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

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

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 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 expression characteristics of human skin epidermal stem cell at different developmental stages, and to explore its biological significance.

METHODSHealth skin samples from 28-32 w fetuses (F group), 4-12 y children (C group), and 35-55 y adult (A group) were harvested, with 10 cases in each group. Epidermis were separated using trypsin digestion and EDTA, and human epidermal stem cells were isolated and purified with type IV collagen attachment method. The monoclonal antibody of integrin beta1 and keratin 19 were used for detection and identification of epidermal stem cells by immunohistochemical staining. Total RNA was extracted from above cells by Trizol one-step method, and were detected by formaldehyde denaturing agarose gel electrophoresis. Probes were prepared and hybridized into cDNA microarray for scanning fluorescent signals and analysis of images, with two-fold differential expression value for screening. Significantly up/down-regulated genes were selected for verification by real time RT-PCR.

RESULTSBy comparing expression profile between A and C groups, a total of 1808 genes with differential expression were detected, including 1089 up-regulated genes and 719 down-regulated genes, and they were classified into 128 categories. Among them, 1462 genes were known (found in GeneBank), 346 genes were unknown. A total of 4534 genes with differential expression were detected between C and F groups, in which 1783 genes were up-regulated and 2751 genes were down-regulated, and they were classified into 216 categories. Among them, 3577 genes were known (found in GeneBank), and 957 genes were unknown. There were 1104 genes with differential expression consistently detected in F, C and A groups, which were classified into 32 categories according to gene function. Among them, 94 genes were consistently up-regulated and 75 genes consistently down-regulated. Test results of real time RT-PCR were in accordance with above-mentioned results.

CONCLUSIONSGene expression profiles of epidermal stem cells cultured in vitro, harvested from fetuses, children, and adult, exhibit obvious difference. This may be closely related to different stages of proliferation and differentiation of human epidermal stem cell and self-repair ability of wound at different developmental stages.

Adult ; Cell Differentiation ; Child ; Child, Preschool ; Epidermis ; cytology ; growth & development ; Epithelial Cells ; cytology ; Fetus ; cytology ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Humans ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; methods ; Stem Cells ; cytology ; Transcriptome

OBJECTIVETo investigate the causes of unintentional injuries in children and to provide reference for reducing the incidence of unintentional injuries in children.

METHODSA retrospective analysis was performed on the clinical data of 924 children with unintentional injuries who were hospitalized in Zhongshan Boai Hospital from January 2008 to December 2010.

RESULTSOf the 924 cases, 26.4% suffered falls, which was the most common cause of injuries, 15.7% suffered burns and scalds, 14.0% were injured by foreign bodies, and 13.1% were injured in traffic accidents. Unintentional injuries occurred mainly among children aged 0-3 years (67%). The proportion of cases in summer (July to September) was significantly higher than in any other season (P < 0.05), and burns were the common causes of injuries in summer. Home was the most common place where unintentional injuries occurred (36.9%). The incidence of traffic accidents was significantly higher in non-local children than in local children (P < 0.001). As for the parents' educational levels, the children whose parents had an education level below junior middle school accounted for the highest proportion of all cases of unintentional injury (45.5%). Injuried cases in rural areas accounted for a significantly higher percentage than in urban areas (P < 0.05).

CONCLUSIONSFalls are the principal cause of unintentional injuries in children. Young children (0-3 years) are most vulnerable to unintentional injuries. Unintentional injuries mostly occur at home. The incidence of traffic accidents is higher in non-local children than in local children. Parents' education levels are associated with the incidence of unintentional injuries. In order to reduce the incidence of unintentional injuries in children, preventive measures must be carried out, and safety education should be enhanced in consideration of children's age, sex and family background.

Accidents ; statistics & numerical data ; Adolescent ; Child ; Child, Preschool ; Educational Status ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Parents ; Retrospective Studies