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*

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

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

It is the purpose of this experimental study to investigate the alterations of the amount of adenosine nucleotides and adenylate energy charge in the acute focal cerebral ischemia of cats utilizing high performance liquid chromatography and to make a comparative study of protective effects of recirculation and combined therapy with mannitol, steroid and barbiturate. Acute focal cerebral ischemia in cats was induced by occlusion of the left middle cerebral artery through the postorbital technique. The experimental animals were divided into four groups according to the duration of occlusion time. The experimental results are obtained as follows: 1) In 1, 3 and 5 hour-occlusion groups, amount of adenosine triphosphate and summation of adenosine nucleotides decreased significantly to 21.4%, 5% & 0%, 44.0%, 29.9% & 10.8% of the sham control, respectively. Also in these groups adenylate energy charge decreased significantly to 62.7%, 38.7% and 30.7% of the sham control, respectively. It was suggested that the longer duration of occlusion time was, the more amount of adenosine triphosphate, summation of adenosine nucleotides and adenylate energy charge decreased significantly. 2) In 1 and 3 hour-occlusion groups, 2 hour-recirculation increased significantly amount of adenosine triphosphate and summation of adenosine nucleotides to 37.4% & 29.4%, and 62.1% & 58.3% of the sham control, respectively. Also in these groups recirculation increased significantly adenylate energy charge to 70.7% and 65.3% of the sham control, respectively. Whereas there was a slight increase of adenylate energy charge after recirculation in 5 hour-occlusion group, but not significant. 3) In the groups of recirculation following 5 hour-occlusion, pretreatment of combination of mannitol and steroid, or mannitol, steroid and barbiturate increased significantly amount of adenosine triphosphate, summation of adenosine nucleotides, and adenylate energy charge to 57.2% or 66.1%, 80.9% or 83.5% and 82.7% or 84.0% of sham control, respectively.
Adenosine ; Adenosine Triphosphate ; Animals ; Brain Ischemia ; Cats ; Chromatography, Liquid ; Mannitol ; Metabolism* ; Middle Cerebral Artery ; Nucleotides

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

BACKGROUND: The information on nasal transport and the metabolism of peptides have been obtained from pharmacokinetic investigations in experimental animals. However, there are no transport and metabolic studies of human nasal epithelial cells. In this study, the permeability characteristics and the metabolic properties of in vitro human nasal cell monolayers were investigated. Material and METHODS: Normal human inferior nasal conchal tissue samples were obtained from patients undergoing endoscopic nasal cavitary surgery. The specimens were cultured in a transwell using an air-liquid interface (ALI) culture, and the transepithelial electrical resistance (TEER) value of the blank filter and confluent cell monolayers were measured. To determine the % leakage of mannitol, 4µmol 14C-labelled mannitol was added and the % leakage was measured every 10 minute for 1 hour. RESULT: Human nasal epithelial cells in the primary culture grew to a confluent monolayer within 7 days and expressed microvilli. The tight junction between the cells was confirmed by transmission electron microscopy. The TEER value of the blank filter, fifth day and seventh day reached 108.5 ohm.cm2, 141 ohm.cm2 and 177.5 ohm.cm2, respectively. Transcellular % leakage of the 14C-mannitol at 10, 20, 30, 40, 50 and 60 minutes was 35.67±5.43, 34.42±5.60, 32.75±5.71, 31.76±4.22, 30.96±3.49 and 29.60±3.68 %, respectively. CONCLUSION: The human nasal epithelial monolayer using ALI using techniques is suitable for a transcellular permeability study. The data suggests that human nasal epithelial cells in as ALI culture technique shows some promise for a nasal transport and metabolism study.
Animals ; Culture Techniques* ; Electric Impedance ; Epithelial Cells* ; Humans* ; Mannitol ; Metabolism ; Microscopy, Electron, Transmission ; Microvilli ; Peptides ; Permeability ; Tight Junctions

The study was designed to examine the effects of pretreatment with mannitol, methyl prednisolone and nimodipine on the acute focal cerebral ischemia in the cats of occlusion of the proximal part of the middle cerebral artery via the postorbital approach. The energy metabolisms of the brain was measured utilizing the high liquid performance chromatography in the brain tissues of cats. The experimental animals were seperated into 3 groups. group I: the sham control group. group II: the recirculation group. group III: the treatment group. There were significant increase in the ATP, GTP, UTP and E.C. levels in focal ischemic cerebral tissues of the treatment group when compared with the recirculation group. It is suggested that pretreatment with the combination of these drugs may prevent the ischemic damage from the acute focal cerebral ischemia by the maintenance of high energy metabolites. However further studies should determine the synergistic pharmacologic mechanisms in this therapeutic strategy.
Adenosine Triphosphate ; Animals ; Brain ; Brain Ischemia* ; Cats ; Chromatography ; Energy Metabolism ; Guanosine Triphosphate ; Mannitol ; Middle Cerebral Artery ; Nimodipine ; Prednisolone ; Uridine Triphosphate

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

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

PURPOSE: The effect of intra-portal infusion of glucose-insulin-potassium (GIK) solution on the energy metabolism during cold preservation was investigated using a small-animal liver transplantation model. METHODS: Fifteen white rats were divided into 3 groups: the group A (feeding group) were fed normally before experiment. The group B (fasting group) and group C (GIK group) were fasted from 3 days before experiment, by which acute nutritional deficiency state was induced. In group A and B, the whole liver was procured after intra-portal perfusion of HTK solution and serial liver biopsies were performed during the cold preservation period with 4degrees C HTK solution. In group C, intra-portal GIK solution infusion for 1 hour preceded liver graft harvest. From the liver tissues, the relative intracellular glycogen contents and the ATP concentration were measured. RESULTS: Relative glycogen contents in group A were 100% at 0 h, 64.6% at 2 h, 54.9% at 4 h, and 16.2% at 8 h; 10.3%, 8.3%, 4.9% and 0%, respectively in group B; 109.2%, 96.9%, 54.2% and 9.7%, respectively in group C. There was a temporary supercharge of ATP level in group C only at 0 h. Apoptosis was less expressed in group C comparing with group A and B. CONCLUSION: Rapid intra- portal infusion of GIK solution could make intrahepatic glycogen content fully restored to the normal level. Considering that intracellular glycogen is the main energy source during immediate post-transplant period, its restoration may contribute to improvement of post-transplant graft function.
Adenosine Triphosphate ; Animals ; Apoptosis ; Biopsy ; Cold Temperature ; Energy Metabolism ; Glucose ; Glycogen ; Humans ; Insulin ; Liver ; Liver Transplantation ; Malnutrition ; Mannitol ; Perfusion ; Potassium ; Potassium Chloride ; Procaine ; Rats ; Transplants