Mixtures of unsaturated lipids, sphingolipids, and cholesterol form coexisting liquid-disordered and sphingolipid and cholesterol-rich liquid-ordered (Lo) phases in water. The detergent Triton X-100 does not readily solubilize Lo domains, but does solubilize liquid-disordered domains, and is commonly used to prepare detergent-resistant membranes from cells and model membranes. However, it has been proposed that in membranes with mixtures of sphingomyelin (SM), 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), and cholesterol Triton X-100 may induce Lo domain formation, and therefore detergent-resistant membranes may not reflect the presence of preexisting domains. To examine this hypothesis, the effect of Triton on Lo domain formation was measured in SM/POPC/cholesterol vesicles. Nitroxide quenching methods that can detect ordered nanodomains with radii >12 Å showed that in the absence of Triton X-100 this mixture formed ordered state domains that melt with a midpoint (= T(mid)) at ∼45°C. However, T(mid) was lower when detected using various fluorescence resonance energy transfer (FRET) pairs. Furthermore, the T(mid) value was Ro dependent, and decreased as Ro increased. Because FRET can only readily detect domains with radii >Ro, this result can be explained by domain radii that are close to Ro and decrease as temperature increases. An analysis of FRET and quenching data suggests that nanodomain radius gradually decreases from ≥150 Å to <40 Å as temperature increases from 10 to 45°C. Interestingly, the presence of Triton X-100 or a transmembrane-type peptide did not stabilize ordered state formation when detected by nitroxide quenching, i.e., did not increase T(mid). However, FRET-detected T(mid) did increase in the presence of Triton X-100 or a transmembrane peptide, indicating that both increased domain size. Controls showed that the results could not be accounted for by probe-induced perturbations. Thus, SM/POPC/cholesterol, a mixture similar to that in the outer leaflet of plasma membranes, forms nanodomains at physiological temperatures, and TX-100 does not induce domain formation or increase the fraction of the bilayer in the ordered state, although it does increase domain size by coalescing preexisting domains.

Despite the importance of lipid rafts, commonly defined as liquid-ordered domains rich in cholesterol and in lipids with high gel-to-fluid melting temperatures (T(m)), the rules for raft formation in membranes are not completely understood. Here, a fluorescence-quenching strategy was used to define how lipids with low T(m), which tend to form disordered fluid domains at physiological temperatures, can stabilize ordered domain formation by cholesterol and high-T(m) lipids (either sphingomyelin or dipalmitoylphosphatidylcholine). In bilayers containing mixtures of low-T(m) phosphatidylcholines, cholesterol, and high-T(m) lipid, the thermal stability of ordered domains decreased with the acyl-chain structure of low-T(m) lipids in the following order: diarachadonyl > diphytanoyl > 1-palmitoyl 2-docosahexenoyl = 1,2 dioleoyl = dimyristoleoyl = 1-palmitoyl, 2-oleoyl (PO). This shows that low-T(m) lipids with two acyl chains having very poor tight-packing propensities can stabilize ordered domain formation by high-T(m) lipids and cholesterol. The effect of headgroup structure was also studied. We found that even in the absence of high-T(m) lipids, mixtures of cholesterol with PO phosphatidylethanolamine (POPE) and PO phosphatidylserine (POPS) or with brain PE and brain PS showed a (borderline) tendency to form ordered domains. Because these lipids are abundant in the inner (cytofacial) leaflet of mammalian membranes, this raises the possibility that PE and PS could participate in inner-leaflet raft formation or stabilization. In bilayers containing ternary mixtures of PO lipids, cholesterol, and high-T(m) lipids, the thermal stability of ordered domains decreased with the polar headgroup structure of PO lipids in the order PE > PS > phosphatidylcholine (PC). Analogous experiments using diphytanoyl acyl chain lipids in place of PO acyl chain lipids showed that the stabilization of ordered lipid domains by acyl chain and headgroup structure was not additive. This implies that it is likely that there are two largely mutually exclusive mechanisms by which low-T(m) lipids can stabilize ordered domain formation by high-T(m) lipids and cholesterol: 1), by having structures resulting in immiscibility of low-T(m) and high-T(m) lipids, and 2), by having structures allowing them to pack tightly within ordered domains to a significant degree.

SUMMARY Borrelia burgdorferi (the agent of Lyme disease) is unusual in that it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). The bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Through ultrastructural, biochemical and biophysical approaches, we show that these cholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi, and in model membranes from B. burgdorferi lipids. The order and size of the microdomains of intact cells and model membranes are temperature sensitive and correlate with the bactericidal activity of CB2. Here we demonstrate the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote.

Lipid rafts in eukaryotic cells are sphingolipid and cholesterol-rich, ordered membrane regions that have been postulated to play roles in many membrane functions, including infection. We previously demonstrated the existence of cholesterol-lipid-rich domains in membranes of the prokaryote, B. burgdorferi, the causative agent of Lyme disease [LaRocca et al. (2010) Cell Host & Microbe 8, 331–342]. Here, we show that these prokaryote membrane domains have the hallmarks of eukaryotic lipid rafts, despite lacking sphingolipids. Substitution experiments replacing cholesterol lipids with a set of sterols, ranging from strongly raft-promoting to raft-inhibiting when mixed with eukaryotic sphingolipids, showed that sterols that can support ordered domain formation are both necessary and sufficient for formation of B. burgdorferi membrane domains that can be detected by transmission electron microscopy or in living organisms by Förster resonance energy transfer (FRET). Raft-supporting sterols were also necessary and sufficient for formation of high amounts of detergent resistant membranes from B. burgdorferi. Furthermore, having saturated acyl chains was required for a biotinylated lipid to associate with the cholesterol-lipid-rich domains in B. burgdorferi, another characteristic identical to that of eukaryotic lipid rafts. Sterols supporting ordered domain formation were also necessary and sufficient to maintain B. burgdorferi membrane integrity, and thus critical to the life of the organism. These findings provide compelling evidence for the existence of lipid rafts and show that the same principles of lipid raft formation apply to prokaryotes and eukaryotes despite marked differences in their lipid compositions.

Some lipid mixtures form membranes containing submicroscopic (nanodomain) ordered lipid domains (rafts). Some of these nanodomains are so small (radius <5 nm) that they cannot be readily detected with Förster resonance energy transfer (FRET)-labeled lipid pairs with large Ro. We define such domains as ultrananodomains. We studied the effect of lipid structure/composition on the formation of ultrananodomains in lipid vesicles using a dual-FRET-pair approach in which only one FRET pair had Ro values that were sufficiently small to detect the ultrananodomains. Using this approach, we measured the temperature dependence of domain and ultrananodomain formation for vesicles composed of various mixtures containing a high-Tm lipid (brain sphingomyelin (SM)) or dipalmitoyl phosphatidylcholine (DPPC)), low-Tm lipid (dioleoylphosphatidylcholine (DOPC) or 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC)), and a lower (28 mol %) or higher (38 mol %) cholesterol concentration. For every lipid combination tested, the thermal stabilities of the ordered domains were similar, in agreement with our prior studies. However, the range of temperatures over which ultrananodomains formed was highly lipid-type dependent. Overall, vesicles that were closest to mammalian plasma membrane in lipid composition (i.e., with brain SM, POPC, and/or higher cholesterol) formed ultrananodomains in preference to larger domains over the widest temperature range. Relative to DPPC, the favorable effect of SM on ultrananodomain formation versus larger domains was especially large. In addition, the favorable effect of a high cholesterol concentration, and of POPC versus DOPC, on the formation of ultrananodomains versus larger domains was greater in vesicles containing SM than in those containing DPPC. We speculate that it is likely that natural mammalian lipids are tuned to maximize the tendency to form ultrananodomains relative to larger domains. The observation that domain size is more sensitive than domain formation to membrane composition has implications for how membrane domain properties may be regulated in vivo.

Purpose: Investigate the activity of high and low molecular weight biomolecules present in the matrix of human calcium oxalate (CaOx) stones not only on the initial mineral phase formation of calcium and phosphate (CaP) but also on its growth and demineralization of the preformed mineral phase. Materials and Methods: Surgically removed renal stones were analyzed by Fourier Transform Infra Red (FTIR) specFourier Transform Infra Red (FTIR) spec-(FTIR) spec-spectroscopy and only CaOx stones were extracted with 0.05M EGTA, 1 mM PMSF and 1% β-mercaptoethanol. Renal CaOx and only CaOx stones were extracted with 0.05M EGTA, 1 mM PMSF and 1% β-mercaptoethanol. Renal CaOx stone extract was separated into > 10 kDa and < 10 kDa fractions by dialysis. Activity of both the fractions along with whole extract was studied on the three mineral phases of CaP assay system. Results: It was interesting to observe that both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones exhibited different roles in the three mineral phases of CaP. Whole extract exhibited inhibitory activity in all the three assay systems; however, mixed (stimulatory and inhibitory) activity was exhibited by the > 10 kDa and < 10 kDa fractions. SDS-PAGE analysis showed bands of 66 kDa, 80 kDa, 42 kDa in whole EGTA extract lane and > 10 kDa fraction lane. Conclusion: Both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones have a significant influence on calcium and phosphate (CaP) crystallization.

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Nephrolithiasis is a common clinical disorder, and calcium oxalate (CaOx) is the principal crystalline component in approximately 75% of all renal stones. It is widely believed that proteins act as inhibitors of crystal growth and aggregation. Acidic amino acids present in these proteins play a significant role in the inhibition process. In this study, interaction of cal-cium oxalate with human phosphate cytidylyltrans-ferase 1(CCT), a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix has been elucidated in silico and involvement of acidic amino acids in the same. As only sequence of CCT is available, henceforth its 3-D structure was modeled via Homology modeling using Prime module of Schrodinger package. Molecular dynamic simulation of modeled protein with solvation was done by mac-romodel (Schrodinger). The quality of modeled pro-tein was validated by JCSG protein structure valida-tion (PROCHECK & ERRAT) server. To analyze the interaction of modeled protein CCT with calcium oxalate along with role played by acidic amino acids, ‘Docking simulation’ was done using MOE–Dock. Interaction between calcium oxalate and CCT was also studied by substituting acidic amino acid in the active sites of the protein with neutral and positively charged amino acids. The in silico analysis showed the bond formation between the acidic amino acids and calcium atom, which was further substantiated when substitution of these acidic amino acids with alanine, glycine, lysine, arginine and histidine com-pletely diminished the interaction with calcium ox-alate