Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.     

Coronary vasoconstrictor influence of angiotensin II is reduced in remodeled myocardium after myocardial infarction

The renin-angiotensin system plays an important role in cardiovascular homeostasis by contributing to the regulation of blood volume, blood pressure, and vascular tone. Because AT(1) receptors have been described in the coronary microcirculation, we investigated whether ANG II contributes to the regulation of coronary vascular tone and whether its contribution is altered during exercise. Since the renin-angiotensin system is activated after myocardial infarction, resulting in an increase in circulating ANG II, we also investigated whether the contribution of ANG II to the regulation of vasomotor tone is altered after infarction. Twenty-six chronically instrumented swine were studied at rest and while running on a treadmill at 1-4 km/h. In 13 swine, myocardial infarction was induced by ligation of the left circumflex coronary artery. Blockade of AT(1) receptors (irbesartan, 1 mg/kg iv) had no effect on myocardial O(2) consumption but resulted in an increase in coronary venous O(2) tension and saturation both at rest and during exercise, reflecting coronary vasodilation. Despite increased plasma levels of ANG II after infarction and maintained coronary arteriolar AT(1) receptor levels, the vasodilation evoked by irbesartan was significantly reduced both at rest and during exercise. In conclusion, despite elevated plasma levels, the vasoconstrictor influence of ANG II on the coronary circulation in vivo is reduced after myocardial infarction. This reduction in ANG II-induced coronary vasoconstriction may serve to maintain perfusion of the remodeled myocardium.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.