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 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

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

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.

OBJECTIVETo investigate the lung function of the normal subjects living in Hebei province and its correlative factors such as living circumstance, age, height, and body weight.

METHODSThe lung volumes and breath capacities of 1,587 normal subjects were tested by portable spirometers (Scope Rotry) from August to October in 2002. The influences of living circumstance, age, gender, height, and body weight on lung functions were observed and analyzed.

RESULTSNo significant difference was found between urban and rural areas in all indexes (P > 0.05); however, significant difference existed between male and female subjects (P = 0.000). The change trends of lung function in male and female subjects were similar. Growth spurt appeared at the age of 12-16 years in male subjects and 12-14 years in female subjects. Vital capacity (VC), forced vital capacity (FVC), and forced expiratory volume in one second (FEV1) reached their peaks at the age of 26-34 years and then decreased with age. Peak expiratory flow (PEF), 25% forced expiratory flow (FEF50%), and 75% forced expiratory flow (FEF75%) appeared at the age of 18 and then went down with age. Both height and weight had a correlation with all the indexes of lung functions, although the influence of height is stronger than weight.

CONCLUSIONSAll the indexes of lung function have correlations with age, height, and weight. Lung function changes with aging, therefore different expected values shall be available for the adolescence, young adults, and middle-aged and old people. This study provides reference values of lung function for normal population.

Adolescent ; Adult ; Aged ; Child ; Female ; Forced Expiratory Flow Rates ; Forced Expiratory Volume ; Humans ; Lung ; physiology ; Male ; Middle Aged ; Peak Expiratory Flow Rate ; Reference Values ; Respiratory Function Tests ; Rural Health ; Sampling Studies ; Urban Health ; Vital Capacity

AIMTo estabilish a rapid and sensitive LC-ESI-ITMSn method for the identification of ephedrine and its main metabolites in rat urine.

METHODSAfter optimizing the detection condition of LC-ESI-ITMSn chromatography and mass spectrometry by using a standard ephedrine, the ionization and cleavage rules of ephedrine in ESI-MS and ESI-MSn modes were summarized, and then serving as the basis for the metabolite analysis of ephedrine in rat urine. Rat urine samples of 0-48 h were collected after ig 10 mg x kg(-1) ephedrine, then the samples were purified through C18 solid-phase extraction cartridge. The purified samples were analyzed by LC-ESI-ITMSn.

RESULTSThe structures of ephedrine 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, three phase I metabolites and the parent drug ephedrine were identified existing in rat urine, but no phase II metabolites were found.

CONCLUSIONThe LC-ESI-ITMSn method is rapid and highly sensitive and sepecific, it is suitable for the identification of ephedrine and its metabolites in rat urine.

Animals ; Chromatography, High Pressure Liquid ; methods ; Ephedrine ; chemistry ; metabolism ; urine ; Male ; Molecular Weight ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods

BACKGROUNDMycoplasma pneumoniae (M. pneumoniae) is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction (PCR) with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally.

METHODSIn this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results.

RESULTSAmong 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment.

CONCLUSIONSThe drug-resistant M. pneumoniae is very common in Beijing, China. Our modified allele-specific PCR analysis can identify erythromycin resistant mutations more rapidly from specimens than any other method currently available. Erythromycin is still effective for treating patients infected with the mutation negative M. pneumoniae, but this treatment fails to work on mutant organisms. This method can facilitate clinicians in selecting appropriate therapy within short timescales.

Alleles ; Anti-Bacterial Agents ; pharmacology ; China ; Drug Resistance, Bacterial ; genetics ; Erythromycin ; pharmacology ; Mycoplasma pneumoniae ; drug effects ; genetics ; Polymerase Chain Reaction ; methods

OBJECTIVETo investigate the feasibility of promoting nerve regeneration by using microcapsulated NGF-expressing cells transplantation.

METHODSThe plasmid pcDNA3. 1 + /NGF, containing rat NGF gene, was transfected into the NIH 3T3 cell by using FuGENE6 transfection reagent. The genetically modified cells expressing NGF gene were enclosed within the alginate-polylysine-alginate (APA) microcapsules and then cultured in vitro. The growth and NGF secretion of the cells were measured periodically. At the same time, these microcapsulated NGF-expressing cells were transplanted into the injured sciatic nerve. The regeneration and functional recovery of the nerve were evaluated in 4 weeks, 8 weeks and 12 weeks after the operation.

RESULTSThe microcapsulated cells had survived and secreted the NGF in three months in vitro. In the group with microcapsulated NGF-expressing cells, the number of the regenerated axons was in large and the nerve fibers were arranged regularly. Compared to other groups, there was less scar , edema and monocytes found at the stoma in the goup. The moter nerve conductive velocity, nerve muscle-action potential and SFI were improved.

CONCLUSIONSThe microcapsulated NGF-expressing cells could significantly enhance the nerve regeneration and reduce inflammatory response of xenograft.

Animals ; Cell Transplantation ; Female ; Male ; Mice ; NIH 3T3 Cells ; Nerve Growth Factor ; biosynthesis ; genetics ; Nerve Regeneration ; Rats ; Rats, Sprague-Dawley ; Transfection

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