In the reductive branch, glycerol is first dehydrated to 3-hydroxypropionaldehyde that is then reduced to 1,3-PD under the consumption of reducing power (NADH). If over-expression of the gene dhaB encoding glycerol dehydrase is achieved,the reducing power will be scarce and 3-hydroxypropionaldehyde will be accumulated,which is disadvantage to produce 1,3-propanediol.The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme[under the consumption of reducing power (NADPH)]and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two gene were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD).Over-expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.The fermentation result on aerobic conversion showed the increase of 20% of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) was obtained compared with Klebsiella pneumoniae.The main by-products,acetic acid and butanediol decreased estrogen receptors 30% obviously.

Abstract: Objective　To investigate the clinical characteristics and diagnosis key points of brain abscess caused by Nocardia asiatica, and provide a clinical basis for diagnosing and treating intracranial infection caused by Nocardia. Methods　A case of pulmonary Nocardia asiatica complicated with brain abscess diagnosed at the Second Affiliated Hospital of Hainan Medical University was selected to analyze the clinical manifestations, cerebrospinal fluid characteristics, pulmonary and cranial imaging features, and treatment plan, and to summarize the diagnosis and treatment experience. Results　The patient was an elderly woman with a history of diabetes, dry cough was the first symptom without fever or headache. At the beginning of the course, it was diagnosed as pulmonary infection and tuberculosis in the local hospital, and received conventional antimicrobial and anti-tuberculosis therapies, but showed no improvement. The patient developed progressive limb weakness, followed by consciousness disorders, and coma. Cerebrospinal fluid (CSF) adenosine deaminase and lactate dehydrogenase were not abnormal, CSF pressure, protein and white blood cells were high, mainly with multiple nuclear cells. CSF glucose and chloride were normal in the early stage of the disease, but decreased significantly in the later stage. Metagenomic analysis of cerebrospinal fluid indicated Nocardia asiatica with a specific sequence number of 537. Lung CT showed exudation, abscess, and cavity in the right lung. Skull MRI scan + enhancement suggested multiple scattered abscesses in both cerebral hemispheres. The abscesses were of different sizes and showed ring enhancement, with extensive surrounding edema, and ventricular compression. After treatment with meropenem, linezolid, and compound sulfamethoxazole tablets, the cerebrospinal fluid recovered, and the lesions in the lungs and intracranial structures improved. Conclusions　Brain abscess caused by Nocardia asiatica is similar to the tuberculous brain in clinical symptoms, cerebrospinal fluid examination, craniocerebral imaging, so we should be alert to the possibility of Nocardia infection in patients with diabetes. At the same time, metagenomic testing of the cerebrospinal fluid can help confirm the diagnosis. The mortality and disability rates of brain abscess caused by Nocardia are high. Early diagnosis and treatment can improve the prognosis.

Animals ; Biological Transport ; Blood-Brain Barrier ; metabolism ; Cell Membrane Permeability ; Chromatography, Liquid ; methods ; Humans ; Membranes, Artificial ; Permeability ; Pharmaceutical Preparations ; metabolism ; Pharmacokinetics ; Quantitative Structure-Activity Relationship ; Skin Absorption

The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).
Escherichia coli ; genetics ; Glycoside Hydrolases ; genetics ; isolation & purification ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Thermotoga maritima ; enzymology

Glutamate dehydrogenase (GDH) is a key enzyme in the biosynthesis of glutamate. The GDHs from Corynebacterium glutamicum S9114 the most commonly used strain in glutamate fermentation, were purified and their molecular structures and properties characterized. The coenzymes were also studied in the hope to increase glutamate production. Cells were harvested at mid-exponential phase by centrifugation and washed with Tris-HCl buffer containing DTT and EDTA (pH 7.5). The cells were then disrupted using a French pressure cell press and the supernatant was collected by centrifugation. The extract was concentrated by 70-fold using the AKTA-100 FPLC system employing a DEAE-cellulose ion exchange column, a hydrophobic interaction chromatography (HIC) and Sephadex G-200 gel filtration. The purified extracts contained NADPH-dependent GDH and NADH-dependent GDH. Both of the enzymes were highly specific for the coenzymes. The molecular masses of the NADPH-dependent GDH and its subunit were 188kD and 32kD respectively, suggesting the enzyme is a homo-hexamer. Our data reported for the first time the presence of NADH- dependent GDH in Corynebacterium glutamicum S9114, similar to other microorganisms containing both GDHs. The NADPH-dependent and NADH-dependent GDH in Corynebacterium glutamicum S9114 may participate in the assimilation and dissimilation of ammonia respectively. The absorptions of NADPH-dependent GDH was very weak at 280nm but very high at 215nm, suggesting a low phenylalanine and tyrosine content in the enzyme.
Chromatography, Gel ; Chromatography, Ion Exchange ; Corynebacterium glutamicum ; enzymology ; Glutamate Dehydrogenase ; isolation & purification ; metabolism ; Molecular Weight ; NADP ; metabolism ; Substrate Specificity

OBJECTIVETo investigate the expression characteristics of the gene of coiled-coil domain-containing protein 70 (Ccdc70) in the mouse testis and its potential role in spermatogenesis.

METHODSUsing expression profile microarray, we screened the mouse testis-specific gene Ccdc70, studied its expression characteristics in the mouse testis by RT-PCR, real-time PCR, Western blot and immunohistochemistry, followed by bioinformatic analysis of the Ccdc70 protein.

RESULTSThe Ccdc70 gene was expressed highly in the testis but lowly in the epididymis of the mice. The Ccdc70 protein was expressed mainly in the spermatocytes and round spermatids of the testis and in the epithelial cells of the epididymis. Bioinformatic analysis showed a structural domain in the Ccdc70 protein, which was highly conserved in mammalian evolution.

CONCLUSIONThe Ccdc70 gene is highly expressed in the mouse testis and mainly in the spermatocytes, round spermatids, and epididymal epithelial cells, which indicates that it is involved in the regulation of spermatogenesis and epididymal sperm maturation.

Animals ; Computational Biology ; Gene Expression Regulation, Developmental ; Male ; Mice ; Proteins ; genetics ; Spermatogenesis ; genetics ; Testis ; metabolism

Diacylglycerol, DAG, because of its multifunctional and nutritional properties, attracted considerable attention recently. Enzymatic synthesis of diacylglycerols from linoleic acid was investigated in a solvent-free reaction in a continuously operated fixed bed reactors containing Lipozyme RM IM. By appropriate manipulation of the fluid-residence time, the relative proportions of the various acylglycerols in the effluent stream can be controlled. In addition, the presence of excess glycerol is effective for the removal of water produced during the esterification reactions. Under the conditions of molar ratio of linoleic acid to glycerol of 0.5, the immoblized enzyme maintained high stability and allowed the reaction to continue for 10 days without significant deterioration in enzyme activity. It was determined that the conversion of fatty acid, content of 1,3-DAG and volume efficiency of reactor reached optima under the conditions: a packaged-bed reactor(with a ratio of packed length to inner diameter of 7.8), reacting temperature at 65 degrees C, molar ratio of linoleic acid to glycerol of 0.5, and feeding flow rate of 1.2 mL/min.
Catalysis ; Diglycerides ; chemical synthesis ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; Lipase ; chemistry

OBJECTIVETo evaluate the effect of snail control through soil pasting mixed with niclosamide.

METHODSFour sites were selected in different epidemic areas in Sichuan province. Soil pasting mixed with niclosamide was carried on, and the dosage was 0 g/m2, 4 g/m2, 6 g/m2, 8 g/m2 and 10 g/m2 respectively. The mortality rate of snail and the density of snail were observed after 7, 15, 30, 90 and 180 days.

RESULTSThe mortality rate of snail was more than 43.3% in blank group after 30 days. The mortality rate of snail was from 75.3% to 100.0% at 4 g/m2 group after 30 days. The mortality rate of snail in 4 g/m2 group was significantly higher than that in the blank group (chi2 = 31.27, P < 0.05). There was no significant difference in the mortality rate of snail among all study groups (chi2 = 1.07, P > 0.05). The decrease rate of snail density was more than 90%. The mortality rate of snail was about 30% higher in Chantu group than Qutu group. The unit cost of Pasting-Mixing Drug with Soil was from 5 to 7 times of spray method, but the total cost was similar for the. two methods at the endpoint of the snail control.

CONCLUSIONThe effect of soil pasting mixed with niclosamide is good, and the dosage of 4-6 g/m2 is suggested in snail control.

Animals ; Molluscacides ; Niclosamide ; Pest Control ; Schistosomiasis ; epidemiology ; prevention & control ; Snails ; Soil

OBJECTIVETo analyze the factors affecting the accuracy of Osaka formula multiparameter ultrasound-based fetal mass estimation, thereby establishing new formulas to improve the accuracy of the estimation.

METHODSA retrospective review was conducted among 519 healthy women with singleton pregnancy. Three days before the delivery (between 37 and 42 weeks' gestation), ultrasonic measurement of the fetal weight and other indices of the fetus was routinely performed. Correlation and multiple linear stepwise regression analysis were used to correct the 3 equations, which, along with Osaka University formula, were used to predict another 219 fetuses' birth weight. The coincidence rate of the predicted value and with the actual birth weight, and the absolute error and relative error were compared between the equations.

RESULTSThe fetal abdominal area (AA) and abdominal circumference (AC) showed the most conspicuous influence on the estimated fetal birth weight, and fetal humerus length (HL) was more sensitive than femur length (FL) for the estimation. Three new regression equations were established, among which the equation 2 (fetal birth weight=1082.859+4.116xAAxHL) showed the best accuracy in clinical prediction.

CONCLUSIONAA,AC and HL are more sensitive indices for estimation of the fetal birth weight, and the equation 2 established in this study still awaits further verification for its clinical value.

Abdomen ; diagnostic imaging ; Anthropometry ; methods ; Female ; Fetal Weight ; Humans ; Humerus ; diagnostic imaging ; Pregnancy ; Reference Values ; Regression Analysis ; Retrospective Studies ; Ultrasonography, Prenatal ; methods

OBJECTIVETo evaluate quality of surveillance and emendate rates of birth and death of population of the Three Gorges area.

METHODSData on the two samples collected were designed based on principle of capture-recapture method. An investigation of missing report of birth and death was conducted in 7061 families selected through stratified random sampling method. We collected and registered the data of birth and death in every family investigated and checked with correlative records reported in disease surveillance system of the Three Gorges area. The missing report rates and the 95% confidence intervals of birth rate and death rate were calculated.

RESULTSThe underreporting rates of birth and death were 13.91% and 15.60% and death of infant was 33.33%. The emended birth rate was 8.92 per thousandth and the 95% confidence interval of birth rate was 8.38 per thousandth-9.45 per thousandth. The emended report rate of death was 6.88 per thousandth and the collectivity 95% confidence interval was 6.37%-7.38 per thousandth.

CONCLUSIONResults showed that the quality of birth and death in the disease surveillance reporting system of Three Gorges area was competent to the quality level of the standard set for national disease surveillance system. The birth and death rates of population in the Three Gorges area were under 10.00 per thousandth.

Adolescent ; Adult ; Aged ; Birth Rate ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant Mortality ; Infant, Newborn ; Male ; Middle Aged ; Mortality ; Population Surveillance ; methods