The genes of maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose tetrahydrolase(MTHase) from Sulfolobus solfataricus ATCC 35092 were amplified using PCR. The expression plasmids, pTrc99a-MTSase and pTrc99a-MTHase, were constructed by inserting these two DNA fragments into E. coli expression vector pTrc99a. The specific activity of MTSase and MTHase in E. coli BL21(DE3) at optimal fermentation conditions reached 31.3U/g (wet cell) and 403U/g (wet cell), respectively. The biotransformation of partially hydrolyzed starch to trehalose catalyzed by MTSase and MTHase was carried out at 75℃ and pH 5.0. The highest yield of trehalose (ca. 53.6%) was gained when the original starch concentration was 15%(w/v) and the DE value was 10.

Cytosine-substituted mildiomycin analogue (MIL-C) was produced effectively by supplementing cytosine into the culture of Streptoverticillium rimofaciens. In order to improve the yield of MIL-C, statistically-based experimental designs were applied to optimize the fermentation medium for S. rimofaciens ZJU 5119. Fifteen culture conditions were examined for their significances on MIL-C production using Plackett-Burman design. The Plackett-Burman design and one-variable-at-a-time design indicated that glucose and rice meal as the complex carbon sources, and peanut cake meal and NH4NO3 as the complex nitrogen sources were beneficial for MIL-C production in S. rimofaciens ZJU 5119. The results of further central composition design (CCD) showed that the optimal concentration of glucose, rice meal and peanut cake meal were 18.7 g/L, 64.8 g/L and 65.1 g/L, respectively. By using this optimal fermentation medium, the MIL-C concentration was increased up to 1336.5 mg/L, an approximate 3.8-fold improvement over the previous concentration (350.0 mg/L) with un-optimized medium. This work will be very helpful to the large-scale production of MIL-C in the future.
Anti-Bacterial Agents ; biosynthesis ; Culture Media ; Cytosine ; analogs & derivatives ; biosynthesis ; Fermentation ; Streptomycetaceae ; metabolism

Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.
Acetates ; pharmacology ; Carbon ; pharmacology ; Cell Division ; drug effects ; Culture Media ; chemistry ; pharmacology ; DNA ; biosynthesis ; genetics ; Escherichia coli ; cytology ; drug effects ; genetics ; Genetic Therapy ; instrumentation ; Genetic Vectors ; biosynthesis ; genetics ; Humans ; Nitrogen ; pharmacology ; Phosphates ; pharmacology ; Plasmids ; biosynthesis ; genetics

Human defensin is a family of cationic antimicrobial peptides in human being. During the last two decades a series of endogenous alpha-and beta-human defensins have been discovered. They are important components of the first barrier in human's body against the invasion of various microorganisms, and they are thought to play an important role in linking the innate and adaptive defense system of human being. The recent advances in the research of human defensins were reviewed, including their discovery, molecular and genetic properties, expression regulation, and mechanisms of antimicrobial activity. The possibility to produce human defensins via genetic engineering was also discussed. And the application outlook of human defensins in medicine and curing patients infected with antibiotics-resistant microbials was presented.
Amino Acid Sequence ; Defensins ; chemistry ; genetics ; metabolism ; physiology ; Genetic Engineering ; Humans ; Molecular Sequence Data ; Sequence Homology, Amino Acid

(R)-chlorprenaline, a selective activator of beta2 receptor and an effective drug for bronchitis and asthma, is industrially prepared from (R)-2'-chloro-1-phenyl-ethanol. In this communication, we describe (1) the identification of Saccharomyces cerevisiae B5 as an effective host for stereoselective reduction of 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol; (2) the presence of ethanol enhances the conversion; and (3) the biochemical factors that effect the yield of the product. Among the four yeast strains capable of reduction 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol we screened, Saccharomyces cerevisiae B5 showed the highest activity and stereoselectivity, and was used for the subsequent study. The effect of the presence of methanol, ethanol, 2-propanol, 1-butanol, glucose, glycerol and lactic acid was first investigated, as it was previously reported that they increased the yield and stereoselectivity of the reaction. The addition of the co-substrate methanol, ethanol, 2-propanol, 1-butanol, glucose and glycerol favored the formation of the 2'-chloroacetophenone to (R)-2'-chloro-1-phenyl-ethanol. Lactic acid inhibited the enzyme activity. Ethanol is the best co-substrate among the seven co-substrates and under the optimum concentration of 5% , the yield of (R)-2'-chloro-1-phenyl-ethanol was increased from 17% to 74%. The oxidation of ethanol regenerates NADH required for the reduction. The effects of the reaction time, pH, cell concentration, substrate concentration and temperature on the reduction were investigated next. The enantiometric excess of (R)-2'-chloro-1-phenyl-ethanol reached 100% under the optimal condition: pH8.0, 25 degrees C and 5% ethanol. The product yield went up with the increasing Saccharomyces cerevisiae B5 concentration and reached 100% when the cell dry weight was 10.75 mg/mL and 2'-chloroacetophenone was 6.47 mmol/L. The yield of (R)-2'-chloro-1-phenyl-ethanol decreased sharply with the increase of substrate concentration, as the high concentration of substrates is toxic to the cell and inhibits the activity of reductases. The aerobic cultivation of the yeast and shaking during the reaction increased the yield of (R)-2'-chloro-1-phenyl-ethanol. The yeast can be reused up to 15 times. This research paves the way for economical preparation of chiral 2'-chloroacetophenone to R-2'-chloro-1-phenylethanol.
Ethanol ; metabolism ; Hydrogen-Ion Concentration ; Oxidation-Reduction ; Phenylethyl Alcohol ; chemistry ; metabolism ; Saccharomyces cerevisiae ; metabolism ; Stereoisomerism ; omega-Chloroacetophenone ; chemistry ; metabolism

OBJECTIVETo evaluate the patients with bone injury in Wenchuan earthquake.

METHODSFrom May 12th to June 15th 2008 the data of 1410 patients with bone injury in Wenchuan earthquake were analyzed to evaluate clinical intervention and remedy-managing experience.

RESULTSThe 1410 patients average age was from 4 to 103 years old. And 744 cases (52.7%) suffered from blunt injuries, 379 cases (26.9%) from buried injuries, 287 cases (20.4%) from falling injuries; And 1317 cases were with fracture, 93 with limbs soft tissue injuries; 261 patients combined with other parts of injuries including 45 cases with paralysis; 66 cases were with crush syndrome, 25 with gas gangrene, 76 with acute kidney failure, 26 with multiple organ failure. And 912 operations were performed including 402 fracture fixation, 224 debridement, 152 debridement and suture, 85 amputation, 29 implant skin, 8 fixation of joint dislocation, 5 surgical flaps transplantation, 4 nerve and tendon suture, 2 arthroscopes, 1 joint replacement. Among the 66 crush syndrome patients, 49 accepted continuous renal replacement therapy, in which 9 cases were bleeding from named arteries and 20 blood vessels were getting embolism. Among the 1410 cases, 1 died from multiple organ failure.

CONCLUSIONSAmong the patients with bone injury in Wenchuan earthquake, the elderly patients are more than the youth; The injuries are always combined with other complications; Opened injuries are polluted severely; It is difficult to deal with the crush syndrome; Paraplegia cases are less, but the amputees are more.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Earthquakes ; Female ; First Aid ; Humans ; Male ; Retrospective Studies ; Wounds and Injuries ; surgery

Adult ; Humans ; Acute Disease ; Leukemia