannitol titer increased 2.6-fold and mannitol dehydrogenase activity increased 2.8-fold. Genetically stable strains constructed in this work could ferment sugars to produce D-mannitol without the addition of antibiotics, inducers and formate, which was favorable for industrial production.
Escherichia coli ; genetics ; metabolism ; Fermentation ; Industrial Microbiology ; methods ; Leuconostoc ; enzymology ; Mannitol ; metabolism ; Mannitol Dehydrogenases ; genetics ; Metabolic Engineering ; methods ; Monosaccharide Transport Proteins ; genetics

Glucose oxidase (GOD) is an important industrial enzyme with many potential applications. In order to increase the production and productivity of GOD by recombinant Pichia pastoris GS115, we investigated the feeding strategies of mixed carbon sources during induction phase, based on results of the optimization of initial cell and methanol concentration on GOD production. The optimal initial cell and methanol concentration were 100 g/L and 18 g/L. During induction phase, the mixed-carbon-sources strategies showed that glycerol, sorbitol or mannitol co-feeding with methanol could enhance GOD production. With mannitol co-feeding (20:1(W/W)), the maximum GOD production and maximum GOD productivity reached 711.3 U/mL and 4.60 U/(mL x h) after an induction period of 156 h. Compared to the control, the enhancements of GOD production and productivity were 66.3% and 67.9%, respectively. Meanwhile, we found an appropriate mannitol co-feeding strategy that would not inhibit the expression of promote. The activity of alcohol oxidase was 8.8 U/g, which was enhanced by 69.2% compared to the control (5.2 U/g). We can use the same optimization process to improve the production of other proteins from recombinant Pichia pastoris by changing the fermentation parameters.
Carbon ; metabolism ; Fermentation ; Glucose Oxidase ; biosynthesis ; Glycerol ; metabolism ; Industrial Microbiology ; Mannitol ; metabolism ; Methanol ; metabolism ; Pichia ; metabolism ; Sorbitol ; metabolism

OBJECTIVETo observe the efficacy of electroacupuncture on sepsis and explore its mechanism.

METHODSFifty cases were randomized into an observation group (26 cases) and a control group (24 cases). The therapeutic programs of anti-infection, anti-shock, respiratory support and nutritional support were provided, but the drugs that might affect gastrointestinal motility were not prescribed in two groups. In the observation group, on the basic treatment as above, electroacupuncture was applied to Zusanli (ST 36), Tianshu (ST 25), Shangjuxu (ST 37), Xiajuxu (ST 39). The excretion ratio of lactulose to mannitol (L/M) in urine and serum D-lactic acid level were detected before and after treatment, as well as the time of target feeding of the patients in two groups. The efficacy was compared between two groups.

RESULTSAfter treatment for 3 days, L/M was (0.083 +/- 0.020) and serum D-lactic acid was (0.155 +/- 0.196) mmol/L in the observation group, which were apparently reduced as compared with (0.123 +/- 0.034) and (0.193 +/- 0.377) mmol/L in the control group respectively (both P < 0.05). The time of target feeding was (93.69 +/- 27.58) h in the observation group, which was shortened apparently than (118.17 +/- 40.28) h in the control group (P < 0.05). The total effective rate was 80.8% (21/26) in the observation group, which was better than 54.2% (13/24) in the control group (P < 0.05).

CONCLUSIONConventional treatment combined with electroacupuncture can improve intestinal permeability in sepsis patients, recover intestinal function as quickly as possible to achieve target feeding.

Acupuncture Points ; Aged ; Aged, 80 and over ; Electroacupuncture ; Female ; Humans ; Intestines ; metabolism ; Lactulose ; metabolism ; Male ; Mannitol ; metabolism ; Middle Aged ; Permeability ; Sepsis ; metabolism ; therapy

The aim of the study is to investigate the effects of concentration, intestinal segments, pH, inhibitors of proteins (P-gp), Na(+)-dependent glucose transporter (SGLT1) on the intestinal absorption of berberine, and to compare intestinal absorption of berberine in combinations. With phenol red as the indicator, in situ single pass intestinal perfusion (SPIP) model was used and intestinal absorption of pure berberine at concentrations of 36.70, 46.17 and 92.33 microg x mL(-1), simulated system of HLJDT (mixture of berberine, baicalin and geniposide), HLJDT with the concentration of berberine 92.33 microg x mL(-1) in perfusion solution of different intestinal segments (duodenum, jejunum, ileum, and colon) were determined by HPLC in combination with diode array detection (DAD). The results indicated that Ka values ofberberine at different concentrations had little significant difference among that obtained after perfusing via duodenum, jejunum, ileum and colon indicating that the absorption of berberine was mainly the passive diffusion. It was also suggested that SGLT1 and P-gp might exert some effects on the absorption of berberine. Ka and Peff values of berberine in a mixture of pure compounds and HLJDT for different intestine segments of rat showed an increasing tendency and was significantly different (P < 0.05) indicating that berberine in a mixture of pure compounds and HLJDT was assimilated better in small intestine. These results indicate that the intestinal absorption of berberine may be affected by compatibility of compounds. Additionally, berberine has wide absorption window and better absorption in colon.
ATP-Binding Cassette, Sub-Family B, Member 1 ; physiology ; Animals ; Berberine ; administration & dosage ; pharmacokinetics ; Colon ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Duodenum ; metabolism ; Ileum ; metabolism ; Intestinal Absorption ; Intestine, Small ; metabolism ; Jejunum ; metabolism ; Male ; Mannitol ; pharmacology ; Perfusion ; Rats ; Rats, Sprague-Dawley ; Sodium-Glucose Transporter 1 ; physiology ; Verapamil ; pharmacology