ABSTRACT
Objective To clone and express the immunodominant domain of the chlamydial proteaselike activity factor(CPAF) from Chlamydophila psittaci(Cps) and evaluated the diagnosing value of the recombinant protein in Cps infection.Methods The immunodominant region epitope of CPAF (CPAFm,A196-A450)from Cps was chosen according to bioinformatics analysis and references.The specific primer was designed and the gene was amplified by PCR and then ligated into a pGEX6p-2 vector.Recombinant protein was induced to express by IPTG and analyzed by SDS-PAGE and Western blot.Indirect EL1SA method of serological diagnosis was established with the reorganization protein as coating antigen.One hundred and eighty sera samples from ducks with respiratory tract infection symptoms were detected with the established indirect ELISA and a commercial ELISA-kit to assess the value of the recombinant protein in serodiagnosis.The results were further identified with Western blot.Results Prokaryotic expression vector pGEX6p-2/CPAFm was constructed and a 54x103 fusion protein was attained.The indirect ELISA method was established with reorganization protein for envelope antigen.Using the indirect ELISA to detect Cps lgG positive and negative reference sera,the sensitivity and specificity were both 100％ (20/20).And the recombinant protein has no cross reaction with either Chlamydophila pneumoniae or Chlamydophila trachomatis.The concordance rate between the indirect ELISA and Western blot to 180 ducks sera samples was 100％,while the concordance rate of the commercial ELISA kit was 77.5％-95.0％.Conclusion The prepared recombinant protein of the CPAF immunodominant region epitope gene from Gps can highly benefit on developing new indirect ELISA as methods to detect specific anti-Cps antibodies.
ABSTRACT
Objective To express and purify Chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,for investigating the effect of its recombinant protein GST-CPAF in inducing human monocytic cells to secrete proinflammatory cytokines and cell apoptosis.Methods The recom-bination expression plasmid pGEX6p-2/CPAF from Chlamydophila pneumoniae was transformed into E.coli.The recombination GST-CPAF was expressed after induction by IPTG,and purified by a agarose gel FF.Human monocytic cells were stimulated by the GST-CPAF to test the production of tumor necrosis factor a(TNF-α)and interleukin-6(IL- 6)by ELISA.Inhibition of cells proliferation with GST-CPAF was assessed by MTT.The THP-1 cell apoptosis stimulated by GST-CPAF was detected by Hoechst33258 fluorescence staining,DNA fragmentation analysis and cell apeptosis was detested bv Annexin V-FITC-propidiuum iodide (PI)staining.Results The recombination protein GST-CPAF was successfully expressed with high level in E.coli,and stimulated human monocytic cells to produce proinflammatory cytokines including TNF-α and IL-6 in a dose-and time-dependent manner.Otherwise,the GST-CPAF inhibited the growth of human monocytic cell in a dose-dependent manner.Apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy,DNA ladders in apoptosis cells were detected after 24 h with the GST-CPAF.Conclusion The GST-CPAF from Chlamydophila pneumoniae can induce the secretion of proinflammatory cytokines TNF-α and IL-6 by human monocytic cells,and inhibited the proliferation of THP-1 cell and apoptosis in vitro.
ABSTRACT
This study compared activity factor, predicted resting metabolic rate (RMR), and nutrient intakes between athletic and non-athletic high school students in Gangwon-do. Fifty soccer players (30 males and 20 females; mean ages 16.7+/-1.0 years and 16.4+/-1.1 years, respectively) and 50 non-athletic (30 males and 20 females; mean ages 17.5+/-0.4 years and 16.4+/-1.1 years respectively) high school students were included. Anthropometric measurements included: weight and height, triceps skinfold, mid-arm circumference, and body fat. Prediction equations consisted of those from the Harris-Benedict, FAO/WHO/UNU, IMNA, Cunningham, Mifflin et al., and Owen et al. A one-day activity diary was collected by interview, and the 24-hour recall method was used to analyze nutrient intakes of subjects. The activity factors of the male and female athletic groups (2.23 and 2.16, respectively) were significantly higher than those (1.52 and 1.46, respectively) of the non-athletic group. There was only a significant difference in RMR by use of the Cunningham's equation between two groups. For the males, almost all nutrient intakes of the athletic group (except carbohydrate, iron, vitamin B1, B6, and niacin) of athletic group were significantly higher than those of the non-athletic group. The female athletic group showed significantly higher nutrient intakes with the exception of most vitamins. These results suggest that assessments of energy balance between energy intake and energy expenditure by employing RMR and activity factors would be useful to prevent and treat obesity in high school athletes. In addition, the Cunningham's equation would be appropriate for predicting their energy needs.
Subject(s)
Female , Humans , Male , Adipose Tissue , Athletes , Energy Intake , Energy Metabolism , Iron , Obesity , Soccer , Sports , Thiamine , VitaminsABSTRACT
This study compared activity factor, predicted resting metabolic rate (RMR), and nutrient intakes between athletic and non-athletic high school students in Gangwon-do. Fifty soccer players (30 males and 20 females; mean ages 16.7+/-1.0 years and 16.4+/-1.1 years, respectively) and 50 non-athletic (30 males and 20 females; mean ages 17.5+/-0.4 years and 16.4+/-1.1 years respectively) high school students were included. Anthropometric measurements included: weight and height, triceps skinfold, mid-arm circumference, and body fat. Prediction equations consisted of those from the Harris-Benedict, FAO/WHO/UNU, IMNA, Cunningham, Mifflin et al., and Owen et al. A one-day activity diary was collected by interview, and the 24-hour recall method was used to analyze nutrient intakes of subjects. The activity factors of the male and female athletic groups (2.23 and 2.16, respectively) were significantly higher than those (1.52 and 1.46, respectively) of the non-athletic group. There was only a significant difference in RMR by use of the Cunningham's equation between two groups. For the males, almost all nutrient intakes of the athletic group (except carbohydrate, iron, vitamin B1, B6, and niacin) of athletic group were significantly higher than those of the non-athletic group. The female athletic group showed significantly higher nutrient intakes with the exception of most vitamins. These results suggest that assessments of energy balance between energy intake and energy expenditure by employing RMR and activity factors would be useful to prevent and treat obesity in high school athletes. In addition, the Cunningham's equation would be appropriate for predicting their energy needs.
Subject(s)
Female , Humans , Male , Adipose Tissue , Athletes , Energy Intake , Energy Metabolism , Iron , Obesity , Soccer , Sports , Thiamine , VitaminsABSTRACT
Middle and high school students in Korea spend a lot of time at school. In other words, many Korean teenagers spend most of their time studying, and have little time for other activities, such as physical workouts. Fewer physical activities, and consumption of many snacks, etc. may lead to overweight or obesity in some teenagers. This study was conducted to find out the activity levels of teenagers' in order to calculate their daily energy expenditures. A total of 1,192 students (461 middle school students and 731 high school students) who lives in either a city or a country area were recruited for this study. One-day Activity Diaries were collected using questionnaires and analyzed by means of SPSS/win. The participating students spent about 7 hours 24 minutes resting including sleeping; 6 hours 28 minutes studying, 4 hours 4 minutes in leisure activities, 1 hour 32 minutes in personal hygiene activities such as washing, dressing and undressing, etc., 50 minutes on transportation : and 23 minutes on household chores such as cleaning, doing the laundry, etc. The average activity factor was 1.49. That of middle school female students was the lowest at 1.47, followed by middle school male students at 1.48, high school female students at 1.49, and high school male students at 1.51. The daily energy expenditure of middle and high school male students was 2,289 kcal and 2,600 kcal, respectively. That of female students was 1,959 kcal and 2,067 kcal, respectively.