To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

Objective To evaluate the diagnostic value of contrast-enhanced digital subtraction MRI in vertebra]metastatic tumors.Methods Forty-four vertebral metastatic tumors in thirty patients were scanned by routine MRI including SE T_1WI,SE T_2WI,STIR and enhanced T_1WI with an injection of Gd-DTPA(0.1 mmol/kg).Digital subtraction was performed between pre-contrast and enhanced T_1 weighted images.All the images of vertebral malignant tumors were evaluated by means of signal intensity ratio(SIR) and nose ratio(NR).The quality of images was also evaluated by comparing subtraction MRI with routine MRI.Results SIR and NR of subtraction MRI was 2.93,0.98 respectively.SIR of routine MRI (enhanced T_1WI,SE T_1 WI,SE T_2WI,STIR)was as follows:1.15,1.16,1.26,1.69.While NR of those was 5.25,3.44,4.56,23.32 respectively.SIR and NR of subtraction MRI images had significant statistical differences from those of routine MRI images(P

Objective To evaluate the effect of pitavastatin on blood glucose in patients with hypercholesterolemia,and to investigate the efficacy of pitavastatin in diabetic patients combined with hypercholesterolemia.Method This study was a 12-week,multi-center,open-label,without parallel-group comparison,phase Ⅳ clinical trail.Results Contrasting to baseline,the prevalences at week 4 and 12 post-treatment of abnormal fasting plasma glucose (FPG) and glycosylated hemoglobin Alc (HbA1c)( FPG:14.2％ vs 14.1％ and 11.0％ ; HbA1c:14.3％ vs 15.1％ and 16.1％ ) in the safety set subjects without diabetes mellitus (DM),as well as in those with DM but not taking glucose-lowering drugs (FPG:7/7 vs 4/7 and 5/7; HbAlc:5/5 vs 4/4 and 5/5) had no significant changes (all P vaules ＞0.05).Contrasting to baseline,the levels of TC [ (6.51±0.94) mmol/L vs (5.12 ±0.93) mmol/L and (4.54 ±1.00) mmol/L],LDL-C [(4.11 ±0.79)mmol/L vs (3.02 ±0.81) mmol/L and (2.51 ±0.70)mmol/L] and TG [2.10(1.53,2.54) mmol/L vs 1.62(1.26,2.00) mmol/L and 1.35(1.10,1.86)mmol/L]at week 4 and 12 post-treatment in the per protocol set 55 subjects with DM were significantly reduced (all P values ＜ 0.05 ) ; 33.3％ of subjects at high risk and 10.0％ of subjects at very high risk had achieved a TC target value; 55.6％ of subjects at high risk and 40.0％ of subjects at very high risk had achieved a LDL-C target value.Conclusion Pitavastatin has a safe effect on blood glucose and it could be used to treat diabetic patients combined with hypercholesterolemia in China.

OBJECTIVETo study the prevalence of asthma and its correlated factors in Zaozhuang area in 2003, to provide a basic consideration for prevention/treatment and control policy.

METHODS6 points were selected by stratified-clusterd-random sampling with a total of 16,725 persons expected, but only 10,610 subjects investigated.

RESULTSIn this survey, 128 asthma cases were identified with a overall prevalence of 1.21%. The prevalence for children was 2.02%, and for adult was 0.90% with the former significantly higher then the latter (chi(2) = 21.39, P < 0.01). Rates for male and female were 1.08%, 1.32% with a ratio of 1:1.22. For 77.97% of children with asthma. The initiative age of asthma was before 7 years old among children while among 36.23% of the adults, it was before 15 years of age. Correlation analysis showed that upper respiratory tract infection (OR = 17.81, 95% CI: 12.25-25.89), cold air exposure (OR = 3.43, 95% CI: 2.41-4.90), stimulation through cooking and by harmful gases (OR = 2.56, 95% CI: 1.80-3.63), allergic materials (OR = 2.74, 95% CI: 1.80-4.17) were main inducing factors. 65.63% of the asthma cases having had history of allergic disease while 25.78% having had family history with the OR of allergic history and family history as 21.69 vs. 73.96.

CONCLUSIONThe epidemic status of bronchial asthma was serious, with an assumption that asthma cases might have reached the number of 43 thousand in Zaozhuang area.

Adolescent ; Adult ; Aged ; Asthma ; epidemiology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Male ; Mass Screening ; Middle Aged ; Prevalence ; Risk Factors

OBJECTIVETo research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of kanamycin.

METHODSFifty wistar rats were randomly divided into four groups: group A (D-galactose group, n = 14) were treated with hypodermic 5% D-galactose (150 mg x kg(-1) x d(-1)) for 8 weeks and then with intraperitoneal saline for 10 days; group B (D-galactose and kanamycin group, n = 14) were given the same dose of D-galactose but kanamycin (500 mg x kg(-1) x d(-1)) instead of saline; group C (kanamycin group, n = 12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n = 10) were given saline only. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and the mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique.

RESULTSThe incidence of mitochondrial DNA 4834 bp deletion mutation was 100% (28/28) in group A, 92.86% (26/28) in group B and 0% in group C or group D. The activity of GSHPX in group A was (59.07 +/- 8.70)U, (63.29 +/- 12. 40)U in group B, (136.67 +/- 9.53)U in group C and (142.10 +/- 7.02)U in group D. The difference between group A and D was significant (P = 0.000) while the difference between group A and B was not significant (P = 0.307), which was similarly as between group C and group D (P = 0.151). ABR threshold was (5.36 +/- 3.08) dB peSPL in group A, (61.79 +/- 11.20) dB peSPL in group B, (34.17 +/- 4.69) dB peSPL in group C and (6.50 +/- 3.37) dB peSPL in group D. No difference was found between group A and D (P = 0.398) while the difference in shift of ABR threshold between group B and group C (or group D) was significant (P = 0.000).

CONCLUSIONSThe mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats present high incidence of mtDNA4834 deletion which can greatly enhance the sensitivity of the inner ear to the kanamycin.

Aging, Premature ; chemically induced ; Animals ; DNA, Mitochondrial ; genetics ; Disease Models, Animal ; Disease Susceptibility ; Ear, Inner ; drug effects ; Galactose ; toxicity ; Kanamycin ; toxicity ; Rats ; Rats, Wistar ; Sequence Deletion

OBJECTIVETo investigate the balance function of the patients with unilateral vestibular hypofunction (UVH) by timed balance tests and static posturography (SPG).

METHODSSixty-five subjects with UVH and 92 healthy subjects were taken the timed balance tests under differential stance including (1) standard Romberg test, (2) feet apart stance test, (3) tandem and (4) unilateral standing tests with eyes open and eyes closed. The average timing that subjects kept balance before falling in each standing conditions was recorded by stopwatch as the timed result. The body sway velocity during the test (1) and (2) were also recorded by the SPG.

RESULTSThe timed results of the tandem and unilateral standing with eyes open and eyes closed in the UVH group were decreased (P < 0.001) compared with the control group. The body sway velocity of the standard Romberg test and foot apart stance with eyes open was not different between the UVH group and control group (P-value was 0.118 and 0.110 for the two tests respectively), and the difference was significant in the eyes closed condition (P < 0.001). For the two groups, the body sway velocity of foot apart standing was decreased than that of the standard Romberg test with eyes open and eyes closed (P < 0.05 or P < 0.001). Significant correlations were not found between the timed results and sway velocity results in both two groups respectively (P > 0.05).

CONCLUSIONSAccording to clinical assessment of balance function in UVH, the tandem and unilateral stance test could provide the additional information about the upright stance to the SPG measurement. The effect of foot position on the results of SPG should been considered in clinic.

Adult ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Neurologic Examination ; Postural Balance ; Vestibular Diseases ; physiopathology

OBJECTIVETo study the maturation effect of CpG2006 and phosphodiester oligonucleotides on leukemia-derived dendritic cells.

METHODSLeukemia cells K562/A02 were induced into dendritic cells by rhGM-CSF and rhIL-4. After 7 days induction, the cell-morphology was observed, the immunophenotype of cells was detected by flow cytometry and the cell function was evaluated by allogeneic mixed lymphocyte reactions, CTL responses and secretion of IL-12 and IL-6. Then a CpG oligonucleotide CpG2006, two synthetic bacterial phosphodiester oligonucleotides A-ODN and T-ODN were added to these leukemia-derived DCs. Three days later, the DCs were re-detected by the above-mentioned methods.

RESULTSAfter induced by CpG2006, A-ODN or T-ODN, the leukemia-derived DCs with typical dendritic morphology were increased. The expressions of CD83, HLA-DR and CD86 were (65.5 +/- 8.4)%, (32.0 +/- 4.3)% and (18.6 +/- 3.2)% respectively in day 7 leukemia-derived DCs, raised to (88.9 +/- 3.6)%, (53.9 +/- 3.2)% and (39.9 +/- 7.3)% respectively after exposing CpG2006 for 3 days; increased to (97.0 +/- 5.3)%, (63.9 +/- 7.3)% and (40.2 +/- 7.4)% respectively after treated by A-ODN; and further increased to (93.26 +/- 4.65)%, (58.3 +/- 5.6)% and (36.2 +/- 6.8)% respectively after treated by T-ODN. These results was markedly different than unaffected cells did. These DCs induced by the above-mentioned three oligonucleotides could upregulate significantly the capacity for stimulating allogeneic T cells. They could also induce CTL to generate specific cytotoxic activity against K562/A02 cells. The secretion of IL-6 and IL-12 was increased remarkably.

CONCLUSIONCpG2006, as well as two phosphodiester oligonucleotides can induce leukemia-derived DCs maturation.

Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Dendritic Cells ; cytology ; drug effects ; Humans ; K562 Cells ; Oligodeoxyribonucleotides ; pharmacology ; Oligonucleotides ; pharmacology

OBJECTIVETo study whether progestogen antagonist mifepristone could reverse multidrug resistance of K562/A02 cells and its mechanisms.

METHODSMTT was used to study the proliferation of K562/A02 cells and sensitivity of K562/A02 cells to ADM after 72 hours treatment with mifepristone. Flow cytometry was used to assay the expression of P-glycoprotein and the mean fluorescent intensity of intracellular daunorubicin. The expressions of apoptosis related proteins (bcl-2, Bax and caspase-3) were assayed by immunohistochemistry and the glucosylceramide synthase mRNA expression by RT-PCR before and after mifepristone treatment.

RESULTSMTT assay revealed that 2.5, 5.0 and 10 micromol/L mifepristone did not affect the proliferation of K562/A02 cells, but enhanced the sensitivity of K562/A02 cells to ADM, by 1. 68-, 4.17- and 10.71- fold increase, respectively. Expression of P-gp in K562/A02 cells was (49.03 +/- 5.32)%, and was decreased to (28.60 +/- 2.13)% (P < 0.01) after 10 micromol/L mifepristone treatment for 72 hours. and intracellular DNR accumulation in K562/A02 was (61.07 +/- 8.61)%, and was increased to (92.72 +/- 3.48)% (P < 0.01). After 10 micromol/L mifepristone treatment, the expression of bcl-2 protein was decreased from (56 +/- 9)% to (37 +/- 6)% (P < 0.05), Bax and caspase-3 proteins was increased from (40 +/- 5)% to (87 +/- 10)% (P < 0.01), and from (36 +/- 7)% to (89 +/- 6)% (P < 0.01) respectively. RT-PCR analysis revealed that expression of glucosylceramide synthase mRNA was higher in K562/A02 than in K562 cells, whereas 10 micromol/L mifepristone significantly down-regulated its expression in K562/A02 cells.

CONCLUSIONMifepristone at 10 micromol/L could dose-dependently reverse the multidrug resistance of K562/A02 cells. The possible mechanisms are related with decreasing the expression of P-gp, regulating the expression of apoptosis related proteins and decreasing the expression of glucosylceramide synthase.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Cell Proliferation ; drug effects ; Daunorubicin ; pharmacokinetics ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Glucosyltransferases ; genetics ; metabolism ; Humans ; K562 Cells ; Mifepristone ; pharmacology ; RNA, Messenger ; genetics ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo establish the visual patch clamp whole-cell recording technique and study the properties and functional significance of muscarinic receptor-mediated currents in rat medial vestibular nucleus neurons (MVNn).

METHODSBrain slices containing the MVN were prepared from fifteen Wistar rats. By combining infrared differential interference contrast (IR-DIC) technique and CCD-Camera system with visual patch clamp whole-cell recording technique, twenty healthy neurons were located and muscarinic receptor-mediated currents in rat MVNn were observed and analyzed.

RESULTSVisual patch clamp technique can be used to make direct localization and to make sure of active neuron. In MVNn, a comparison of the current-voltage relationships before and during the application of muscarine, which revealed an increase in the slope of the I-V curve and the reversal potential for this response lay at (-88.4 +/- 4.9) mV (x +/- s), indicates that the activation of muscarinic cholinergic receptors leads to a decrease in potassium current. The test in the voltage sensitivity of the muscarine-induced response, which showed that the effect had a linear current-voltage relationship and reversed at (-86.7 +/- 3.5) mV, indicates that the potassium current blocked by muscarine corresponds to the voltage-insensitive leak potassium current.

CONCLUSIONSVisual patch clamp technique, which was considered better than blind patch clamp technique, can improve the success of sealing process. By the analysis of muscarinic receptor-mediated currents, the data provide support that muscarinic cholinergic mechanisms play a prominent role in the modulation of the excitability of MVNn and also offer a new idea for the efficacy of anticholinergic drugs.

Animals ; Electrophysiological Phenomena ; Neurons ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Receptors, Muscarinic ; physiology ; Vestibular Nuclei ; physiology

OBJECTIVETo study the membrane properties of rat medial vestibular nucleus (MVN) neurons and their firing responses to simulated input signals of peripheral vestibular system, and to discuss how the intrinsic membrane properties contribute to physiologic functions in central vestibular system.

METHODSBy using infrared differential interference contrast technique, whole-cell recordings were made from rat MVN neurons under direct observation. On the basis of their averaged action potential shapes, the MVN neurons were classified. Linear and non-linear currents were put into the neurons to simulate the input signals of peripheral vestibular system. The differences of intrinsic membrane properties and firing response dynamics were observed between two types.

RESULTSThe discharge activities were recorded in MVN neurons, which remained in low Ca2+-high Mg2+ artificial cerebrospinal fluid (ACSF). Neurons are classified as type A (33%) characterized by a single deep after-hyperpolarization (AHP) and A-like rectification, or type B (63%) characterized by double AHP, and another two neurons with all or none of the characters. The passive membrane properties were not significantly different between type A and type B neurons, while part of active membrane properties was significantly different. Both type A and B neurons well responded to simulated current inputs, but disparities existed in response range and firing dynamics.

CONCLUSIONSThe discharge activities of MVN neurons were initiated by their intrinsic membrane properties. Most MVN neurons were classified as type A and B, while several showed unrepresentative firing properties. Linear and nonlinear inputs evoked a heterogeneous range of firing responses. The differences of response range and firing dynamics between neurons may determine their different physiological functions.

Action Potentials ; Animals ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Vestibular Nuclei ; cytology ; metabolism ; physiology