Profiling cells and body fluids--chromatography and two-dimensional electrophoresis as complementary techniques

Recent results on the use of gas chromatography-mass spectrometry, high-performance liquid chromatography with diode array detector, amino acid analysis and high-resolution two-dimensional (2-D) electrophoresis to study body fluids and cells in health and disease are surveyed. The chromatography profiling techniques are particularly suitable for the diagnosis of inborn errors of metabolism, with DNA technology as a complementary tool for prenatal diagnosis. Both chromatography and electrophoresis were utilized to study pre-diagnostic sera from the JANUS serum bank and to classify certain bacteria. Protein profiling by 2-D electrophoresis was employed to identify marker proteins associated with the metastatic properties of cloned cancer cells. The electrophoretic technique is also appropriate as a preparative tool for isolating sufficient amounts of marker proteins for microsequencing of amino acids. Chromatography and protein profiling were complementary tools for evaluating the toxicity and mutagenicity of environmental samples in a new test utilizing living human leukocytes and fibroblasts.     

PRIMARY DNA DAMAGE, HPRT MUTATION AND CELL INACTIVATION PHOTOINDUCED WITH VARIOUS SENSITIZERS IN V79 CELLS

DNA strand breaks and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants were measured in parallel in photochemically treated (PCT) cells and compared at the same level of cell survival. Chinese hamster fibroblasts (V79 cells) were either incubated with the lipophilic dyes tetra(3—hydroxyphenyl)porphyrin (3THPP) and Photofrin II (PII), the anionic dye meso -tetra(4—sulfonatophenyl)porphine (TPPS₄) or the cationic dye meso -tetra( N -methyl-4-pyridyl)porphine ( p -TMPyPH₂ before light exposure. In the cells, the lipophilic dyes were localized in membranes, including the nuclear membrane, while the hydrophilic dyes were taken up primarily into spots in the cytoplasm. In addition, the hydrophilic TPPS₄ was distributed homogeneously throughout the whole cytoplasm and nucleoplasm. According to the HPRT mutation test, the mutagenicity of light doses survived by 10% of the cells was a factor of six higher in the presence of 3THPP than of PII, whereas for X-rays it was a factor of three higher than for PCT with 3THPP. Light exposure in the presence of the hydrophilic dyes TPPS₄ and p -TMPyPH₂ was not significantly mutagenic. There was no correlation between the induced rates of HPRT mutants and of DNA strand breaks. Thus, TPPS₄ was the most efficient sensitizer with regard to DNA strand breaks when compared at the same level of cell survival, followed by 3THPP, PII and p -TMPyPH₂. Hence, the rate of DNA strand breaks cannot be used to predict the mutagenicity of PCT.     

Potential of capillary electrophoresis, tandem mass spectrometry and coupled capillary electrophoresis-tandem mass spectrometry as diagnostic tools

Urine and blood samples from patients with known metabolic disorders have been analyzed by CE, MS-MS and CE-MS-MS. For the identification of defects in acylcarnitine metabolism, blood spots on filter paper were analyzed using an MS-MS "neonatal screening" approach. Direct CE-MS-MS analysis was used for the analysis of urine samples from patients with different metabolic disorders, including galactosemia, neuroblastoma, Zellweger syndrome, propionic acidemia and alcaptonuria. The sensitivity of the CE-MS-MS method was increased by use of multiple reaction monitoring.     

Chemical, antioxidant and antimicrobial investigations of Pinus cembra L. bark and needles

The chemical constituents and biological activity of Pinus cembra L. (Pinaceae), native to the Central European Alps and the Carpathian Mountains, are not well known. The aim of the present work was to examine the phenolic content, antioxidant and antimicrobial effects of hydromethanolic extracts of Pinus cembra L. bark and needles. Bark extract had higher concentrations of total phenolics (299.3 vs. 78.22 mg gallic acid equivalents/g extract), flavonoids (125.3 vs. 19.84 mg catechin equivalents/g extract) and proanthocyanidins (74.3 vs. 12.7 mg cyanidin equivalents/g extract) than needle extract and was more active as a free radical scavenger, reducing agent and antimicrobial agent. The EC?? values in the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) and reducing power assays were 71.1, 6.3 and 26 mg/mL for bark extract and 186.1, 24 and 104 mg/mL for needle extract, respectively. In addition, needle extract showed ferrous ions chelating effects (EC?? = 1,755 μg/mL). The antimicrobial effects against Staphylococcus aureus, Sarcina lutea, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans were assessed by the agar diffusion method. Both extracts (4 mg/well) were active against all the microorganisms tested; bark extract showed higher inhibition on all strains. These results indicate that Pinus cembra L. bark and needles are good sources of phytochemicals with antioxidant and antimicrobial activities.