Cathepsin B from Helicoverpa armigera (HCB) belongs to the group of cysteine proteinases. HCB is proved being involved in the degradation of yolk proteins during embryonic development,which is an acidic preferring enzyme and is resistant to SDS. The expression of the proenzyme may offer a model for investigating the activation of the enzyme. The HCB gene was constructed into pPIC9K and expressed in Pichia pastoris KM71 strain . After induction by methanol, HCB was expressed and secreted into the medium. The molecular weight of the recombinant procathepsin B was determined as about 38 kDa. The expressed product was confirmed to be HCB by immunoblotting assay using specific rabbit anti-HCB polyclonal antibody. The activity of the product was assayed by in situ hydrolysis (gelatin-SDS-PAGE). These results showed that HCB with proteolytic activity was expressed in P. pastoris KM71. This proenzyme can be used for further research on the activation of the proenzyme or industrial production.

Cirrhosis with portal hypertension is a common disease which has a significant impact on the quality of patients' life. Esophagogastric devascularization (EGDV) has been demonstrated to be an effective method to treat portal hypertension, however certain complications are associated with it. The purpose of this study was to evaluate the effectiveness and clinical outcome of the selective EGDV (sEGDV) for the treatment of portal hypertension. The study was conducted prospectively from Jan. 1 2011 to Dec. 31, 2012, and 180 patients were randomized to the sEGDV group (n=90) or the non-sEGDV (n-sEGDV) group (n=90). Patients' demographics, preoperative lab test results and operative details were comparable between the two groups. Postoperative and short-term complications were analyzed in two groups. There was statistically significant difference (P<0.01) in the PVF reduction between the two groups. Post-operative complications showed no statistically significant difference between the two groups in the incidence of bleeding, ascites, acute portal vein thrombosis, fever and hepatic encephalopathy. Mortality between two groups was comparable. The incidence of splenic fossa effusion after the surgery was lower in sEGDV group than in n-sEGDV group. There were no significant differences in the short-term follow-up data such as esophageal varices and portal hypertensive gastropathy (P>0.05). It is suggested that sEGDV is a safe, simple and effective surgical procedure. It has both the advantages of the shunt and devascularization because it preserves body's voluntary diversion. With the advantage of low incidence of postoperative complications, it is an ideal surgical approach for the treatment of portal hypertension.

Adolescent ; Adult ; Ear, Middle ; diagnostic imaging ; Female ; Humans ; Imaging, Three-Dimensional ; Male ; Mastoid ; diagnostic imaging ; Middle Aged ; Tomography, X-Ray Computed ; Young Adult

OBJECTIVETo observe the effect of staged Chinese herbal medicinal therapy combined with chemotherapy in treating patients with non-small-cell lung cancer (NSCLC) of stage III or IV.

METHODSAdopting prospective randomized controlled multi-centered method, the 116 patients enrolled were assigned to the treated group (n = 60) and the control group (n = 56). The control group was treated by chemotherapy alone, while the treated group treated by chemotherapy combined with Chinese herbal medicine, i.e. Kangliu Zengxiao Decoction (KLZX) was administered in the chemotherapy stage, followed with Feiyanning Decoction (FYN) in the stage after ending the chemotherapy. The survival time, Karnofsky score, main clinical symptoms and adverse reactions, etc. were observed.

RESULTSThe median survival time was 15.57 months in the treated group, which was higher than that in the control group (11.17 months, P< 0.01). The improvements in tumor related symptoms such as fatigue, dyspnea, etc. in the treated group were better than those in the control group (P <0.05). In addition, adverse reactions such as leucopenia, digestive reaction (such as nausea and vomiting) in the treated group were less than those in the control group (P <0.05).

CONCLUSIONChinese herbal medicine combined with chemotherapy shows favorable effect in improving quality of life and prolonging survival time on patients with advanced NSCLC.

Aged ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Phytotherapy ; Prospective Studies ; Single-Blind Method ; Treatment Outcome

AIMTo analyze if the response factors of different aminoglycoside antibiotics detected by evaporative light-scattering detector (ELSD) are the same. If they are, then ELSD can be applied to the quality analysis of this class of antibiotics.

METHODSThe response factors of five different aminoglycosides (amikacin, sisomicin, netilmicin, etimicin and vertilmicin) detected by ELSD were determined by using a Diamonsil C18 column (150 mm x 4.6 mm, 5 microns) as analytical column and 0.2 mol.L-1 trifluoroacetic acid-methanol (94:6) as mobile phase at a flow rate of 0.6 mL.min-1, the temperature of the drift tube was set at 110 degrees C, and the flow of carrier gas at 2.80 L.min-1. Detector responses (A) and the amount of injection of each substance (m) were fitted to the logarithmic regression: logA = blogm + loga.

RESULTSThe linear regression equation obtained were amikacin: Y = 1.46X + 5.07, gamma = 0.9997; sisomicin: Y = 1.51X + 5.03, gamma = 0.9997; netilmicin: Y = 1.52X + 4.88, gamma = 1.000; etimicin: Y = 1.46X + 4.85, gamma = 0.9999; vertilmicin: Y = 1.41X + 4.90, gamma = 0.9998. The differences between them were negligible.

CONCLUSIONDifferent aminoglycosides can give the same responses with ELSD detection. So, the HPLC-ELSD methods can be applied to the analysis of impurities, the control of the ratio of multi-components drug and the determination of new substances by using another substance as reference, etc.

Aminoglycosides ; analysis ; Anti-Bacterial Agents ; analysis ; Chromatography, High Pressure Liquid ; methods

Objective: To assess changes of liver function in HIV-positive children with/without HBV/HCV co-infection after 1 year of highly active antiretroviral therapy ( HARRT ) . Methods: Seventy-eight pediatric AIDS patients with HBV/HCV co-infection,19 pediatric AIDS patients with HBV co-infection and 44 pediatric AIDS patients without HBV/HCV co-infection who received HAART at least for 1 year were enrolled .HIV-1 viral load was quantitatively detected using a standardized reverse transcriptase-polymerase chain reaction assay , and blood cells were determined by three-color flow cytometry . Anti-HCV antibody and HBsAg was detected using an enzyme-linked immunosorbent technique , and ALT, AST and TBIL were detected by automatic biochemical analyzer .Results: After 1 year-HAART, the viral load was decreased to the lowest limit of detection in 90 .34% patients ( t=2 .61 , P<0 .01 ) , and CD4 +T cell counts were increased from 170 .187 ±132 .405/μl to 796 .014 ± 158 .491/μl ( t=3 .17 , P<0 .01 ) .The levels of ALT and AST were elevated ( t=2 .02 , P <0 .05 ) , while the ALT and AST levels in patients receiving nevirapine (NVP) based HAART increased from 18.28 ±13.74 U/L and 24.23 ±8.09 U/L to 55.35 ±22.40 U/L and 69.97 ±26.72 U/L, respectively(t=3.80,t=4.11;Ps<0 .01 ) .The increment of ALT and AST in NVP based HAART were significantly higher than that in the efavirenz based HAART (ALT:46.28 ±13.35 U/L vs 37.70 ±15.25 U/L and AST:19.53 ±7.23 U/L vs 1.25 ±0.21 U/L, respectively; t=4.53, t=5 .79;Ps<0 .01 ) , particularly in patients co-infected with HIV/HBV/HCV ( ALT:54 .32 ±22 .85 U/L vs 16 .89 ±14 .42 U/L and AST:41 .71 ±19 .26 U/L vs -3 .44 ±15.59 U/L, respectively; t=3.42, t=2.98, Ps<0.01).Conclusion: HARRT can repress HIV-1 replication effectively , but it also cause the damage of liver function , especially in patients with HBV and/or HCV co-infection.

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.
Gene Products, gag ; genetics ; Gene Products, pol ; genetics ; Human T-lymphotropic virus 1 ; genetics ; isolation & purification ; Humans ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

Objectives To evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube or L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.
Methods The expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.
Results The expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.
Conclusion MyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Chemical and Drug Induced Liver Injury ; blood ; immunology ; prevention & control ; Concanavalin A ; adverse effects ; Cytokines ; blood ; Dexamethasone ; pharmacology ; Disease Models, Animal ; Female ; Hepatocytes ; drug effects ; pathology ; Liver ; drug effects ; immunology ; pathology ; Male ; Mice ; Mice, Inbred ICR ; Peroxidase ; blood ; Protective Agents ; pharmacology ; Random Allocation ; Saponins ; pharmacology ; Triterpenes ; pharmacology

OBJECTIVETo analyzed retrospectively two groups of patients with newly diagnosed multiple myeloma (MM) receiving bortezomib and dexamethasone (VD) regimen and vincristine combined with pirarubicin and dexamethasone and melphalan(VADM) regimen.

METHODSTwenty-four patients were enrolled in a group of VD, receiving bortezomib 1.3mg/m(2) on days 1, 4, 8, 11 and dexamethasone 20mg on days 1-4 intravenously of every 21-day cycle. EBMT Standard was used to evaluate the efficacy and NCI-CTC V3.0 was used to decide the adverse effect. Thirty matched patients with newly diagnosed MM who received VADM were used as control group, receiving vincristine 0.4 mg/d and pirarubicin 9 mgxm(-2)xd(-1) and dexamethasone 20 mg/d and melphalan 12 mg/d on days 1 - 4 intravenously of every 28 day cycle.

RESULTSWith a median follow-up of 10.5 months in VD group, there were 87.5% patients (21/24) responded, including 12 cases (50.0%) of complete remission (CR) or near complete remission (nCR). The total response rate (RR) was 76.7% in VADM group, with no significant difference in VD group (P = 0.483). CR + nCR rate was significantly higher in VD group than in VADM group (10%) (P = 0.001). RR and CR + nCR of light chain patients in VD group were significantly higher than in VADM group (P = 0.025 and 0.040, respectively). The median time to response and to best response were significantly shorter in VD group than in VADM group. In VD group, the RR of 8 patients with renal dysfunction was 87.5%, and that of 16 with normal renal function was 75% (P = 0.631). There was no significant difference in adverse effects between patient with renal dysfunction and normal function (P > 0.05). The main adverse effects in VD group were fatigue (66.7%), diarrhea (58.3%), peripheral neuropathy (54.2%), thrombocytopenia (29.2%), infection (29.2%), fever (25.0%) and constipation (25.0%). Most of the adverse effects were mild (grade 1 - 2) and could be relieved by symptomatic treatments. The most common adverse event in VADM group was neutropenia (83.8%), infection (35.5%), vomiting (35.5%), loss of hair (32.5%) and thrombocytopenia (16.2%).

CONCLUSIONVD has higher CR + nCR rate compared with VADM and can be tolerant in most patients. VD is safe in patients with renal inadequacy.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Boronic Acids ; administration & dosage ; adverse effects ; Bortezomib ; Dexamethasone ; administration & dosage ; adverse effects ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; drug therapy ; Pyrazines ; administration & dosage ; adverse effects ; Retrospective Studies ; Treatment Outcome