Effect of genistein on the gene expressions of androgen generating key enzymes StAR, P450scc and CYP19 in rat ovary.

In this investigation, the effects of genistein (GEN) on the expression of steroidogenic genes such as steroidogenic acute regulatory protein (StAR), side-chain cleavage enzymes (P450scc) and cytochrome P450 aromatase (CYP19) were assessed. For this study, forty young female Sprague Dawley (SD) rats at aged 2-3 months (200±20 g) and forty aged female SD rats aged 10-12 months (490±20 g) were selected. Also, based on weight they were divided into a negative control group (NC), three different GEN dose groups, which received GEN of 15, 30, 60 mg/kg, and a positive control group (PC). The experiment lasted 30 days. Concentrations of serum hormones were determined by Enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of StAR, P450scc and CYP19 were determined by Real-Time PCR and western blot techniques. It was observed that 30-60 mg/kg GEN could increase the expression of androgen generating key enzymes in the young rat ovary. GEN also significantly increased progesterone and E2 levels in the serum of aged rats and reduced the levels of LH and FSH in the serum of both young and aged rats. Compared with young rats, the effect of GEN on the ovary of aged rats was stronger and a lower dose of GEN (15 mg/kg) showed an obvious effect on these indicators. GEN influenced both estrogen level and indicators associated with estrogen and androgen transformation processes, which indicates that GEN can impair the growth and maturation of the ovary.

Quantifying serum antiplague antibody with a fiber-optic biosensor.

The fiber-optic biosensor, originally developed to detect hazardous biological agents such as protein toxins or bacterial cells, has been utilized to quantify the concentration of serum antiplague antibodies. This biosensor has been used to detect and quantify the plague fraction 1 antigen in serum, plasma, and whole-blood samples, but its ability to quantify serum antibodies has not been demonstrated. By using a competitive assay, the concentration of serum antiplague antibodies was ascertained in the range of 2 to 15 microgram/ml. By making simple dilutions, concentrations for 11 serum samples whose antiplague antibody concentrations were unknown were determined and were found to be in good agreement with enzyme-linked immunosorbent assay results. The competitive assay method could be used to effectively determine the exposure to plague of animals or humans or could be applied to other diseases, such as hepatitis or AIDS, where the presence of antibodies is used to diagnose infection.

Fiber-optic fluorometric sensing of polymerase chain reaction-amplified DNA using an immobilized DNA capture protein.

A fiber-optic assay for amplified DNA products has been developed. Modifications of the DNA capture strategy described previously by Kemp et al. [Proc. Natl. Acad. Sci. USA 86, 2423-2427 (1989)] were made that allowed selective binding of DNA labeled during the amplification process to the sensing surface of fused silica fibers. The gene for a chimeric protein composed of the IgG-binding beta 2 subdomain of streptococcal protein G fused with the DNA binding domain of yeast GCN4 was constructed, and this PG/GCN4 protein was overexpressed in Escherichia coli. The purified protein was noncovalently bound to IgG-modified fibers utilizing strong and specific interactions between the protein G beta 2 domain and goat IgG that had been covalently immobilized on the fiber surface. Nanomolar concentrations of amplified DNA labeled with the fluorophore tetramethylrhodamine and the AP-1 consensus nucleotide sequence recognized by GCN4 (5'-ATGACTCAT) were rapidly and selectively bound within the evanescent zone of multimode laser-illuminated fibers. Signal from unincorporated fluorescent PCR primer was negligible. Individual fibers could be used for multiple sequential assays, since the fluorescent double-stranded DNA was rapidly and completely stripped from their surfaces with high salt solutions, leaving the IgG-PG/GCN4 DNA binding complex intact to accept another PCR sample.

Detection of Yersinia pestis fraction 1 antigen with a fiber optic biosensor.

A fiber optic biosensor was used to detect the fraction 1 (F1) antigen from Yersinia pestis, the etiologic agent of plague. The instrument employs an argon ion laser (514 nm) to launch light into a long-clad fiber and measures the fluorescence produced by an immunofluorescent complex formed in the evanescent wave region. This sensing area is a short section (12.5 cm) at the end of the optical fiber from which the cladding has been removed and in which the silica core has been tapered. Capture antibodies, which bind to F1 antigen, were immobilized on the core surface to form the basis of the sandwich fluoroimmunoassay. The ability to detect bound F1 antigen was provided by adding tetramethylrhodamine-labeled anti-plaque antibody to form fluorescent complexes. The evanescent wave has a limited penetration depth (< 1 lambda), which restricts detection of the fluorescent complexes bound to the fiber's surface. The direct correlation between the F1 antigen concentration and the signal provided an effective method for sample quantitation. This method achieved a high level of accuracy for determining F1 antigen concentrations from 50 to 400 ng/ml in phosphate-buffered saline, serum, plasma, and whole blood, with a 5-ng/ml limit of detection. Subsequent blind studies, which included serum samples from patients, yielded results in good agreement with measurements by enzyme-linked immunosorbent assay. A major advantage of the fiber optic biosensor is that results can be generated within minutes while isolating the user from hazardous samples. These factors favor development of this biosensor into a facile and rapid diagnostic device.