A comparative study on the extraction of membrane-bound bilirubin from erythrocyte membranes using various methods

In this study, we used three different methods for the extraction of membrane-bound bilirubin (EMB) from erythrocyte membranes. Use of 2.5% albumin, pH 7.4, for elution of EMB resulted in only 34% of the total EMB which was estimated after the solubilization of bilirubin-loaded erythrocyte membranes (BLEMs) with 1% SDS. On the other hand, incubation of BLEMs with 38 mM sodium carbonate solution containing 5 mM EDTA, pH 11.0, yielded 77% of the total EMB. Application of Fog\u27s reaction for the estimation of EMB directly on the BLEMs resulted in the estimation of 75% of the total EMB. These results suggest that either of the above methods, i.e. use of albumin or high pH, or direct Fog\u27s reaction cannot estimate the total EMB correctly. Increase in ionic strength from 0.15 to 0.45 did not release any EMB from erythrocyte membranes. Therefore, the best method for the estimation of total EMB is the solubilization of membrane with 1% SDS followed by Fog\u27s reaction method. Copyright (C) 1999 Elsevier Science B.V

Elevated Intracellular Calcium Triggers Recruitment of the Receptor Cross-talk Accessory Protein Calcyon to the Plasma Membrane

Calcyon is called a cross-talk accessory protein because the mechanism by which it enables the typically Gs-linked D1 dopamine receptor to stimulate intracellular calcium release depends on a priming step involving heterologous Gq-linked G-protein-coupled receptor activation. The details of how priming facilitates the D1R calcium response have yet to be precisely elucidated. The present work shows that calcyon is constitutively localized both in vesicular and plasma membrane compartments within HEK293 cells. In addition, surface biotinylation and luminescence assays revealed that priming stimulates a 2-fold increase in the levels of calcyon expressed on the cell surface and that subsequent D1R activation produces further accumulation of the protein in the plasma membrane. The effects of priming and D1R agonists were blocked by nocodazole implicating microtubules in the delivery of calcyon-containing vesicles to the cell surface. Accumulation of calcyon in the plasma membrane correlated well with increased intracellular calcium levels as thapsigargin mimicked, and 2-aminoethoxydiphenylborane abrogated, the effects of priming. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII) also blocked the effects of priming and D1R agonists. Furthermore, expression of constitutively active forms of the kinase bypassed the requirement for priming indicating that CaMKII is a key effector in the Ca2+ and microtubule-dependent delivery of calcyon to the cell surface

Calcium-induced bilirubin-dependent hemolysis of human erythrocytes

Human erythrocytes, preincubated with different concentrations of calcium chloride (0.17-1.67 mM) showed hemolysis after addition of bilirubin (72 μM). Hemolysis was observed only when cells were incubated first with calcium followed by bilirubin and not vice versa. This hemolysis was found to be dependent upon several factors such as concentration of bilirubin, time of incubation of erythrocytes with calcium and time of incubation of bilirubin with the calcium-loaded erythrocytes. Inclusion of EDTA in the incubation medium reduced the percentage hemolysis to a significant extent. Involvement of activated oxygen species in hemolytic process seems to be unlikely as inclusion of sodium azide and catalase did not prevent hemolysis. A comparison of other bivalent cations such as Ba2+, Mg2+, Mn2+ and Cu2+ with Ca2+ for their ability to hemolyse cells in presence of bilirubin shows that Ba2+ and Mg2+ are ineffective, whereas both Mn2+ and Cu2+ induce hemolysis both in the absence as well as in the presence of bilirubin. However, their mechanism of hemolysis is different from that of calcium-induced hemolysis. Formation of calcium-induced hydrophobic aggregates of phospholipid molecules in erythrocyte membrane may open the new binding sites for bilirubin on these membranes which may perturb the membrane conformation

Erythrocytes from healthy smokers bind more bilirubin than the erythrocytes from healthy nonsmokers

Cigarette smoking is an adverse prognostic factor for health. Its damaging effects on many enzymatic and cellular activities are well known. The present study was carried out to evaluate whether there is a difference in the binding of bilirubin to the erythrocytes from healthy smokers and non smokers. The results suggest that the binding of bilirubin to the erythrocytes from healthy smokers as well as in vitro smoked erythrocytes is significantly higher than that of healthy non-smokers

Public Perception of Financial Incentives During COVID-19: A Case Study of the Ready-Made Garment Sector in Bangladesh

In the midst of the COVID-19 pandemic, researchers are considering resilience factors for global industry to provide public policy experts with field insights. Financial incentives are one tool used to support industry. This article focuses on the ready-made garment sector suggesting there is a relationship between incentives and workers in this industry, specifically when facility owners anticipate incentives from the government. The primary findings are that sector respondents are aware incentives by the government will be provided, that additional support allocations can be used for alternative uses by the owners, and that the sector is recovering by utilizing resilience factors