In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508 u/mL, which is 81% that of the wild type lipase PEL-GS (627 u/mL) and 55% that of random-mutant PEL-ep8-GS (924 u/mL). The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the Tm of the double-mutant lipase is 41.0 degrees C, 2.3 degrees C higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability.
Electroporation ; Enzyme Stability ; Hot Temperature ; Lipase ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutant Proteins ; metabolism ; Penicillium ; enzymology ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo observe the clinical efficacy difference between segmentation scraping and conventional acupuncture for cervical spondylosis (CS) so as to provide effective treatment method.

METHODSEighty-five cases of cervical type of CS were randomly divided into a scraping group (44 cases) and an acupuncture group (41 cases). The segmentation scraping therapy was used in the scraping group. The scraping group was treated with focusing on scraping the head and joint part of neck and occiput in the upper cervical spine injury, and focusing on scraping the lower section of cervical and shoulder in the lower cervical spine injury, once every seven days, totally for 3 times. In the acupuncture group, Fengchi (GB 20),Wangu (TE 5), Tianzhu (BL 10),Neck-Jiaji (EX-B 2), etc. were selected,once daily,for 15 days. The visual analogue scale (VAS) was used to evaluate the immediate analgesic effect after the first treatment and the clinical efficacy was observed after the end of treatment.

RESULTSAfter the first treatment, the score of VAS was decreased significantly in the scaping group (P < 0.01), but there was no significant difference in the acupuncture group compared with those before treatment (P > 0.05); the score of VAS in the scaping group after the first treatment was lower than that in the acupuncture group (3.66 +/- 0.74 vs 5.43 +/- 0.35, P < 0.01). Compared with before treatment, the scores of VAS were decreased significantly after treatment in two groups (both P < 0.01), but without significant difference between two groups (P > 0.05); the effective rate was 95.5% (42/44) in the scaping group and 87.8% (36/41) in the acupuncture group, the curative effects were similar (P > 0.05).

CONCLUSIONBoth of scraping and acupuncture therapies have good analgesic effect for cervical spondylosis, and overall effects are similar, but the immediate analgesic effect of scraping thrapy is better than that of conventional acupuncture.

Acupuncture Points ; Acupuncture Therapy ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Neck Pain ; therapy ; Spondylosis ; therapy ; Treatment Outcome

OBJECTIVETo analyze the distribution features of systemic vascular resistance (SVR) disorder in healthy subjects in Hebei province.

METHODSSVR and systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), pulse pressure (PP), heart rate (HR), cardiac output (CO), cardiac index (CI), stroke volume (SV), stroke index (SI), left ventricular ejection time (LVET), left cardiac work (LCW) and cardiovascular function were determined with Bioz. com Cardio Dynamics. Total cholesterol (TC), triglyceride (TG) and influencing factor were tested by automated hematology analyzer in 999 healthy subjects living in Hebei province.

RESULTSSVR disorder gradually increased with the ageing in healthy subjects and the prevalences of SVR disorders was higher among females than among males (P < 0.001). SVR was positively correlated with SBP, DBP, MAP, PP, LVET, body mass index (BMI), TC, and TG, and was inversely associated with HR, CO, CI, SV, SI, LCW, and arterial compliance (AC). CO and MAP were independent influencing factors with SVR.

CONCLUSIONSVR disorder is associated with age, sex, and blood pressure in populations in Hebei province and may be a marker of vascular injury.

Adolescent ; Adult ; Age Factors ; Aged ; Blood Pressure ; Body Mass Index ; Cardiac Output ; Female ; Hemodynamics ; physiology ; Humans ; Male ; Middle Aged ; Risk Factors ; Sex Factors ; Vascular Capacitance ; Vascular Diseases ; physiopathology ; Vascular Resistance

OBJECTIVETo assess the efficacy and safety of recombinant human thrombopoietin (rhTPO) on chemotherapy-induced thrombocytopenia in patients with solid tumor.

METHODSIn this randomized crossover self-controlled multi-center clinical trial, 154 patients with solid tumor were randomly divided into two groups (group A 77 cases and group B 77 cases). All patients were given the same two cycles of chemotherapy. In group A, the first cycle was treated cycle, in which patients were given rhTPO, while the second cycle was non-treated cycle as a control. In group B, the first cycle was non-treated cycle as a control, while the second cycle was treated cycle. RhTPO 1.0 microg/(kg x d) was administered subcutaneously 6-24 hours after chemotherapy for the longest 14 days. Laboratory tests included complete blood counts, urinalysis, serum chemistry, coagulant test, chest radiography, and electrocardiogram. Serum samples were screened for anti-rhTPO antibodies.

RESULTSIn both group A and group B, platelet decrease and duration had no significant difference between the treated cycle and non-treated cycle. Platelet count was higher in the treated cycle, than in the non-treated cycle: [minimal mean platelet count (64.4 +/- 45.4) x 10(9) cells/L and (52.4 +/- 30.9) x 10(9) cells/L (P=0.000), maximal mean platelet count (263.9 +/- 142.5) x 10(9) cells/L and (148.9 +/- 67.7) x 10(9) cells/L (P=0.000)]. Duration of thrombocytopenia was shorter in the treated cycle than in the non-treated cycle [days with platelet count < 50 x 10(9) cells/L, (2.5 +/- 3.9) and (3.7 +/- 5.7) (P=0.04); days with platelet count recovered > or = 75 x 10(9) cells/L, (10.3 +/- 8.7) and (14.0 +/- 8.9) (P=0.000), and days with platelet count recovered > or = 100 x 10(9) cells/L, (15.9 +/- 10.5) and (21.1 +/- 9.5) (P=0.000)]. The need for platelet transfusion was not significantly reduced in treated cycle. The effects of rhTPO on WBC, Hb, hepatic function, renal function, and coagulant function were not found. Transient low-titer non-neutralizing antibody was developed in one patient. Therapy with rhTPO was tolerated by all patients. Mild side effects were observed in individual patients, including fever, dizziness, and chill. Conclusion Administration of rhTPO after chemotherapy can significantly reduce the degree and duration of thrombocytopenia and promote platelet recovery. Therapy with rhTPO seems to be safe.

Adolescent ; Adult ; Aged ; Antineoplastic Agents ; adverse effects ; Cross-Over Studies ; Female ; Humans ; Lung Neoplasms ; blood ; drug therapy ; Male ; Middle Aged ; Neoplasms ; blood ; drug therapy ; Platelet Count ; Recombinant Proteins ; administration & dosage ; therapeutic use ; Thrombocytopenia ; chemically induced ; drug therapy ; prevention & control ; Thrombopoietin ; therapeutic use

Objective To evaluate diagnosis and preoperative management of cases with interrupted aortic arch(IAA)in infancy.Methods Fifty-three infants who were admitted to our hospital from Jan.2001 to Nov.2007 were involved.Clinical data,findings of echocardiogram(Echo),spiral CT,MRI,angiocardiography,preoperative management,surgical repair and postoperative outcome were analyzed retrospectively.Results There were 38 boys and 15 girls,weighted 2.18-10.0(4.32?1.60)kg,aged 1 day to 12(3.05?3.53)months,of which 50.94% were neonates.Symptoms at presentation were 90.57% with tachypnea and 83.02% with difficulty in feeding.Eighty three point zero two percentage cases had different degree of congestive heart failure,37.74% of which were in grade Ⅲ heart function.All cases had weakened femoral pulse.All cases were performed Echo,38 cases of them diagnosed as IAA,6 cases as IAA or severe coarctation of aorta(CoA);they were diagnosed as IAA by CT,and 9 as severe CoA who were diagnosed as IAA via CT or operation.Thirty-three cases were performed CT,of which 15 underwent surgical repair,cardiovascular abnormalities revealed by CT were the same as those in surgical findings.Three cases were taken MRI,and 7 cases were performed angiocardiography.According to the results of Echo,CT,MRI,angiocardiography and surgical findings,35 cases were type A,15 cases were type B and 3 cases were type C.Preoperative treatment included maintaining patent ductus areriosis,management of heart failure and supportive treatment.After proper preoperative management of medication,most cases with congestive heart failure were improved.Twenty-six cases underwent surgical repair,16 survived,10 died du-ring perioperative stage.Main cause of death was severe low cardiac output.Conclusions Value of Echo in diagnosis of IAA is limi-ted.Combination of Echo with CT or MRI is a convenient and safe way to diagnose IAA,it can replace the traditional method of Echo combined with angiocardiography.Proper preoperative management is helpful to patients with IAA to pass to surgical repair,and makes for successful operation.

AIMTo identify the main metabolites of stachydrine in rat.

METHODSThe ionization, cleavage and chromatographic characteristics of stachydrine were studied by using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometry (HPLC-ESI/MS) for the first time. These characteristics of stachydrine were used as the basis for the analyses of metabolites in rat urine. The 0 - 24 h urine samples of rats after ig 25 mg x kg(-1) stachydrine were collected and purified by using C10 solid-phase extraction cartridge, and then analyzed by HPLC-ESI/MS to identify stachydrine and its metabolites.

RESULTSThe parent drug (stachydrine), 6 phase I metabolites (N-demethyl, dehydrogenation, ring-oxidation) and 2 phase II metabolites (glycine conjugates of 2 ring-oxidation products) were identified existing in rat urine.

CONCLUSIONThe presented method was proved to be sensitive, rapid, high selective and specific for the identification of stachydrine and its metabolites in rat urine.

Animals ; Chromatography, High Pressure Liquid ; methods ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Proline ; analogs & derivatives ; isolation & purification ; metabolism ; urine ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Stachys ; chemistry

AIMTo establish a rapid and sensitive LC-MSn method for the identification of trigonelline and its main metabolites in rat urine.

METHODSAfter optimizing the detection conditions of LC-MSn chromatography and mass spectrometry using trigonelline, its ionization and cleavage in ESI-MS and ESI-MSn modes were summarized, then serving as the basis for the metabolite analysis of trigonelline in rat urine. The 0-48 h urine samples of rats were collected after iv 8 mg x kg(-1) trigonelline, then, the samples were purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by LC-MSn.

RESULTSThe structures of trigonelline metabolites were elucidated according to the changes of the molecular weights of the metabolites (deltaM) and their cleavage pattern in ESI-ITMSn. As a result, two phase I metabolites and the parent drug were identified existing in rat urine, and two phase II metabolites were identified.

CONCLUSIONThe LC-MSn method is rapid and high sensitive and specific, it is suitable for the identification of trigonelline and its metabolites in rat urine.

Alkaloids ; chemistry ; isolation & purification ; metabolism ; Animals ; Chromatography, High Pressure Liquid ; methods ; Hypoglycemic Agents ; chemistry ; isolation & purification ; metabolism ; Male ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Trigonella ; chemistry

OBJECTIVETo investigate the clinical significance of micrometastasis detection in sentinel lymph nodes (SLN) from patients with early cervical carcinoma.

METHODSThirty patients with early cervical carcinoma were studied to identify SLN intraoperatively using methylene blue. One lymph node was removed randomly from palpable SLN and other pelvic lymph nodes (nSLN) in each patient, so 268 lymph nodes were collected and cut into two halves, one half of the lymph node was used to analyze the expression of cytokeratin 19 (CK19) mRNA by real-time fluorescence quantitative polymerase chain reaction to determine the presence of micrometastasis, the other half was examined by routine histology with HE staining.

RESULTS67 SLNs were detected in 28 cases (93.3%). Pelvic lymph nodes of 6 cases were confirmed pathological metastasis. The sensitivity of SLN detection was 66.7%, the accuracy rate was 96.4%, and the false negative rate was 16.7%. Among 268 lymph nodes (including 9 lymph nodes with pathological metastasis) detected by real-time fluorescence quantitative polymerase chain reaction, 68 lymph nodes were pathological negative but had micrometastasis, accounting for 26.3% (68/259) in pathologically negative lymph nodes. Among 24 patients with pathological negative lymph nodes, 16 cases had micrometastasis, accounting for 66.7% in those patients. Among 16 patients with micrometastasis, SLN of 3 cases were negative, but nSLN were micrometastasis, so the SLN false-negative rate rose to 18.2%. There were no significant relationships between pelvic lymph nodes micrometastasis and perivascular space involvement, deep stromal invasion and tumor grade (all P > 0.05). The micrometastasis rate of nSLN in patients with SLN micrometastasis was 100%, significantly higher than that in the patients with SLN non-micrometastasis (27.3%, P < 0.01).

CONCLUSIONSReal-time fluorescence quantitative polymerase chain reaction is a sensitive method to detect SLN micrometastasis. SLN micrometastasis may be an effective complement to SLN pathology to predict nSLN metastasis. Pelvic lymph nodes micrometastases have no significant relationship with pathological risk factors in cervical cancer and prognosis of patients.

Early Detection of Cancer ; methods ; Female ; Humans ; Lymphatic Metastasis ; diagnosis ; Neoplasm Micrometastasis ; diagnosis ; Neoplasm Staging ; Prognosis ; Sentinel Lymph Node Biopsy ; Uterine Cervical Neoplasms ; diagnosis

BACKGROUNDNuclear factor-kappa B (NF-kappaB) is elevated in regulating transcription of many cytokines and inflammatory mediators. The purpose of this study was to investigate the activation and the significance NF-kappaB in lipopolysaccharide (LPS) induced keratitis.

METHODSLPS induced keratitis model was based on Wistar rats. At 0.5, 1, 3, 6, 12, 24 or 72 hours after LPS treatment, the rat corneas were observed with a slit lamp microscope, then the rats were sacrificed and their corneas were excised for routine histological analysis. The expression of NF-kappaB was detected with immunohistochemical staining. The change of tumour necrosis factors-alpha (TNF-alpha) mRNA expression was identified by reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSHistological findings demonstrated that LPS treated corneas showed significant changes in corneal structure. Corneal edema, pronounced inflammatory cells infiltration and inordinate collagen fibres were observed. Immunohistochemical results showed that the expression of NF-kappaB and its activation obviously increased after LPS treatment compared with the normal group and control group. Positive cells could be observed at 0.5 hour and peak expression of NF-kappaB was observed between 3 hours and 12 hours after infection, but returned to or approached normal level by 72 hours. RT-PCR showed that the level of TNF-alpha mRNA began to increase 0.5 hour after LPS treatment, peaked at 6 hours and then subsided by 72 hours. NF-kappaB had a positive correlation with the expression of TNF-alpha mRNA (r = 0.964, P < 0.01), both NF-kappaB and TNF-alpha had a strong positive correlation with the degree of inflammatory response in LPS treated corneas (r = 0.929, P < 0.01; r = 0.587, P < 0.05, respectively).

CONCLUSIONSThe activation of NF-kappaB was increased in LPS treated corneas and was elevated in LPS induced keratitis by promoting overexpression of TNF-alpha mRNA. NF-kappaB may play an important role in the pathogenesis of LPS-associated keratitis in rats.

Animals ; Aqueous Humor ; cytology ; Epithelium, Corneal ; physiology ; Immunohistochemistry ; Keratitis ; chemically induced ; pathology ; Lipopolysaccharides ; toxicity ; NF-kappa B ; analysis ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; genetics ; physiology

OBJECTIVETo study the relationship of inspiratory capacity (IC) with body mass index (BMI), fat free mass index (FFMI), and fat mass index (FMI) in adults with normal lung function.

METHODSBased on a survey on the Chinese physiological constants conducted in Heilongjiang during 2008, we obtained the data of 2,050 adults (921 men and 1,129 women aged 19-81 years) with normal lung function and grouped them according to the standard of classification of body weight recommended by Working Group on Obesity of China. Statistical analysis was performed using Pearson correlation analysis, one-way analysis of variance,and multi-factor regression analysis.

RESULTSAfter the influences of age and gender were adjusted, IC was positively correlated with BMI, FFMI, and FMI in adults aged 19-81 years (all P= 0.000) (the correlation coefficients were 0.320, 0.303, and 0.204, respectively). The means of IC, FFMI, and FMI in obesity group were significantly higher than those in overweight group normal weight group, and underweight group, were significantly higher in overweight group than in normal weight group and underweight group, were significantly higher in normal weight group than in underweight group (all P = 0.000). Multiple stepwise regression analysis showed that factors including age, gender, height, FFMI, and FMI influenced IC, and while height, FFMI, and FMI were positively correlated with IC, gender and age were negatively correlated with IC.

CONCLUSIONSBMI, FFMI, and FMI are positively correlated with IC in adults with normal lung function. The elevation of IC derived from BMI may attribute to FFMI and FMI, and FFMI has a higher influence on IC than FMI does.

Adult ; Aged ; Aged, 80 and over ; Analysis of Variance ; Body Composition ; physiology ; Body Mass Index ; China ; Female ; Humans ; Inspiratory Capacity ; Male ; Middle Aged ; Regression Analysis ; Young Adult