Relatively High Bone Mineral Density in Chinese Adolescent Dancers Despite Lower Energy Intake and Menstrual Disorder

Objective The effect of dietary restriction, intense exercise and menstrual dysfunction on bone mineral density remains controversial. The aim of this study was to assess the skeletal health status and relationship between bone mineral density and nutrient intake, menstrual status, estrogen level and other factos in Chinese adolescent dancers. Methods Sixty dancers and 77 healthy controls underwent measurements of bone density, body composition, and estrogen level. Nutrient intake, menstrual status and physical activity were assessed with questionnaires. The correlation between these factors were analyzed. Results The dancers under study had a significantly lean body mass index （18.3±1.4 kg/m^2 vs. 21.7±3.1 kg/m^2）, lower percentage of body fat （0.25±0.05 vs. 0.34±0.04） and later age at menarche （14.0±0.9 y vs. 13.0±1.3 y）, and the estrogen level, daily calorie and fat intake in them were also lower than in the controls. All the dancers undertook intensive physical activity every day and up to 69% of them suffered from irregular menarche. Yet they had relatively high BMD and BMC of the total body and legs than the controls after adjusting for BM1 and age. Site-specific BMD was positively correlated to BMI, body composition and training hours per week and negatively correlated to the age at menarche and menstrual frequency. Conclusions The relatively high BMD and BMC of the dancers at the total body and legs were probably caused by high levels of weight-bearing physical activity. To ameliorate disordered eating, especially low energy intake might be helpful to prevent the Triad and to improve the bone health in adolescent dancers.

Preparation of Monoclonal Antibody Against HPT and Its Application to Detecting Marker Protein in Genetically Modified Rice

To produce the monoclonal antibodies （mAbs） against hygromycin B phosphotransferase （HPT） and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice （GM rice）. Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected （80-150ng/mL）. But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.

Correlation of Serum Leptin Level with Bone Mineral Density and Bone Turnover Markers in Chinese Adolescent Dancers

Objective To investigate plasma leptin concentrations in adolescent female dancers and to determine whether leptin has some effects on their bone mineral density （BMD） and bone turnover markers. Methods Sixty dancers aged 15-17 years and 77 healthy controls were enrolled in the study. Bone mineral density （BMD） and body composition were detected by dual energy X-ray absorptiometry. Serum leptin concentrations were measured by radioimmunoassay （RIA）. Two bone turnover markers, bone-specific alkaline phosphatase （BAP） and tartrate-resistant acid phosphatase（TRACP）, were determined by ELISA. Results The dancers had a lower fat mass and a lower leptin level than the controls, while they had a relatively higher BMD of the total body and legs after adjustment for BMI and age. The levels of bone resorption and formation of markers were higher in the dancers than in the controls. Leptin was positively correlated with BMI, body weight, fat mass, and percentage of body fat. In dancers, Leptin was positively correlated with the BMD of the total body and the left leg. However, after adjustment for BMI, no correlation of serum leptin concentrations with BMD values was found in either dancers or controls. Nor correlation was found between leptin and bone turnover markers after adjustment for BMI. Conelusion The leptin profile is different between the controls and the dancers with a lower BMI and a lower fat mass. Circulating plasma leptin level depends on BMI and is not a direct determinant of BMD in Chinese adolescent dancers.

Purification and Immunity Analysis of Recombinant 6His-HPT Protein Expressed in E.coli

To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from E.coli and to prepare the polyclonal antibody (pAbs) against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transfered into E.coli DH5?for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E.coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E.coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). Conclusion HPT protein can be expressed and purified from E.coli by a relatively simple method, which has high immunity activity.