Structural effects of neutral lipids on the divalent cation-induced interactions of phosphatidylserine - containing bilayers

As Ca2+ and phosphatidylserine (PS) are known to induce the adhesion of bilayer vesicles and form collapsed multibilayer structures in vitro, it was the aim of this study to examine how that interaction and the resultant structures might be modified by neutral lipid species. X-ray diffraction data from multilamellar systems suggest that phosphatidylcholine (PC) and diacylglycerol (DG) might be in the collapsed phase up to a concentration of -30 mole % and that above this concentration these neutral lipids may modify Ca2+-induced bilayer interactions. Using large unilamellar vesicles and long incubations in excess Ca2+ to ensure equilibration, similar preliminary results were again obtained with PC, and also with phosphatidylethanolamine (PE). A combination of X-ray diffraction, thin-layer chromatography, density gradient centrifugation and freeze-fracture electron microscopy, used in conjunction with an osmotic stress technique, showed that (i) -30 mole % PC can be accomodated in the Ca(DOPS)2 phase; and (ii) higher PC levels modify Ca2+-induced bilayer interactions resulting in single lamellar phases of larger dimension and reduced tendency for REV collapse. Importantly, the data suggest that PC is dehydrated during the rapid collapse process leading. to Ca(DOPS)2 formation and exists with this dehydrated phase. Similar results were obtained using PS isolated from bovine brain. Preliminary studies using two different phosphatidylethanolamine (PE) species indicated accomodation by Ca(DOPS)2 of -25-30 mole 0/0 PE and bulk phase separation, of species favouring a non-bilayer phase, at higher levels. Significantly, all PS/PE vesicles appear to undergo a complete Ca2+-induced collapse, even with contents of up to 90 mole % PE. These data suggest that PE may have an important role in fusion mechanisms in vivo. In sum the data lend both structural and stoichiometric evidence for th~ existence of laterally segregated neutral lipid molecules within the same bilayers as PS domains exposed to Ca2+.

Isoflavone exposure throughout suckling results in improved adult bone health in mice

Exposure to isoflavones (ISO), abundant in soy protein infant formula, for the first 5 days of life results in higher bone mineral density (BMD),greater trabecular connectivity and higher fracture load of lumbar vertebrae (LV) at adulthood. The effect of lengthening the duration of exposure to ISO on bone development has not been studied. This study determined if providing ISO for the first 21 days of life, which more closely mimics the duration that infants are fed soy protein formula, results in higher BMD, improved bone structure and greater strength in femurs and LV than a 5-day protocol. Female CD-1 mice were randomized to subcutaneous injections of ISO (7 Q1 mg kg/body weight/day) or corn oil from postnatal day 1 to 21. BMD, structure and strength were measured at the femur and LV at 4 months of age, representing young Q2 adulthood. At the LV, exposure to ISO resulted in higher (P,0.05) BMD, trabecular connectivity and fracture load compared with control (CON). Exposure to ISO also resulted in higher (P,0.05) whole femur BMD, higher (P,0.05) bone volume/total volume and Q3 lower (P,0.05) trabecular separation at the femur neck, as well as greater (P,0.05) fracture load at femur midpoint and femur neck compared with the CON group. Exposure to ISO throughout suckling has favorable effects on LV outcomes, and, unlike previous studies using 5-day exposure to ISO, femur outcomes are also improved. Duration of exposure should be considered when using the CD-1 mouse to model the effect of early life exposure of infants to ISO.

EARLY LIFE EXPOSURE TO GENISTEIN AND DAIDZEIN DISRUPTS STRUCTURAL DEVELOPMENT OF REPRODUCTIVE ORGANS IN FEMALE MICE

In mice, exposure to isoflavones (ISO), abundant in soy infant formula, during the first 5 d of life alters structural and functional development of reproductive organs. Effects of longer exposures are unknown. The study objective was to evaluate whether exposure to a combination of daidzein and genistein in the first 10 compared to 5 d of life results in greater adverse effects on ovarian and uterine structure in adult mice. Thirteen litters of 8–12 pups were cross-fostered and randomized to corn oil or ISO (2 mg daidzein + 5 mg genistein/kg body weight/d) for the first 5 or 10 d of life. The 10-d protocol mimicked the period when infants are fed soy protein formula (SPF) but avoids the time when suckling pups can consume the mother’s diet. Body and organ weights and histology of ovaries and uteri were analyzed. There were no differences in the ovary or uterus weight, number of ovarian follicles, number of multiple oocyte follicles, or percent of ovarian cysts with 5 or 10 d of ISO intervention compared to respective controls. The 10-d ISO group had higher body weights from 6 d to 4 mo. of age and a higher percent of hyperplasia in the oviduct than the respective control. Lower numbers of ovarian corpus lutea and a higher incidence of abnormal changes were reported in the uteri of both ISO groups compared to their respective controls. Five- and 10-d exposure to ISO had similar long-lasting adverse effects on the structures of ovaries and uterus in adult mice. Only the 10-d ISO exposure resulted in greater body weight gain at adulthood.

The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)

Human Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITPα and PITPβ. PITPs are believed to be lipid transfer proteins based on their capacity to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named retinal degeneration B (RdgBα). The PITP domain of RdgBα shares 40 % sequence identity with PITPα and has been shown to possess PI and PC binding and transfer activity. The detailed molecular mechanism of ligand transfer by the human PITPs and the Drosophila PITP domain remains to be fully established. Here, we investigated the membrane interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in comparison to PITPα as determined by the FRET assay. Interestingly, DPI analysis also demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound PITPs. In addition, the FRET studies demonstrated the significance of membrane curvature in the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA) which have smaller head groups, the transfer rate increased. In contrast, when the accepting vesicles contained phosphoinositides which have larger head groups, the transfer rate was diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not appear to influence the ligand transfer rate of PITPs. Both DPI and FRET examinations revealed that the PITP domain of RdgBα was able to bind to membranes. However, the RdgBα PITP domain appears to be a poor binder and transporter of PC.