Objective To evaluate the improvement of diagnostic accuracy with background region of interest(ROI)rectification for 99mTc-DTPA renography in patients with GFR≤plasma sampling method). Methods Thirty-three patients(age＞20 years,male/female=13/20)dose of 111 MBq/0.5 ml of 99mTc-DTPA was injected into an antecubital vein.The background ROI was selected below the kidney(Gates method,method a)or around the kidney(method b),then these two different GFR(GFRa,GFRb)were automatically estimated by computer.Meanwhile,3 ml blood samples were collected 2 h and 4 h after injection respectively,and radioactivity of 1 ml plasma was measured.GFR was calculated by dual plasma sampling method(GFRp)and the results were all standardized with the body surface area.The accuracies and correlations of GFRa and GFRb were compared to GFRp respectively. Results The correlation coefficients were ra=0.602 and rb=0.834.The median of difference of GFRa and GFRb was 8.33,-4.41.The median of absolute difference of GFRa and GFRb was 8.33,4.49.The accuracies within±15%,±30%and±50%of GFRa were 24.2%,30.3%and 48.5%,and those of GFRb were 33.3%,51.5%and 81.8%.Conclusion The background ROI around kidney can obviously improve the diagnostic accuracy of 99mTc-DTPA renography in patients with severe renal insufficiency.

By engineering the subsite +1 of cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans, we improved its maltodextrin specificity for 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) synthesis. Specifically, we conducted site-saturation mutagenesis on Leu194, Ala230, and His233 in subsite +1 separately and gained 3 mutants L194N (leucine --> asparagine), A230D (alanine --> aspartic acid), and H233E (histidine --> glutamic acid) produced higher AA-2G yield than the wild-type and the other mutant CGTases. Therefore, the 3 mutants L194N, A230D, and H233E were further used to construct the double and triple mutations. Among the 7 obtained combinational mutants, the triple mutant L194N/A230D/H233E produced the highest AA-2G titer of 1.95 g/L, which was increased by 62.5% compared with that produced by the wild-type CGTase. Then, we modeled the reaction kinetics of all the mutants and found a substrate inhibition by high titer of L-AA for the mutants. The optimal temperature, pH, and reaction time of all the mutants were also determined. The structure modeling indicated that the enhanced maltodextrin specificity may be related with the changes of hydrogen bonding interactions between the side chain of residue at the three positions (194, 230 and 233) and the substrate sugars.
Ascorbic Acid ; analogs & derivatives ; chemistry ; Glucosyltransferases ; genetics ; metabolism ; Hydrogen Bonding ; Kinetics ; Mutagenesis, Site-Directed ; Paenibacillus ; enzymology ; Polysaccharides ; chemistry ; Protein Engineering ; Substrate Specificity ; Temperature

Objective To study the efficacy and complications of reconstruction of failed urethro-plasty for hypospadias with pedicle flap , bladder mucosa , buccal mucosa , and biological patch . Methods 23 patients were enrolled from Jul .2005 to Dec.2011.8 patients, with good local skin condition , were performed with pedicle flap urethroplasty .The other 15 patients, with bad local skin condition or with long segment urethral stricture , were performed with free grafts , including 6 bladder mucosa , 7 buccal muco-sal and 2 biological patch. Results Of the 23 cases, 7 cases were cured by one phase operation .There were 16 (16/25) cases had complications.3 (3/16) cases were failed because of serious infection (2 pedi-cle flap, 1 bladder mucosa ) .The failed cases were implemented with urethroplasty 6 months later by the buccal mucosa installments operation .4 (4/16) cases had solitary urethral fistula (1 pedicle flap, 2 bladder mucosa, and 1 buccal mucosal), who were successfully treated with simple fistula repair 3 to 6 months later. 9 ( 9/16) cases had urethral stricture ( 2 pedicle flap , 3 bladder mucosa , 3 buccal mucosal , and 1 biologi-cal patch graft ) , who were treated with urethral sound and got good result .We had reconstructed the urethra using mucosa graft onlay urethroplasty .All of the 23 patients were followed up with an average of 14.5 ( 6-24) months.23 cases were satisfied with the stretched penis , urination and no need to expand the urethra more than 6 months.3 cases were not satisfied with penile appearance .After communication, these patients did not require a further penis orthopedic surgery . Conclusions Pedicle flap, bladder mucosa , buccal mucosa and biological patch can be used in urethral repair and construction of failed urethroplasty for hypos -padias.Urethral sound dilation plays an important role in hypospadias repair .

Cutinase (EC 3.1.1.74) is a kind of hydrolase capable of catalyzing the cleavage of ester bonds of cutin to release fatty acids. Cutinase displayed hydrolytic activity not only toward cutin but also a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Besides its hydrolytic activity, cutinase also showed synthetic activity and transester activity. Therefore, cutinase was evaluated as a versatile lipolytic enzyme used in food and chemical industry. Recently, it is found that cutinase has potential use in cotton bio-scouring and synthetic fibers modification. Cutinase is the most important enzyme in clean production of textile industry.
Amino Acid Sequence ; Carboxylic Ester Hydrolases ; chemistry ; genetics ; metabolism ; Catalysis ; Environmental Pollution ; prevention & control ; Molecular Sequence Data ; Textile Industry

We reviewed the microbial production of alkaline polygalacturonate lyase (PGL) and its application in the clean production of textile industry. Currently PGL is mainly produced by microbial fermentation and Bacillus sp. is an ideal wild strain for PGL production. Microbial PGL production was affected by many factors including the concentration and feeding mode of substrate, cell concentration, agitation speed, aeration rate, pH and temperature. Constructing the recombinant strain provided an effective alternative for PGL production, and the concentration of PGL produced by the recombinant Pichia pastoris reached 1305 U/mL in 10 m3 fermentor. The recombinant Pichia pastoris had the potential to reach the industrial production of PGL. PGL can be applied in bio-scouring process in the pre-treatment of cotton. Compared with the traditional alkaline cooking process, the application of PGL can protect fiber, improve the bio-scouring efficiency, decrease energy consumption and alleviate the environmental pollution. The future research focus will be the molecular directed evolution of PGL to make PGL more suitable for the application of PGL in bio-scouring process to realize the clean production of textile industry.
Alkalies ; Bacillus subtilis ; metabolism ; Environmental Pollution ; prevention & control ; Fermentation ; Industrial Microbiology ; Pichia ; genetics ; metabolism ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Textile Industry

Objective To investigate the feasibility of Antituberculotic?loaded bone cement (ATLBC) prepared by mix?ing the anti?TB drugs Rifampicin (RFP), Isoniazid (INH), Pyrazinamid (PZA), Moxifloxacin (MFX) with Palacos R PMMA bone cement in Total Joint Arthroplasty treatment for Joint Tuberculosis. Methods Forty grams of Palacos R bone cement powder without antibiotics was mixed with 1 or 2 grams of RFP, INH, PZA and MFX respectively. According to ISO 5833:2002 stan?dard, 8 groups of ATLBC standard test specimen were prepared as experiment group and Palacos R PMMA bone cement with?out antibiotics was prepared as control group. Physical properties (such as the average dough time, curing time, maximum tem?perature), mechanical strength (such as the compressive strength, the bending resistance strength, the modulus of elasticity) and the concentrations of eluant drug in different time points of ATLBC were detected. Results In RFP (1 g), RFP (2 g), INH (1 g) and INH (2 g) group, the average dough time and curing time were longer than those in control group, which exceeded the standard scope of ISO, while the average maximum temperature was significantly lower than that in control group. The INH ( 1 g) group and INH (2 g) group hardened after mixing for 14 days. The RFP (1 g) group and RFP (2 g) group hardened after mixing for 30 days. Twenty minutes after mixing and hardening, the compressive strength, bending resistance strength and modulus of elastic?ity were significantly lower than the specified values of ISO standard. The physical properties and mechanical strength in PZA ( 1 g) group, PZA (2 g) group, MFX (1 g) group, MFX (2 g) group and control group were in accordance with the specified values of ISO standard, and they hardened after 20 minutes. In RFP (1 g) group, RFP (2 g) group, INH (1 g) group, INH (2 g) group, PZA (1 g) group, PZA (2 g) group, MFX (1 g) group and MFX (2 g) group, the concentration of eluant could maintain for 3 days, 7 days, 90 days, 90 days, 45 days, 60 days, 60 days and 60 days respectively. Conclusion RFP and INH mixing with Palacos R PMMA bone cement can hinder the aggregation of bone cement so they are unsuitable for preparing ATLBC. PZA and MFX mixing with Palacos R PMMA bone cement do not affect the physical properties of bone cement, with excellent mechanical strength and elu?tion properties. Because the minimal inhibitory concentration of PZA is higher and its antimicrobial activity maintains shorter time, while MFX maintains longer time in antimicrobial activity, it's more suitable for the preparation of ATLBC.

Trypsin as an important serine protease has been widely used in food, pharmaceutical and tanning industries. In this study, we successfully expressed trypsin (cloning from Streptomyces griseus ATCC10137) in Streptomyces lividans TK24 and comparatively investigated its enzymatic properties. Specifically, applying S. griseus ATCC 10137 genome as template, we obtained the sprT gene and sub-cloned it into the expression plasmid pIJ86, generating the recombinant strain S. lividans TK24/pIJ86-sprT. When cultivated in R2YE and SELF, the activity of rSGT reached 9.21 U/mL and 8.61 U/mL, respectively. Meanwhile, the results of the enzymatic analysis showed that rSGT exhibited a higher acid tolerance and a higher specificity to hydrolyze amide bonds compared with bovine trypsin (BT). In addition, Zn2+ and organic solvents up-regulated esterase and amidase of rSGT. Taken together, the results obtained herein provide meaningful information for further modification of rSGT and its industrial application.
Fermentation ; Recombinant Proteins ; biosynthesis ; genetics ; Streptomyces griseus ; enzymology ; Streptomyces lividans ; genetics ; metabolism ; Trypsin ; biosynthesis ; genetics

We studied the overproduction of catalase (CAT) by Bacillus sp.WSHDZ-01 by oxidative stress via the feeding of ethanol and the pulse addition of H2O2. By adding 2.0% (V/V) ethanol to the culture broth, the intracellular CAT activity reached 11 151 U/mL, which was 2.5 times than that of the control (4 450 U/mL in flask). By adding 0.3% (V/V) H2O2, more extracellular CAT secreted to the culture broth, and the ratio of extracellular CAT to the total CAT increased to 27%. Based on these results, an oxidative stress strategy combining the ethanol feeding and the pulse addition of H2O2 was developed. With this strategy, the ratio of extracellular CAT to the total CAT reached 82.5%, increased by 18.6% than that of the control (without ethanol and H2O2 addition). CAT production increased to 28 990 U/mL, which was 95.5% higher than the control (14 830 U/mL in 3 L fermentor). The fermentation time decreased to 42 h, which was much shorter than that of adding ethanol or H2O2, and CAT productivity reached 470 U/(mL x h) while the control achieved 396.4 U/(mL x h).
Bacillus subtilis ; drug effects ; enzymology ; physiology ; Catalase ; biosynthesis ; Culture Media ; pharmacology ; Ethanol ; pharmacology ; Hydrogen Peroxide ; pharmacology ; Oxidative Stress

Glucosamine (GlcN), also called amino sugar, is a compound derived from the substitution of a hydroxyl group of glucose molecule with an amino group. GlcN finds a wide-range of applications in health food and pharmaceutical industries. In our previous research, a recombinant Escherichia coli-glms-gnal was constructed for the efficient production of GlcN and N-acetylglucosamine (GlcNAc), the latter can be readily deacetylated to GlcN under mild acidic conditions. However, the results indicated that the titer of GlcN and GlcNAc decreased significantly due to the transportation of GlcN and GlcNAc from the culture broth to the inside of cells. To alleviate or block the transportation process, nagE gene (encoding for the GlcNAc-specific transporter) and manX gene (encoding for the mannose transporter) were knocked out with the Red homologous recombination method, and two engineered strains, E. coli-glms-gna1-delta nagE (with nagE gene deletion) and E. coli-glms-gna1-delta nagE-delta manX (with nagE and manX genes deletion), were successfully constructed. The two strains were cultured in a 7-L fermentor for the production of GlcN and GlcNAc. The maximal GlcN concentration of control strain E. coli-glms-gnal reached 4.06 g/L, and the maximal GlcNAc concentration reached 41.46 g/L. The maximal GlcN and GlcNAc concentration of E. coli-glms-gna1-delta nagE reached 4.38 g/L and 71.80 g/L, respectively, which were 1.08-fold and 1.70-fold of those of E. coli-glms-gnal, respectively. The maximal GlcN and GlcNAc concentration of E. coli-glms-gnal-delta nagE-delta manX reached 4.82 g/L and 118.78 g/L, respectively, which were 1.20-fold and 2.86-fold of those of E. coli-glms-gnal, respectively. These results suggested that the deletion of nagE and manX could significantly increase the extracellular accumulation of GlcN and GlcNAc. The results obtained here maybe useful for the microbial GlcN production in an industrial scale.
Acetylglucosamine ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Gene Knockout Techniques ; Glucosamine ; biosynthesis ; genetics ; Repressor Proteins ; genetics

In order to increase the production and productivity of alkaline polygalacturonate lyase (PGL), we studied the mixed carbon sources feeding strategies during the induction phase by recombinant Pichia pastoris GS 115. Glycerol, sorbitol or lactic acid co-feeding with methanol all enhanced the PGL production. Among all the feeding strategies, the sorbitol co-feeding strategy was most significant. By using this strategy, the PGL activity and productivity reached 1593 U/mL and 16.7 U/(mL-h). Compared to the control, the enhancements of PGL activity and productivity were 84.6% and 45.2% respectively, when we set the sorbitol feeding rate at 3.6 g/(h x L).
Carbon ; metabolism ; Culture Techniques ; Methanol ; metabolism ; Pichia ; enzymology ; genetics ; growth & development ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sorbitol ; metabolism