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 compare the short-time effect of 3 different regimens of neoadjuvant chemotherapy (NACT) in patients with FIGO stage Ⅰb2-Ⅱb cervical squamous carcinoma.Methods A total of 50 patients with FIGO stage Ⅰb2-Ⅱb cervical squamous carcinoma were divided into 3 groups:systemic chemotherapy group (n=13) ,trans-arterial chemotherapy group (TAC,n=19) ,trans-arterial chemoembolization group (TACE,n=18) .After 1-3 periods of NACT,all patients received surgical operation.Tumor response and reduction ratio after NACT,side effects,hemorrhage volume in surgery and bad prognostic factors (including intraluminal tumor thrombi,pelvic lymph node metastasis,parametrial involvernent,positive surgical margin,ovary metastasis) of operation sample were statistically analyzed.Results Tumor response,reduction ratio and hemorrhage volume in surgery were significantly better in TAC group and TACE group than those in systemic chemotherapy group (P＜0.05) ,but no significant difference was found between TAC group and TACE group (P＞0.05) .The incidence rate of intraluminal tumor thrombi and lymph node metastasis was lower in TACE group than in systemic chemotherapy group and TAC group,but there was no statistical difference.No difference of parametrial involvement,positive surgical margin,ovary metastasis and side effects was found among 3 groups.Conclusion For stage Ⅰb2-Ⅱb cervical squamous carcinoma,preoperative TAC and TACE have more advantages than systemic chemotherapy.In comparison with TAC,TACE is expected to reduce the incidence rate of bad prognostic factors such as intraluminal tumor thrombi and pelvic lymph node metastasis.

OBJECTIVETo establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.

METHODSThe femoral heads from the same SD rats of two-month old were divided into control group and experimental group respectively. They were cultured with DMEM medium plus 10% fetal bovine serum or DMEM medium plus 10% fetal bovine serum plus 50 ng/ml IL-1β for three days. Femoral heads were fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and cut into slices. Specimens were stained with Toluidine blue and Safranine O-Fast Green FCF. The protein expression levels of type II collagen, MMP13, Sox9 and ADAMTS5 were analyzed by immunofluorescence.

RESULTSBoth the Toluidine blue and Safranine O staining were pale in the margin of femoral heads which were stimulated with IL-1β for three days compared to that in control group. The Fast Green FCF staining was positive at the edge of the femoral head in experimental group, which indicated that cartilage became degenerated. The expression levels of both type H collagen and Sox9 were decreased significantly while the expression levels of MMP13 and ADAMTS5 were increased in experimental group.

CONCLUSIONThe model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.

Animals ; Cartilage Diseases ; genetics ; metabolism ; Collagen Type II ; genetics ; metabolism ; Disease Models, Animal ; Femur Head ; metabolism ; Humans ; In Vitro Techniques ; Interleukin-1beta ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; SOX9 Transcription Factor ; genetics ; metabolism

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

OBJECTIVETo investigate the application value of high-frequency color Doppler ultrasonography in the diagnosis of polyorchidism.

METHODSWe retrospectively studied 6 cases of polyorchidism diagnosed by ultrasound, 5 of which were pathologically confirmed, and analyzed the sonographic and clinical findings.

RESULTSAll the 6 cases were triorchidism, 4 located in the scrotum, and 2 in the same groin with indirect hernia. The supernumerary testis was linked to the epididymis in 3 cases, 1 accompanied with repeated epididymis and vas deferens, and 2 connected to no vas deferens and epididymis. Color Doppler ultrasonography revealed blood flow signals in 4 cases, but not in the other 2.

CONCLUSIONPolyorchidism has typical sonographic appearances, and high-frequency color Doppler ultrasonography plays an important role in its diagnosis.

Adolescent ; Adult ; Child ; Child, Preschool ; Humans ; Male ; Retrospective Studies ; Testicular Diseases ; diagnostic imaging ; Testis ; abnormalities ; diagnostic imaging ; Ultrasonography, Doppler, Color

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

OBJECTIVETo observe the efficacy of Chinese herbs for supplementing qi and activating blood circulation (CHSQABC) on patients with acute coronary syndrome (ACS) and type 2 diabetes mellitus (DM) after successful percutaneous coronary intervention (PCI).

METHODSIn this ChiCTR-TRC-00000021, a total of 281 ACS patients complicated with type 2 DM after successful PCI were randomly assigned to the Western medicine treatment group (the control group, treated by routine Western medicine treatment) and the combined treatment group (the treatment group, treated by CHSQABC + routine Western medicine treatment). Patients in the combined treatment group took Xinyue Capsule (2 pills each time, 3 times per day) and Compound Chuanxiong Capsule (2 pills each time, 3 times per day for half a year and 1-year follow-ups). Primary endpoints covered incidence of death, nonfatal myocardial infarction (MI), ischemia-driven revascularization, and secondary endpoints included stroke, heart failure, and rehospitalization for ACS. At the same time scores for blood stasis syndrome (BSS) and the incidence of angina pectoris were evaluated before treatment, at month 1, 3, 6, 9, and 12 after treatment.

RESULTSThe incidence of ischemia-driven revascularization was obviously less in the treatment group than in the control group (P < 0.05). No patient had nonfatal MI in the treatment group, while 5 patients in the control group had it. The incidence of non-fatal MI showed an obvious lowering tendency in the treatment group, but with no statistical difference when compared with that in the control group (P > 0.05). Four patients readmitted to hospital in the treatment group, while 12 patients readmitted. There existed obvious tendency in the treatment group, but with no statistical difference when compared with that in the control group (P > 0.05). The incidence of angina was significantly lower in the treatment group at month 6, 9, and 12 than that at month 1 , but it was lower in the control group at 9 months (P < 0.05). The incidence of angina was 15. 4% in the treatment group, obviously lower than that in the control group (26.2%, P < 0.05). Compared with before treatment, scores for BSS were obviously lowered in the treatment group at 1, 3, 6, 9, and 12 months of treatment and in the control group at 3, 6, 9, and 12 months of treatment (P < 0.05). It was obviously lower in the treatment group than in the control group at 3, 6, 9, and 12 months of treatment (P < 0.01).

CONCLUSIONAdministration of CHSQABC combined routine Western medicine treatment could reduce the event of revascularization and post-PCI recurrent angina, and improve scores for BSS of ACS patients complicated with DM after PCI.

Acute Coronary Syndrome ; complications ; surgery ; therapy ; Angina Pectoris ; Combined Modality Therapy ; Diabetes Mellitus, Type 2 ; complications ; therapy ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Incidence ; Medicine, Chinese Traditional ; Myocardial Infarction ; Percutaneous Coronary Intervention ; Qi

Alkaline amylase is one of alkaline enzymes with optimum pH in the alkaline range, and it could keep stability and efficiently hydrolyze starch under alkaline conditions. Alkaline amylase finds wide applications in textile, detergent, pharmaceutical, food and other fields. Alkaline amylases could be produced by alkaliphilic microorganisms. In this work, the advances of alkaline amylase production and applications were reviewed.
Amylases ; biosynthesis ; chemistry ; Enzyme Stability ; Hydrogen-Ion Concentration

OBJECTIVETo study the influence of Xinqin tablets on guinea-pig nasal hypersensitivity.

METHOD2,4-Toluene Diisocyanate (TDI) was selected as antigen and used in nose to establish guinea-pig allergic rhinitis. The effects of Xinqin tablets on symptoms of nasal hypersensitivity in guinea-pigs, histamine content of nasal mucosa and activity of nitric oxide synthase (NOS) were examined.

RESULTXinqin tablets could significantly relieve the pathological symptoms of nasal hypersensitivity in guinea-pig, reduce histamine content of nasal mucosa and inhibit the activity of nitric oxide synthase.

CONCLUSIONXinqin tablets have significant effect on nasal hypersensitivity, and prevent the occurrence of allergic rhinitis.

Animals ; Asarum ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Guinea Pigs ; Histamine ; metabolism ; Male ; Nasal Mucosa ; drug effects ; metabolism ; pathology ; Nitric Oxide Synthase ; metabolism ; Phytotherapy ; Plants, Medicinal ; chemistry ; Rhinitis, Allergic, Perennial ; chemically induced ; metabolism ; pathology ; Scutellaria ; chemistry ; Toluene 2,4-Diisocyanate