Objective To construct prokaryotic expression vector of hepatitis C virus NS5ATP1 gene, and to induce its expression in E. coli. To purify the fusion protein and obtain its polyclonal antibody from immunized New Zealand rabbits. Methods The NS5ATP1 gene, which was cut from the vector pGBKT7-NS5ATP1, which was self-constructed by the authors, was cloned into plasmid pET32a(+) to construct the pET32a(+)-NS5ATP1 prokaryotic expression vector. It was proved that the recombinant plasmid was constructed correctly by sequencing. And then the expression vector was transformed into the competent E. coli DH5? and BL21. After being induced with IPTG, the NS5ATP1 fusion protein was expressed and analyzed with SDS-PAGE and Western blot. The transformed bacteria were fragmented by ultrasonic and then separated by SDS-PAGE. The fusion protein formed inclusion body. They were then purified and re-natured through Ni+ affinity column chromatography. The purified pET32a(+)-NS5ATP1 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and immunizing potence of polyclonal antibody were evaluated by Western blot and ELISA. Results After transferring pET32a(+)-NS5ATP1 plasmid into DH5? and BL21 and induced with IPTG, the NS5ATP1 fusion protein of about 56kD was highly expressed. SDS-PAGE analysis showed that the fusion protein products were mainly in inclusion body and expressed in the highest level at 4.5h of induction. The purified protein and polyclonal antibody were obtained successfully. ELISA manifested the titer of polyclonal antibody was over 1∶512 000. The high specificity was testified by Western blot. Conclusions The successful expression and purification of NS5ATP1 fusion protein and the preparation of NS5ATP1 specific polyclonal antibody will be valuable for the study on the biological function of NS5ATP1.

Objective To report filaminopathy with novel insertion mutation in a Chinese family.Methods Total 19 patients from successive 5 generations involved in an autosomal dominant family. The detailed clinical manifestations had been described (Chinese Journal of Neurology, 2008, 41:751-755).The filamin C gene sequencing was performed in 3 patients, 5 family members without symptoms and 50 normal persons. The amplified fragments of the exon 18 in filamin C gene were cloned into pBluesripts vectors, then sequenced and identified with capillary electrophoresis. Results 18-nucleotide deletion and 6-nucleotide insertion were identified in the exon 18 of filamin C gene. The mutation caused the disturbance of the seventh immunoglobulin-like domain in filamin C, leading to the instability of dimmers of filamin C.Another 2 patients in the family had same mutation while 5 family members without symptoms and 50 normal controls were normal. Conclusion The novel nucleotide deletion-insertion in exon 18 of filamin C gene causes filaminopathy. This disease can appear in non-Nordic race.

objective To investigate the clinicopathological features of mini-cancer of the stomach. Method In this study,out of 296 early gastric cancer cases there were 34 cases of early gastric cancer in which tumor diameter was≤10 mm,among those there were 5 cases with tumor size≤2 mm and 29 cases of the size 2～10 mm. Result Mini-cancer accunted for 2% of all early gastric cancers in this series:All these mini-cancers were of intramucosal cancer(100%),while that took up to 45%in control group in which tumors were between≥2 mm and≤10 mm:Tumors were high or moderately differentiated pathologically in 100%of mini-cancers and 55%in control group.None of mini-cancer patients had lymph node metastasis,however,1 of 29 patients in control group had lymph node metastasis.Both groups had no blood vessel and lymphatic vessel invasion:The differentiation concordance rate between superficial lesions and invasive fronts in mini-cancer was 100%,higher than 86%in control group. Conclusion Gastric mini-cancer is usually of high differentiation,low tumor invasion and low rate of lympy node metastasis than control group.Endoscopic therapy is applicable for most gastric mini-cancers.

Objective To investigate the effects of low-dose of hydrocortisone on circulating thymus-dependent lymphocyte (T lymphocyte) apoptosis in patients with septic shock. Method fifty-seven patients with septic shock admitted into ICU from January 2006 to January 2009 were prospectively randomized (random number) to treatment group and control group. Another 20 healthy volunteers and 18 patients with sepsis alone were included as external control groups.The patients of treatment group and control group were treated with low-dose of hydrocortisone and placebo,respectively. Samples of peripheral blood were taken from healthy volunteers and patients 0 hr,24 hrs,48 hrs,72 hrs and 168 hrs after onset of the disease to determine the circulating T lymphocyte apoptosis by using the assays of Annexin V and flow cytometry. Least significant difference t -test was used for multiple comparisons. Results The percentage of Annexin V-positive CD4+ T lymphocytes in the primary stage was (11.01 +4.52)% in septic shock patients, (4.41 + 1.45)% in healthy volunteers, and (7.87 + 3. 82)% in patients with sepsis alone. And in the initial setting, the percentage of Annexin V-positive CD4+ T lymphocytes in the septic shock patients was higher than that in healthy volunteers ( P < 0.05) and in patients with sepsis alone ( P < 0.05). The percentage of Annexin V-positive CD8 + T lymphocytes at the beginning was (11.33+19.62)% in septic shock patients, (9.62+8.32)% in healthy volunteers, and (13.09+ 15.84)% in patients with sepsis alone (P > 0.05 between three groups). The percentages of Annexin V-positive CD4+ T lymphocytes in control group after 24 his, 48 hrs and 72 hrs were(13.51+6.85)%, (19.39 + 6.63)% and (15.33+ 6.21)%, respectively. And the percentages of Annexin V-positive CD4+ T lymphocytes in treatment patients after 24 hrs, 48 hrs and 72 hrs were (17.4 + 7.21)%, (22.61 + 5.64)%, and (25.73 + 6.91)%, respectively. The percentage of Annexin V-positive CD4+ T lymphocytes in septic shock patients was higher than that in control groups ( P < 0.05). The percentages of Annexin V-positive CD8+ T lymphocytes in control group after 24 hrs, 48 hrs and 72 hrs were (11.49+ 11.73)%, (12.74+ 10.39)% and (13.28+ 16.6)%, respectively, and in the treatment group, those were (9.49 + 8.9)%, (15.32+18.17)% and (13.68+16.84)%, respectively (P >0.05 between two groups). In the meantime, the percentages of Annexin V-positive CDS'1' T lymphocytes in control group and in treatment group were (12.72+ 19.69)% and (13.88 + 13.28)%, respectively (P >0.05). Conclusions Low-dose of hydrocortisone could induce CD4+ T lymphocyte apoptosis and has no effects on CD8+ T lymphocyte apoptosis when it is used to treat septic shock.

Objective To evaluate plasma natriuretic peptide brain (BNP) levels in elderly male patients with type 2 diabetes and primary osteoporosis.Methods A total of 122 elderly male patients with type 2 diabetes were divided into 3 groups according to bone mineral density(BMD):normal group (41 cases),osteopenia group (40 cases) and osteoporosis group (41 cases),and another 33 age matched healthy subjects as control group.Plasma BNP levels were determined by ELISA.Results Plasma BNP levels in osteoporosis group [(1.95 ± 0.49) pmol/L] and osteopenia group [(1.64±0.48) pmol/L] were significantly elevated compared with that in normal group [(1.32±0.38) pmol/L] and control group [(1.26±0.39) pmol/L] (all P＜0.01).There was a statistical difference between osteoporosis group and osteopenia group (t=3.539,P＜0.05),and also between normal group and control group (t=2.726,P＜0.05).Plasma BNP levels had negative correlation with BMD of 2na-4th lumbar vertebra (r=-0.366) and femoral neck (r=-0.375),body mass index (r=-0.288) and estrodiol (E2) (r=-0.352) (all P＜0.05); while had a positive correlation with parathyroid hormone (PTH) (r=0.353,P＜0.05).Conclusions With BMD declining,plasma BNP levels are elevated in elderly male type 2 diabetes,which may be related to the compensatory increase in PTH and the decrease in estradiol.

A new organic-soluble co-reactant of N,N,N’,N’-tetrakis(propyl)-pentanediamine (TPPD) was synthesized and co-immobilized with tris ( 2, 2’-bipyridine ) ruthenium ditetrakis ( 4-chlorophenyl ) borate ([Ru(bpy)3][4-(Clph)4B]2) on an indium tin oxide (ITO) electrode for electrochemiluminescent (ECL) study. The hydrophobicity of TPPD prevented its leakage from the electrode surface, thus the long-term stability of the ECL electrode was enhanced. The TPPD/[Ru(bpy)3][4-(Clph)4B]2 modified-ITO electrode exhibited strong, stable and reproducible ECL signal. The ECL signal could be quenched efficiently by phenol with a quenching efficiency of 95. 4% in the presence of 0. 1 mmol/L phenol, demonstrating the potential of the modified electrode in determination of phenolic compounds. The transient-state of the electrogenerated chemiluminescence reaction was also investigated, which revealed that the TPPD/[Ru(bpy)3][4-(Clph)4B]2 modified-ITO electrode had a longer lifetime than conventional TPA-Ru(bpy)2+3 co-reactant system.

Objective: To study the quality and transdermal properties of matrine microemulsion-based hydrogel (MBH) to provide basis for the development of the preparation.Methods: The stability of MBH was observed at 4 ℃ for 3 months and the changes of particle appearance, viscosity, pH and matrine content were observed.The transdermal permeation of MBH was investigated by a dual chamber permeation and diffusion device with excised mouse skin as the barrier.Taking rabbits as the experimental subjects, the irritation of MBH to the normal skin and damaged skin was investigated.Results: The appearance, viscosity, pH and matrine content of MBH at 4 ℃ in 3 months did not change significantly.In vitro transdermal test showed that MBH had a good penetration rate on mouse skin, and no skin irritation occurred after single or multiple administrations.Conclusion: MBH has good stability and high rate of transdermal penetration without skin irritation, which is a promising drug delivery system of matrine with good application prospects.

Objective To study the value of application of histamine provocation test and airway resistance measurement in diagnosis and therapeutic efficacy in preschool children with asthma.Methods Histamine provocation test and airway resistance measurement by the Italian MEFAR MB3 provocating instrument and Germen Microloop lung function instrument for 42 cases who were diagnosed as asthmatic(27 patients with bronchial asthma and 15 cases of cough variant asthma)and 21 healthy cases was compared,and the differences between the 2 groups and the value of therapeutic efficacy were analyzed.Results The resistance ratio of respiratory tract of control group was(97.11?9.09)%,which in asthma and cough variant asthma group was(229.37?57.48)% and(248.80?76.80)%.There was significant difference between the 3 groups(F=48.466 P

An organic solvent tolerant isolate A213 originating from soil samples were successfully isolated via direct plating method using 10g/L of toluene as the sole carbon source and transparent cycle plate assay method.It was identified as Yarrowia based on its characteris- tics.The results in shake flask cultivation showed that the suitable tipase producing media were(g/L):yeast extract 40,vegetable oil 10, MgSO_4?7H_2O1,KH_2PO_4 5.Under optimal culture conditions (27℃and pH 6.5 ),the maximal lipase activity could reach 67.8 IU/ mL The optimal pH and temperature for the hydrolysis of p-nitrophenyl acetate by crude lipase were pH6.5 and 40℃The enzyme was sta- ble under 70℃and pH 5.5～8.5.Then isolate A213 was found to produce the lipase which can synthesize L-ascorbyl palmitate in tert-amyl alcohol validated by the thin-layer chromatography.

To establish a convenient and rapid method for identification of Pseudostellariae Radix by molecular identification, the rDNA-ITS sequences of Pseudostella riaheterophylla and its adulterants had been aligned to find out specific fragment. The specific primers against the fragment were designed and the PCR amplification conditions were optimized. The fluorescence reaction of the PCR products colored by 100 x SYBR Green I was observed under UV. The concentration of reaction buffer included 5.5 μL DNA Taq polymerase premix, 10 pmol Tzs-2F and 10 pmol Tzs-2R, 20-80 ng template DNA, and plus double sterile distilled water to 25 μL. The PCR thermal profile was as follows: predenaturation at 95 degrees C for 1 min, followed by 30 cycles of denaturation at 95 degrees C for 5 seconds, primer annealing and extension at 56 degrees C for 15 seconds, then it was extension at 72 degrees C for 30 seconds. The fluorescence reaction of Pseudostellariae Radix showed green fluorescence, while adulterants had not. Extraction, amplification DNA and all steps of molecular identification could be completed successfully in 40 minutes. The approach could amplify DNA template of Pseudostellariae Radix specificity, and its product with 1 μL 100 x SYBR Green I could engender green fluorescence under UV. The method was simple and accurate, so it could be used for identification of Chinese traditional medicine.
Caryophyllaceae ; classification ; genetics ; DNA Primers ; genetics ; Drug Contamination ; prevention & control ; Plant Roots ; classification ; genetics ; Polymerase Chain Reaction ; methods