2-DE analysis of a new human cell line EM-G3 derived from breast cancer progenitor cells and comparison with normal mammary epithelial cells.

We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized clonal cell line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 cells may represent progenitors of the mammary epithelial cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 line with proteins from populations of normal mammary epithelial cells (NME), and determined the phenotype of both types of cells. NME cells are a mixture of both main cell types in breast epithelia, myoepithelial and luminal cells. The EM-G3 breast cancer cell line has a unique basal-like phenotype. We identified proteins that are differently expressed in these cells. Cytokeratin 16, cytokeratin 19, squamous cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intracellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 cells. The proteins up-regulated in EM-G3 cells may represent potential protein markers of mammary epithelial cells progenitors and may be important in early phase of carcinogenesis.

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora.

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.

Analytical monitoring of trinitrotoluene metabolites in urine by GC-MS. Part I. Semiquantitative determination of 4-amino-2,6-dinitrotoluene in human urine.

A routine procedure for the monitoring of occupational exposure to 2,4,6-trinitrotoluene (TNT) based on the semiquantitative determination of 4-amino-2,6-dinitrotoluene (4A2,6-DNT) in urine samples by gas chromatography (GC-MS) was developed. For calibration purposes, a standard sample of 4-amino-4,6-dinitrotoluene was prepared. Urine samples were collected from munition workers in a military plant in Hradec Kralove district. The samples were extracted into ethyl acetate, and analyzed by GC-MS without previous derivatization. The GC was equipped with a DB1701 column (30 m, 0.25 mm, 0.25 microm) for optimum separation. 4A2,6-DNT was detected at concentrations above the limit of quantitation (0.2 mg/L). This simple method is sufficient for the screening of high degree of TNT exposure and constitutes the first step of a broad project focused on this occupational medicine problem.

Dracophane, a metacyclophane derivative from the resin of Dracaena cinnabari Balf.

Dracophane, a novel structural derivative of metacyclophane, was isolated from the resin of Dracaena cinnabari Balf. The structure of this compound was determined by spectroscopic methods to be 3,12,21-trihydroxy-1,10,19-tris(4-hydroxyphenyl)-5,14,23-trimethoxy[3.3.3]metacyclophane.