PubMed · 5555271
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L M Allen, H W Allen, J J Quilligan. 1971. Proposal for improved techniques in electron microscopy.. https://doi.org/10.1016/0022-5193(71)90044-0
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Although the phosphorous radioisotope 32P is routinely used in Maxam-Gilbert sequencing, it presents disadvantages that adversely affect safety and ease of use, resolution, and DNA degradation rates. Here, we introduce a new protocol utilizing the alternative isotope 35S for 3'-end-labeling DNA restriction fragments. In our method, plasmid DNA is labeled with [35S]dATPalphaS and 7 Sequenase Version 2.0. We have shown that bands on Maxam-Gilbert sequencing gels are sharp with extremely low background. In addition, a single labeling reaction produces DNA sufficient for 80 sequencing lanes, and the labeled DNA can be utilized for prolonged periods of time without significant degradation. We have further demonstrated the utility of our 35S-end-labeling procedure by successfully mapping the sequence-specificity of DNA damage induced by photoexcited riboflavin. Overall, we have shown that 35S can be used as a safe and practical alternative to 32P in the 3'-end-labeling of DNA restriction fragments.
The effects of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6) and interferon gamma (IFNgamma) were studied on the activity of type 2,5'-deiodinase and on the binding of [125I] T(4) to proteins in human thyroid cytosolic (supernatant) and membrane (pellet) fractions. The activity of thyroid type 2,5'-deiodinase was measured by iodothyronine outer ring deiodinase assay. The binding of [125I] T(4) to the proteins of thyroid cytosolic and membrane fractions was determined by autoradiography. The results showed that thyroid type 2,5'-deiodinase activity could be detected also in the cytosolic fraction, not only in the membrane. TNFalpha, IL-6 and IFNgamma could inhibit the type 2 deiodinase activity in vitro. The dose-dependent binding of [125I] T(4) to the proteins of 29, 66 and 200 kDa could be observed both in the cytosolic and membrane fractions. TNFalpha and IFNgamma inhibited the binding of [125I] T(4) to the two characteristic proteins of type 2,5'-deiodinase, to proteins of 29 and/or 200 kDa, both in the cytosolic and membrane fractions. We conclude that TNFalpha, IL-6 and IFNgamma can inhibit the activity of type 2,5'-deiodinase. Furthermore, TNFalpha and IFNgamma can still also decrease the binding of [125I] T(4) to the enzyme in vitro. It can be suggested that the increased level of these cytokines can be one of the reasons in the induction of the clinical symptoms in nonthyroidal illness associated with low levels of T(3).
In human vascular smooth muscle cells endothelin-1, acting at both endothelin A and endothelin B receptors, has been demonstrated to be both a potent vasoconstrictor and mitogen. Our aim was to study the functional expression of endothelin receptors in human radial artery smooth muscle using both native tissue and cultured cells (RASMCs). Radial artery smooth muscle cells were cultured from arterial explants and loaded with the calcium fluorescent dye fura-2. Cells responded to endothelin-1 and a variety of other vasoconstrictors with rises in cytoplasmic calcium ([Ca2+]c). Arterial rings responded to endothelin-1 with an increase in tension. The response of both cells and arterial rings to endothelin-1 was characterized using the selective endothelin A receptor antagonist BQ123 and the endothelin B receptor antagonist BQ788. The RASMCs were found to express [Ca2+]c responses consistent with the expression of only the endothelin A receptor. Endothelin-1-mediated vasoconstriction in radial artery rings was unaffected by BQ788 but was completely blocked by BQ123. Using the selective radioligands [125I]-PD151242 and [125I]-BQ3020 and a combination of in vitro receptor autoradiography and isolated cell preparations, endothelin A receptors were confirmed to be present on RASMCs and on arterial sections, whereas endothelin B binding was barely detectable on native smooth muscle and on RASMCs.