Protein fouling is a critical problem for ultrafiltration. In this study, we adopted bovine serum albumin (BSA) as a model protein and polysulfone membrane as a typical ultrafiltration membrane. We then investigated the factors of the protein denaturation and aggregation, such as stirring shear stress and intermolecular exchange of disulfide during ultrafiltration, and discussed the BSA fouling mechanism. Fourier transform-infrared analysis revealed that magnetic stirring did not cause any difference in the secondary structural change of BSA gel-like deposits on the ultrafiltration membrane. BSA aggregates were collected from BSA gel-like deposits on the ultrafiltration membrane by centrifugation. Polyacrylamide gel electrophoresis in SDS analysis of BSA aggregates proved that the major binding of the BSA aggregates involved intermolecular disulfhydryl binding and that capping the free thiol group in BSA molecules with cysteine induced a remarkable decrease in the amount of the BSA aggregates during ultrafiltration. We concluded that one of the main factors in the BSA aggregation during ultrafiltration is the intermolecular exchange of disulfide through cysteinyl residue. We also found that the BSA aggregation caused a decrease in alpha-helix from 66% to 50% and an increase in beta-sheet from 20% to 36%, which was presumably because the cysteine residues associated with the intermolecular disulfide bonds had been located in alpha-helices.

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The properties of liposomes are known to be influenced by many factors. Especially, the stability of liposomes is effected by the change of the phase transition temperature (T,) of lipids. In this work, the keto-enol tautomerism of benzoylacetoanilide (BAA) was used to measure the T,. When BAA is added to the liposome suspensions, the ketonic form exists in bulk water and the enolic form in bilayers of the liposomes. Moreover, when the temperature for the system is changed, the change of the ratio of the enolic to ketonic absorances can be expected to show a sharp peak at a temperature corresponding to the T, of lipids. Therefore, the T, value for liposomes can be estimated from the change of the absorbance ratio caused by the change of the temperature for the system. Three kinds of phosphatidylcholines were used as the standard lipids, and these data were used for determination of the T, of hydrogenated egg lecithins.Further, additional effects of cholesterol in liposomes were investigated in the same way. These results indicated that the T, values of hydrogenated egg lecithins decreased linearly with the increase of iodine values, and the T, values for most liposomes dropped suddenly above the concentration of about 15 mol ?6 cholesterol. This suggests that above this concentration cholesterol molecules are incorporated homogeneously into the bilayers and then the T, values decrease due to formation of the mixed layers such as a palisade type between cholesterol and lipid molecules. Consequently, this method for T, determination can be applied even in the diluted aqueous solution with concentration below 0.15% of lipids and is very simple and useful in comparison with common methods.

Pseudohypoaldosteronism type 1 (PHA1) is a rare condition characterized by neonatal salt loss with dehydration, hypotension, hyperkalemia, and metabolic acidosis, despite elevated plasma aldosterone levels and PRA. Two modes of inheritance of PHA1 have been described: an autosomal dominant form and an autosomal recessive form. An autosomal recessive form manifests severe life-long salt wasting resulting from multiple mineralocorticoid target tissue such as sweat, salivary glands, the colonic epithelium, and lung. Contrary, an autosomal dominant PHA1 manifests milder salt wasting that gradually improves with advancing age. Recently, in one sporadic and four dominant cases, four different mutations including two frame shift mutations, two premature termination codons, and one splice site mutation in the mineralocorticoid receptor (MR) gene were identified. We studied the molecular mechanisms of one Japanese family with a renal form of PHA1. PCR and direct sequencing of the MR gene identified a heterozygous point mutation changing codon 924 Leu (CTG) to CCG (Pro) (L924P) in all affected members. COS-1 cells were transfected with expression vectors for either wild type or the mutant MR-L924P receptors, together with the reporter plasmid (glucocorticoid response element tk-CAT). Aldosterone increased CAT activity in cells expressing wild-type receptor, but had no effect in cells expressing the mutant receptors. These results suggest that mineralocorticoid resistance in this family is due to a missense mutation in the MR gene. To our knowledge, this is the first case of the missense mutation of the MR gene in renal PHA1.