Differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The results showed that well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733+/-0.101 mm in IEF direction, and 0.925+/-0.207 mm in SDS-PAGE direction. For tumor tissue, a total of 1241+/-88 spots were detected, 987+/-65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190+/-72 spots were detected, and 875+/-48 spots were matched with an average matching rate of 73.5%. A total of 864+/-34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduction. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.
Amino Acid Sequence ; Bronchi ; pathology ; Carcinoma, Squamous Cell ; genetics ; pathology ; Databases as Topic ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis, Polyacrylamide Gel ; Epithelial Cells ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Image Processing, Computer-Assisted ; Isoelectric Focusing ; Lung Neoplasms ; genetics ; pathology ; Molecular Sequence Data ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

OBJECTIVETo apply pulse magnetic acupuncture at scalp acupoints to treat acute cerebral infarction and to explore the mechanism.

METHODSA pulse magnetic acupuncture group, a routine acupuncture group and a static magnetic acupuncture group were set up, 30 cases in each group. Their clinical therapeutic effects were observed.

RESULTSThe cured-markedly effective rate was 80.0% in the pulse magnetic acupuncture group and 70.3% in the routine acupuncture group with no significant difference between the two groups (P>0.05), which were significant difference with 36.6% in the static magnetic acupuncture group (P<0.01).

CONCLUSIONPulse magnetic acupuncture and routine acupuncture at scalp acupoints have same therapeutic effect on acute cerebral infarction, which is superior to that of static magnetic acupuncture.

Acupuncture Points ; Acupuncture Therapy ; Cerebral Infarction ; therapy ; Humans ; Scalp ; Stroke ; therapy

OBJECTIVETo analyze the forces of rotational wall vessel (RWV) bioreactor on small tissue pieces or microcarrier particles and to determine the tracks of microcarrier particles in RWV bioreactor.

METHODSThe motion of the microcarrier in the rotating wall vessel (RWV) bioreactor with both the inner and outer cylinders rotating was modeled by numerical simulation.

RESULTSThe continuous trajectory of microcarrier particles, including the possible collision with the wall was obtained. An expression between the minimum rotational speed difference of the inner and outer cylinders and the microcarrier particle or aggregate radius could avoid collisions with either wall. The range of microcarrier radius or tissue size, which could be safely cultured in the RWV bioreactor, in terms of shear stress level, was determined.

CONCLUSIONThe model works well in describing the trajectory of a heavier microcarrier particle in rotating wall vessel.

Bioreactors ; Computer Simulation ; Microspheres ; Motion ; Porosity ; Rheology ; Rotation ; Stress, Mechanical ; Tissue Engineering ; methods

The purpose of this article is to express the UDP-glucose 4-epimerase from Acinetobacter baumannii AB0057, characterize its enzymatic properties and analyze its structure and function.The epimerase gene was constructed into pET-28a expression vector and heterologously expressed in BL21(DE3).Enzyme activity was assayed using high performance liquid chromatography.The structure and key residues were analyzed by phylogenetic analysis, sequence alignment, homology modeling and molecular docking.Results indicated that the recombinant enzyme Gne1 was expressed at a molecular weight of 38.9 kD, with an optimum temperature of 44 °C and an optimum pH of 6.0 .Michaelis-Menten parameters KM and kcat were (1.227 ± 0.082 4) mmol/L and (82.64 ± 3.562) × 10-3?min-1, respectively.This enzyme belongs to NADB_Rossmann superfamily and UDP_G4E_1_SDR_e subgroup with typical GXGXXG and YXXXK sequence motifs.The N-terminal structural domain bound to NAD, while the C-terminal structural domain bound to substrate, and the catalytic key sites were S125 and Y150.The current work verified the epimerase activity of Gne1, explained its sequence and structural features, revealed its binding mode with substrates and cofactors, and analyzed the key residues, which provides a basis for protein engineering to improve the epimerase activity and then use biological enzymatic method to synthesize rare functional sugars.

OBJECTIVETo analyze the association between hypertension and tobacco exposure Luoping county of Yunnan province and estimate the direct cost attributable to hypertension .

METHODSUsing Probability proportional to size (PPS) sampling method, 5000 rural residents aged over 18 years were selected from 12 townships in Luoping county, Yunnan province in April 2011, from which 4611 subjects completed the survey. Self-designed questionnaires were used to collect general information, smoking status, costs for outpatient consultation, inpatient, treatment, medication, travel, accommodation and extra-nutrition caused by hypertension as well as the information of health-related behavior. Their height, weight, waist circumference, hip circumference and blood pressure were measured. The direct economic burden of hypertension was calculated. Chi-square (χ(2)) test was used to compare gender differences of hypertension prevalence, smoking and passive smoking. And t test was used to compare the differences of direct economic burden of hypertension among different gender, smoking and passive smoking status. Multivariable logistic regression model was used to analyze the influence factors of hypertension.

RESULTSAmong the 4611 subjects, the age was (46.90 ± 16.74) years old. Male accounted for 49.8% (2294/4611) and female 50.2% (2317/4611) . The smoking rate was higher in males (75.7% (1736/2294)) than in females(1.6% (38/2317)) (χ(2) = 2669.21, P < 0.01). The passive smoking rate was lower in males (10.0% (230/2294)) than in females (46.2% (1070/2317)) (χ(2) = 744.27, P < 0.05). Non-tobacco exposure rate in males (14.3% (328/1537)) was lower than in females (52.2% (1209/1537)) (χ(2) = 744.37, P < 0.05) . The risk of hypertension in smokers and passive smokers were higher than those without tobacco exposure, OR (95%CI) was 1.41 (1.15-1.71) (P < 0.05) and 1.31 (1.07-1.63) (P < 0.05) respectively. The per capita direct cost of hypertension was (3444.09 ± 3067.83) Yuan. Of this, tobacco exposure (4552.46 ± 3189.05) Yuan was higher than non-tobacco exposure (1907.71 ± 1383.94) Yuan (t = -3.81, P < 0.05) . Moreover, smokers were (6951.71 ± 3422.87) Yuan higher than passive smokers (3128.09 ± 2083.17) Yuan (t = 3.19, P < 0.05) and males (5827.39 ± 3240.50) Yuan were higher than females (2633.03 ± 2569.01) Yuan (t = 3.22, P < 0.05) . The total direct costs of hypertension attributable to smoking and SHS was 41 million and 38 million, respectively.

CONCLUSIONBoth smoking and SHS had significant impact on prevalence and economic burden of hypertension in Luoping county. Implementing effective strategies to control tobacco exposure is useful to reduce the economic burden of hypertension in the study region.

Adolescent ; Adult ; Aged ; China ; epidemiology ; Cost of Illness ; Female ; Humans ; Hypertension ; economics ; epidemiology ; Male ; Middle Aged ; Rural Population ; Tobacco Smoke Pollution ; economics ; statistics & numerical data ; Young Adult

OBJECTIVETo study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB).

METHODSThe bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation.

RESULTSThe results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones.

CONCLUSIONSThe developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D) interactions among cells and is suitable for osteopath expansion in vitro.

Animals ; Bioreactors ; Cell Culture Techniques ; instrumentation ; Cell Enlargement ; Culture Media ; Glucose ; metabolism ; Hydrogen-Ion Concentration ; Lactic Acid ; metabolism ; Osmolar Concentration ; Osteoblasts ; cytology ; metabolism ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the protective effect of L-carnitine (LC) combined with sildenafil on the reproductive endocrine function of male rats with diabetes mellitus (DM).

METHODSA total of 40 male SD rats were randomly divided into five groups, group A taken as normal controls, and groups B, C, D and E made into DM models by injection of streptozotocin at 65 mg/kg. Then the rats in groups A and B were treated with normal saline, C with sildenafil at 5 mg per kg per d, D with LC at 300 mg per kg per d, and E with sildenafil at 5 mg per kg per d plus LC at 300 mg per kg per d, all via gastric gavage for 6 weeks, followed by determination of the levels of testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the serum of the rats.

RESULTSAfter 6 weeks of treatment, the T, FSH and LH levels were (25.25 +/- 2.67) nmol/L, (5.78 +/- 0.61) IU/L and (625.21 +/- 43.45) ng/L in group A, (9.63 +/- 1.71) nmol/L, (1.98 +/- 0.42) IU/L and (479.89 +/- 27.62) ng/L in group B, (18.98 +/- 3.07) nmol/L, (5.08 +/- 0.33) IU/L and (586.57 +/- 31.72) ng/L in group C, (16.18 +/- 2.65) nmol/L, (4.63 +/- 0.30) IU/L and (540.78 +/- 25.52) ng/L in group D, and (23.65 +/- 2.66) nmol/L, (5.59 +/- 0.48) IU/L and (621.53 +/- 36. 40) ng/L in group E. The three parameters were significantly lower in B than in the other four groups (P < 0.01), and so were they in C and D than in A and E (P < 0.05), but showed no significant differences either between C and D (P > 0. 05) or between A and E (P > 0.05).

CONCLUSIONSix-week medication of either sildenafil or LC alone could increase the levels of T, FSH and LH in the serum of DM rats, but the combination of the two had an even more obvious increasing effect, which indicates a still better protective effect on the reproductive endocrine function of diabetic male rats.

Animals ; Carnitine ; adverse effects ; therapeutic use ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Drug Therapy, Combination ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Piperazines ; administration & dosage ; therapeutic use ; Purines ; administration & dosage ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Sildenafil Citrate ; Sulfones ; administration & dosage ; therapeutic use ; Testosterone ; blood

For constructing pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6, the ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified by RT-PCR, and transiently transfected into 293T cells by calciumphosphate co-precipitation. After 48 h, 293T cells were collected and surveyed by flow cytometry examination. The result indicated that the expression level of the E protein that mediated by the M protein was higher than that of the E protein expressed independently. Then pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6 were respectivly co-transfected into 293T cells with pHIT60 (include MuLV structural genes,namely gag and pol) and pHIT111 (retroviral genome, containing LacZ as a reporter). The supernatants were harvested 48 h post-transfection,and the analysis of the characteristic of the pseudotyping virions was performed by Western blot and infection test. The result indicated that the E proteins were expressed on the virions, and incorporated into the retroviral virions. Infection test were performed on Marc-145 and PAM, all the cells infected were Lac Z positive. These results indicated the pseudotype virions of MuLV-E and MuLV-E/M were infectious, and higher infectivity was achieved by MuLV-E/M.
Flow Cytometry ; Leukemia Virus, Murine ; genetics ; Plasmids ; Porcine respiratory and reproductive syndrome virus ; genetics ; Viral Envelope Proteins ; genetics ; physiology ; Viral Matrix Proteins ; genetics ; physiology ; Virion ; genetics

OBJECTIVESTo investigate the efficacy of by combining a 12-week course of lamivudine in those HBeAg-positive hepatitis B patients receiving peginterferon alfa-2a (peg-IFN alpha-2a) therapy.

METHODSA total of 58 patients initiated a 52-week course of peginterferon alfa-2a were enrolled and divided into 3 groups. The patients with HBV DNA undetectable or HBeAg negative at week 12 were divided into group A, in this group treatment continued to week 52 with peg-IFN alpha-2a alone; The rest patients were divided into group B1 and B2, in group B1, lamivudine was combined at a course of 12 weeks, while in group B2 treatment continued to week 52 with peg-IFN alpha-2a alone. Clinical responses were assessed at week 52.

RESULTS8 out of 58 patients achieved undetectable HBV DNA or HBeAg loss at week 12 and divide into group A. In this group the HBV DNA loss rate, HBeAg seroconversion rate, HBsAg loss rate and ALT normalization rate were 100% (8/8), 75% (6/8), 0% (0/8) and 100% (8/8) respectively at the end of treatment. In this group the HBV DNA loss rate, HBeAg seroconversion rate, HBsAg loss rate and ALT normalization rate were 100% (8/8), 75% (6/8), 0% (0/8) and 100%(8/8) respectively at the end of treatment. The rest 50 patients without early response to peg-IFN alpha-2a at week 12 were divided into group B1 (24 patients enrolled) and B2 (26 patients). At the end of treatment, the HBV DNA loss rate, HBeAg seroconversion rate, HBsAg loss rate and ALT normalization rate in Group B1 were 50% (12/24), 38% (9/24), 4% (1/24) and 63% (15/24) respectively, and 31% (8/26), 27% (7/26), 0% (0/26) and 35% (9/26) respectively in group B2.

CONCLUSIONThose patients with early responses to peg-IFN alpha-2a therapy can achieve high clinical responses at the end of 52-week treatment. The combining therapy of lamivudine for a course of 12-weeks can improve the clinical responses for the patients without early responses to peg-IFN alpha-2a.

Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Drug Therapy, Combination ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Interferon-alpha ; therapeutic use ; Lamivudine ; therapeutic use ; Male ; Pilot Projects ; Polyethylene Glycols ; therapeutic use ; Recombinant Proteins ; Treatment Outcome ; Young Adult