Synthesis and biological activity of L-tyrosine-based PPARgamma agonists with reduced molecular weight.

A series of PPARgamma agonists were synthesized from L-tyrosine that incorporated low molecular weight N-substituents. The most potent analogue, pyrrole (4e), demonstrated a K(i) of 6.9nM and an EC(50) of 4.7nM in PPARgamma binding and functional assays, respectively. Pyrrole (4e), which is readily synthesized from L-tyrosine methyl ester in four steps, also demonstrated in vivo activity in a rodent model of Type 2 diabetes.

Identification of a series of oxadiazole-substituted alpha-isopropoxy phenylpropanoic acids with activity on PPARalpha, PPARgamma, and PPARdelta.

A series of oxadiazole-substituted alpha-isopropoxy phenylpropanoic acids with dual agonist activity on PPARalpha and PPARgamma is described. Several of these compounds also showed partial agonist activity on PPARdelta. Resolution of one analogue showed that PPARalpha and PPARgamma activity resided in mainly one enantiomer, whereas PPARdelta activity was retained in both enantiomers.

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry.

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears.