A modified rapid microprocedure for the determination of VE in serum, was described. VE reduced Fe3+ to Fe2+ and then the Fe2+ combined with bathophenanthroline to form a pink complex -which could be determined spect-rophotometrically. As little as 0.3ml (or 0.2ml) of serum or plasma sample was sufficient for determination. The reproducibility of this method was good. The CV was 4.1% (n=15). The recoveries of added VE (0.8, 0.2 and 1.6 mg/dl) were 86.3-102.5%. The mean value and SD of 99 normal adults were 1.15+ 0.31mg/dl. This spectrophotometric method was simple and could be used in common, laboratories. Some affecting frctors were discussed.

BACKGROUND: As a kind of polypeptide, angiotensin-converting enzyme (ACE) inhibitory peptide can lower the blood pressure of human body through restraining the formation of angiotensin Ⅱ.OBJECTIVE: To investigate the influence of AGE inhibitory peptide on endothelial cell proliferation and endothelin expression in cultured human umbilical vein cells based on cellular and molecular levels in order to provide the experimental evidences for ACE inhibitory peptide to be the potential blood pressure-lowering health food.DESIGN: Repeated measures design.SETTING: School of Bioscience and Bioengineering, South China University of Technology; School of Applied Chemistry and Biological Technology,Shenzhen Polytechnic.MATERIALS: The experiment was carried out in the Institute of Applied Chemistry and Biological Technology, Shenzhen Polytechnic from September 2004 to March 2005. The AGE inhibitory peptide was provided by the Institute of Applied Chemistry and Biological Technology, Shenzhen Polytechnic. Under certain circumstance, 15 μ mol/L of the inhibitor was needed to decrease half of the AGE activity. The human umbilical vein endothelial cells of the 4th generation were cultured randomly in 7 groups with different concentrations: medium group, 150, 300 and 600 mg/L ACE inhibitory peptide groups, captopril group, norepinephrine(NE) group, and ACE inhibitory peptide+NE group.METHODS: ①The endothelial cells were cultured as recommended. The medium was M199+FBS(0.15, v/v)+penicillin(10 000 U/mL)+streptomycin (100 mg/L). After cellular fusion, the cells were carried on the passage with the ratio of 1:2. The 4th generation cells were used for experiment. ②M199(0.15, v/v) was contained in each group. ACE inhibitory peptides were added to make the final concentration 150, 300 and 600 mg/L in the 150, 300 and 600 mg/L ACE inhibitory peptide groups respectively. Captopril was added to make the final concentration 10-5 mol/L in the captopril group. NE was added to make the final concentration 100 μg/L in the NE group. ACE inhibitory peptide and NE were added to make the final concentration 300 mg/L and 100 μg/L in the ACE inhibitory peptide+NE group respectively. ③The state of cell growth was determined with cytometry. The contents of endothelial cells in the medium with different culture times were determined with radioimmunoassay. The expression of endothe lin mRNA was determined with reverse transcriptase-polymerase chain reaction. The expression of cellular endothelin protein was determined with immunohistochemical method.MAIN OUTCOME MEASURES: ①The influence of ACE inhibitory peptide on endothelial cell proliferation. ②The influence of ACE inhibitory peptide on the endothelin mRNA and endothelin protein.RESULTS: ①The influence of ACE inhibitory peptide on endothelial cell proliferation and endothelin secretion: Compared with the medium group,in the captopril and 150, 300, 600 mg/L ACE inhibitory peptide groups,the growth of endothelial cells was restrained and the endothelin content in the medium was lowered(P ＜ 0.01 or 0.05). NE could promote the growth of endothelial cells and the secretion of endothelin, but the cell density and endothelin content after treatment with ACE inhibitory peptide were similar to those in the medium group (P ＞ 0.05). ②The influence of ACE inhibitory peptide on the expressions of endothelin mRNA and protein in endothelial cells: Compared with the medium group, the expressions of endothelin mRNA and protein might be lowered in the captopril and 150,300, 600 mg/L ACE inhibitory peptide groups(P ＜ 0.01 or 0.05). The expressions of endothelin mRNA and protein could be up-regulated by NE.The gene expression after treatment with ACE inhibitory peptide was similar to that in the medium group(P ＞ 0.05).CONCLUSION: The ACE inhibitory peptides of different dosages can all restrain the growth of endothelial cells, lower the endothelin content, decrease the expression of endothelin gene and resist NE improved growth and secretion of endothelial cells in umbilical vein cell effectively.

Objective To observe the changes of the endothelium-dependent vasodilatation(EDF)and serum vascular endothelial growth factor(VEGF)in the patients with impaired glucose tolerance(IGT)and type 2 diabetes mellitus(DM).Methods 30 IGT patients,30 type 2 DM patients and 33 normal subjects were divided into3 groups. Fasting glucose(FPG),fasting insulin(FINS),serum superoxide dismutase(SOD),maleie. dialdehyde(MDA)and VEGF were measured after 12 hours overnight fast. Oral 75g glucose tolerance test(OGTT)was performed. The inner diameter of braehial artery was assessed by a high resolution ultrasound system before and after reactive hyperemia. EDF was calculated as the percent change in brachial artery diameter 1 minute after reactive hyperemia compared with baseline. Results In the IGT group and DM group, EDF was significantly lower than that in NGT group(both P＜0.01),and EDF in the DM group was significantly lower than that in the IGT group(P＜0.01).SOD in the IGT group and DM group were significantly lower than that in the NGT group(both P＜0.01),but MDA in reverse(both P＜0.01).Compared with the IGT group, SOD in DM group was significantly lower(P＜0.01),but MDA was significantly higher(P＜0.01).VEGF was progressively increased in the NGT,IGT, DM groups. The difference between the two groups was significant(both P＜0.01).Stepwise regression analysis showed that EDF was positively related to SOD(r=0.418,P＜0.01,n=93),and negatively related to HOMA-IR and VEGF(r=-0.553,-0.221,both P＜0.01,n=93).VEGF was negatively related to SOD(r=-0.552,P＜0.01,n=93).Conclusion EDF is impaired in IGT patients while the impairment in DM patients becomes more marked. Insulin resistance, VEGF,SOD and MDA are closely related to the impairment of EDF in IGT and type 2 DM.

Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.

Objective To study the changes of proteome expression in brain tissues from rats with severe traumatic brain injury(sTBI). Methods Total protein of brain tissues were obtained at days 3,7 and 14 for two-dimensional gel electrophoresis to screen and identify differential protein spots.Results We screened 17 differential protein spots that were involved in cellular metabolism,stress and inflammatory reaction. Conclusion Some differential proteins involved in sTBI can be found by twodimensional gel electrophoresis.

Objective To judge injury severity of severe traumatic brain injury (sTBI) by using surface enhanced laser desorption-ionization (SELDI) protein chip technique. Methods Serum sam-ples from sTBI patients were used to detect expression of differential proteins by protein chip CM10 and SELDI to analyze the correlation between expression peak intensity and GCS. Results We obtained 101 protein peaks, with statistical difference upon expression of 27 protein peaks, when negative correla-tion was found between two peaks ( m/z 4 972 and m/z 5 322 ) and GCS score and positive correlation be-tween six peaks (m/z 3 941, m/z 4 295, m/z 8 714, m/z 8 792, m/z 14 020 and m/z 28 148) and GCS score. Conclusion SELDI protein chip technique may become a new and objective detection method in judging injury severity of sTBI.

Objective: To determine the antibody titer in the serum of allergic rabbits after the injection of 1, 3-di-caffeoylquinic acid contained in Shuanghuanglian injection.Methods: The complete antigen was prepared by incubating the suspected small molecular hapten 1, 3-di-caffeoylquinic acid contained in Shuanghuanglian injection with the serum from the normal rabbits.The specific antibody was obtained in the immunized rabbits.The antibody titers of antiserum were measured by ELISA kits.Indirect competitive ELISA was used to determine serological specificity, and the obtained data was used to plot the inhibition curves.The content of IgE antibody in the antiserum of rabbits was detected by rabbit immunoglobulin E (IgE) ELISA kits.Results: The antibody titer (A) of 1,3-di-caffeoylquinic acid was 2 times higher than that of the negative control, which indicated its potential allergenicity.The regression equation was I=0.170 6 lg C + 0.317 5 , which was with the correlation coefficient of r=0.985 4 , the detection limit of IC 10 =57.40 μg·ml-1 and the half inhibitory concentration of IC 50 =8.732 0 mg·ml-1.Furthermore, the exogenous IgE antibody was produced in the rabbits.Conclusion: The results indicated that the hapten substance 1,3-di-caffeoylquinic acid in Shuanghuanglian injection was allergenic under the present experimental conditions.

Objective To evaluate the choice of early diagnosis method of primary ureteral neoplasms in or-der to improve the ratio of clinical diagnosis. Methods 28 cases with primary ureteral neoplasms were retrospectively analyzed. Ultrasonic examination, IVU, retrograde urogram, spiral CT, MRI, ureteroscopy and exfoliative cell examina-tion of urine were compared in this study. Results The most useful methods of detecting tumors preoperation were retrograde urogram, spiral CT, MRI, ureteroseopy. All the 28 patients underwent surgical treatment. Among them, nephroureterectomy and bladder cuff or partial resection were performed in 19 cases. Postoperative pathology showed transitional cell carcinoma in 27 cases,and adenoma in 1 case. 8 cases were T1-2 tumours. Of the 14 cases during 1990 ～1999 period, 1,5,3,2,2 and 1 cases had survival time of 1,2,3,4,5 and 6 years ,respectively. Of the 14 cases during 2000～2007,4 were lost to follow-up;2 survived for 3 years and 2 for 1 year;the other 6 who have survived near 5 years have been followed till now. Conclusions To improve the early diagnosis rate,B-ultrasonic examination, IVU,retrograde urogram,3D spiral CT and MRI examination were necessary in the early stage. The patients should be opeiated as early as possible after diagnosis.

Objective To observe the effects of electro-acupuncture (EA) at “Weizhong” (BL40) on expressions of Fas and FasL in rat models with lumbar disc degeneration; To discuss its mechanism of action.MethodsThirty SD rats were randomly divided into sham-operationgroup, model group and EA group, with 10 rats in each group. The sham-operation group was treated with sham operation to incide local skin; the model and EA groups established rat model of lumbar disc degeneration by puncturing the annulus fibrosus. Four weeks after modeling, rats in the sham-operation and model groups received fixed treatment under identical condition, and rats in the EA group were treated with EA at “Weizhong” (BL40) in 20 minutes, once a day, for 4 weeks. After treatment, Western blot and RT-PCR technology were used to test the protein and mRNA expression levels of Fas and FasL.ResultsThe expressions of Fas and FasL in the model group were higher than those in the sham-operation group (P<0.05); protein and mRNA expression levels of Fas and FasL in the EA group were lower than those in the model group (P<0.05). ConclusionEA at “Weizhong” (BL40) can reduce the expressions of Fas and FasL to inhibit the development of lumbar disc degeneration.

Salmonella enterica serovar Choleraesuis strain C500 is a live attenuated vaccine that has been widely used in Chi‐na for over 50 years to prevent piglet paratyphoid .However ,as C500 is obtained by chemical methods ,the genetic background of this strain remained unclear .In this study ,we compared the genomic differences between the virulent reference strain C 78‐2 and C500 by suppression subtractive hybridization combined with the mirror orientation selection method (MOS‐SSH ) .Six genes (asr ,ydgF ,ydgD ,ydgE ,rpoS ,and ptsG) were lost in C500 strain .Using real‐time PCR analysis ,we demonstrated that the genes regulated by rpoS ,a vital transcriptional regulator playing an important role in Salmonella infection ,were downregulated in C500 .Additionally ,the virulence of the rpoS mutant strain C78‐2ΔrpoS was 100 000 times lower than the parental strain in BALB/c mice .So loss of rpoS gene is the major factor leading to the attenuation of C500 strain .