Interfacial hybridization kinetics of oligonucleotides immobilized onto fused silica surfaces

Fused silica optical fibers have been used in an intrinsic mode optical configuration as biosensors for fluorescence based detection of hybridization of nucleic acids. In this work, the kinetics of hybridization of single-stranded oligonucleotides that were covalently immobilized were studied. The probe DNA was dT₂₀, and the target was Fluorescein-labeled non-complementary (dT₂₀) or complementary (dA₂₀) oligonucleotide. Chronofluorimetric monitoring of the adsorption and hybridization processes was used to investigate oligonucleotide films of different density, in different salt concentrations, at temperatures of 25 and 40 °C, with the concentration of the target DNA being 0.005–0.1 μM. Mathematical models based on first- and second-order Langmuir adsorption have been examined to describe both the adsorption and the hybridization processes. Experimental data were processed using the models, and the hybridization kinetics were calculated. Hybridization kinetics on these optical fiber DNA sensors was found to be up to three orders faster than results presented for a number of other experiments using different immobilization chemistries.     

The role of glutathione in dopaminergic neuronal survival

An increased production of reactive oxygen species is thought to be critical to the pathogenesis of Parkinson's disease. At autopsy, patients with either presymptomatic or symptomatic Parkinson's disease have a decreased level of glutathione in the substantia nigra pars compacta. This change represents the earliest index of oxidative stress in Parkinson's disease discovered to this point. This study compares the sensitivity of dopaminergic and nondopaminergic neurons in dissociated mesencephalic cultures to the depletion of glutathione. We have found that dopaminergic neurons are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. The possibility that dopaminergic neurons have a higher baseline glutathione level than nondopaminergic neurons is suggested by measurements of levels of cellular glutathione in a parallel system of immortalized embryonic dopaminergic and nondopaminergic cell lines. We also examined the role of glutathione in 1-methyl-4-phenylpyridinium toxicity. Decreasing the glutathione level of dopaminergic neurons potentiates their susceptibility to 1-methyl-4-phenylpyridinium toxicity, although 1-methyl-4-phenylpyridinium does not deplete glutathione from primary mesencephalic cultures. Our data suggest that although a decreased glutathione content is not likely to be the sole cause of dopaminergic neuronal loss in Parkinson's disease, decreased glutathione content may act in conjunction with other factors such as 1-methyl-4-phenylpyridinium to cause the selective death of dopaminergic neurons.     

Determination of 5-methoxypsoralen in suntan cosmetics

A method for the determination of 5-methoxypsoralene in suntan-cosmetics is described. After liquid chromatographic clean-up, the final extract is screened by twodimensional thin layer chromatography. For positive extracts 5-methoxypsoralene is analysed quantitatively by capillary gas chromatography, using 5-alpha-cholestane as an internal standard and flame ionization detection (FID). The results were confirmed by gas chromatography-mass spectrometry (GC-MS). The recoveries for suntan-cosmetics of emulsion type were 70% and for tanning oils 90%. Detection limits of the method were 0,1 to 0,5 mg/kg, depending on the sample type. By application of this method 5-methoxypsoralene was detected in 6 of the 21 cosmetic products at levels up to 28 mg/kg.     

Clenbuterol and anabolic steroids: a previously unreported cause of myocardial infarction with normal coronary arteriograms

During the last 10 years, several cases of myocardial infarction associated with anabolic steroid use have been reported. Postulated mechanisms to explain this association have included changes in lipid levels, the fibrinolytic system, and platelet aggregation. Clenbuterol is a beta 2-agonist with anabolic properties that has not been seen previously with myocardial infarction. We report a case of myocardial infarction in an otherwise healthy 26-year-old body-builder who recently used clenbuterol and anabolic steroids. In this case, synergistic effects of the two agents seem likely to have played a role in the infarct. The normal coronary arteriograms before any anticoagulant or thrombolytic therapy strongly suggest coronary spasm as the mechanism of the infarct.     

Kinetics of receptor-mediated radiotoxicity of 16alpha-[125I]-iodostradiol-3,17beta

AIM: The radiocytotoxic effects in estrogen receptor (ER) containing MCF-7 cells of a mamma carcinoma were investigated following incubation with [125I]E ranging from 1 h to 24 h. METHODS: The receptor status of the cells was confirmed by immunohistochemical staining. The accumulation of [125I]E in MCF-7 cells was tested in the presence and absence of radioinert E and [127I]E and in ER-negative cells in comparison to ER-positive cells. The subcellular distribution was investigated in 0.25 M Saccharose by ultra centrifugation. The radiocytotoxicity was assessed in ER-positive and negative cells by a standard colony forming assay after incubating with [125I]E (1.85 kBq/ml-55.5 kBq/ml) for 1, 2, 4, 8, 12, and 24 h. RESULTS: A significant cytotoxicity was observed only when ER-rich MCF-7-cells were incubated with [125I]E alone. The maximal cytotoxic effect was a reduction of survival fraction to 20-25%. This was achieved at radioactivity concentrations > 37 kBq/ml. Maximal effect was seen after 8 h incubation, extension of incubation time did not further increase toxicity. CONCLUSION: The results suggest that the radioactivity was bound to ER. Through their nuclear localization radioestrogens tagged with radionuclides emitting very low energy electrons (Auger electrons) bear potential for therapy by ER-mediated deposition of lethal doses of ionizing radiation to single cells without affecting neighbouring cells. But, instead of 125I the shorter-living 123I shall be used for labelling because the deciding radiation effectes occur within the first 8 h.     

Electrokinetically controlled DNA hybridization microfluidic chip enabling rapid target analysis

Biosensors and more specifically biochips exploit the interactions between a target analyte and an immobilized biological recognition element to produce a measurable signal. Systems based on surface nucleic acid hybridization, such as microarrays, are particularly attractive due to the high degree of selectivity in the binding interactions. One of the drawbacks of this reaction is the relatively long time required for complete hybridization to occur, which is often the result of diffusion-limited reaction kinetics. In this work, an electrokinetically controlled DNA hybridization microfluidic chip will be introduced. The electrokinetic delivery technique provides the ability to dispense controlled samples of nanoliter volumes directly to the hybridization array (thereby increasing the reaction rate) and rapidly remove nonspecific adsorption, enabling the hybridization, washing, and scanning procedures to be conducted simultaneously. The result is that all processes from sample dispensing to hybridization detection can be completed in as little as 5 min. The chip also demonstrates an efficient hybridization scheme in which the probe saturation level is reached very rapidly as the targets are transported over the immobilized probe site enabling quantitative analysis of the sample concentration. Detection levels as low as 50 pM have been recorded using an epifluorescence microscope.