Detection of liposomal cholesterol and monophosphoryl lipid A by QS-21 saponin and Limulus polyphemus amebocyte lysate

Liposomes containing cholesterol (Chol) have long been used as an important membrane system for modeling the complex interactions of Chol with adjacent phospholipids or other lipids in a membrane environment. In this study we utilize a probe composed of QS-21, a saponin molecule that recognizes liposomal Chol and causes hemolysis of erythrocytes. The interaction of QS-21 with liposomal Chol results in a stable formulation which, after injection into the tissues of an animal, lacks toxic effects of QS-21 on neighboring cells that contain Chol, such as erythrocytes. Here we have used liposomes containing different saturated phospholipid fatty acyl groups and Chol, with or without monophosphoryl lipid A (MPLA), as model membranes. QS-21 is then employed as a probe to study the interactions of liposomal lipids on the visibility of membrane Chol. We demonstrate that changes either in the mole fraction of Chol in liposomes, or with different chain lengths of phospholipid fatty acyl groups, can have a substantial impact on the detection of Chol by the QS-21. We further show that liposomal MPLA can partially inhibit detection of the liposomal Chol by QS-21. The Limulus amebocyte lysate assay is used for binding to and detection of MPLA. Previous work has demonstrated that sequestration of MPLA into the liposomal lipid bilayer can block detection by the Limulus assay, but the binding site on the MPLA to which the Limulus protein binds is unknown. Changes in liposomal Chol concentration and phospholipid fatty acyl chain length influenced the detection of the liposome-embedded MPLA.     

Urban-rural contrasts in explanatory models and treatment-seeking behaviours for stroke in Tanzania

Stroke is an emerging problem in sub-Saharan Africa, about which little is known since most research to date has been based on retrospective, hospital-based studies. This anthropological work, designed to complement a large community-based project on stroke incidence, focuses on local understandings and treatment-seeking behaviours in urban (Dar-es-Salaam) and rural (Hai) areas of Tanzania. Semi-structured interviews (n=80) were conducted with 20 stroke patients, 20 relatives of stroke patients, ten traditional healers, and 30 other local residents. In contrast to common expectations, and literature that finds witchcraft beliefs to be most common in rural areas, stroke in urban Dar was widely believed to emanate from supernatural causes (demons and witchcraft), while in rural Hai, explanations drew mostly on 'natural' causes (hypertension, fatty foods, stress). These different beliefs and explanatory models fed into treatment-seeking behaviours. The first option in Hai was hospital treatment, while in Dar-es-Salaam, where belief in demons led to hospital avoidance, it was traditional healers. In both sites, multiple treatment options (serially or simultaneously) were the norm. Analysis of patient and carer narratives suggested that causation beliefs outweighed other factors, such as cost and distance, in shaping effective treatment. Three policy implications are drawn. First, as other studies have also shown, it is important to engage with, rather than dismiss, local explanations and interpretations of stroke. Stroke awareness messages need to take into account the geographical and belief systems differences. Developing an understanding of explanatory models that recognizes that local beliefs arise from dynamic processes of social interaction will be critical to designing effective interventions. Second, there is a clear role for multiple healing systems with possibility of cross-reference in the case of a chronic, disabling condition like stroke, since biomedical treatment cannot offer a 'quick fix' while traditional healers can help people come to terms with their condition. Third, issues of communication between health services and their patients are particularly critical.     

Two kinds of physician‐assisted death

I argue that the concept ‘physician‐assisted suicide’ covers two procedures that should be distinguished: giving someone access to humane means to end his own life, and taking co‐responsibility for the safe and effective execution of that plan. In the first section I explain the distinction, in the following sections I show why it is important. To begin with I argue that we should expect the laws that permit these two kinds of ‘assistance’ to be different in their justificatory structure. Laws that permit giving access only presuppose that the right to self‐determination implies a right to suicide, but laws that permit doctors to take co‐responsibility may have to appeal to a principle of mercy or beneficence. Actually this difference in justificatory structure can to some extent be found in existing regulatory systems, though far from consistently. Finally I argue that if one recognizes a right to suicide, as Oregon and other American states implicitly do, and as the European Court of Human Rights has recently done explicitly, one is committed to permit the first kind of ‘assistance’ under some conditions.     

Membrane cholesterol uptake by recombinant lipoproteins

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.     

Formulations of single or multiple H. pylori antigens with DC Chol adjuvant induce protection by the systemic route in mice. Optimal prophylactic combinations are different from therapeutic ones

The ability to induce a protective response against Helicobacter pylori infection has been investigated by systemic immunization of mice with urease formulated with the cationic lipid DC Chol. This compound acts both as a formulating agent and as an adjuvant and induces a balanced Th1/Th2 response shown to be more effective for protection in our previous studies. Urease-DC Chol induced a significant protection in prophylaxis but not in therapeutic immunization. The protection level was between 1.5 and 2 log reduction of bacterial density measured by quantitative culture compared to unimmunized-infected mice. In parallel, the protective efficacy of other H. pylori antigens formulated in a similar way and administered with DC Chol was tested. These antigens were tested alone or in combination in prophylactic and therapeutic regimens. Some combinations of antigens induced a better prophylactic or therapeutic activity than urease alone (0.5-1.5 log further reduction in prophylaxis and therapy respectively, P<0.05). The combinations that induced the best protection were different in prophylaxis and therapy. In conclusion, DC Chol provides a convenient and efficient method to formulate different antigens even when they are present in non-compatible buffers initially. Moreover, the results obtained in protection against H. pylori with such formulations should lead the way to future clinical trials.     

Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.     

Genes,environment and responsibility for violent behavior: “Whatever genes one has it is preferable that you are prevented from going around stabbing people”

For the legal system to function effectively people are generally viewed as autonomous actors able to exercise choice and responsible for their actions. It is conceivable that genetic traits associated with violent and antisocial behavior could call into question an affected individual's responsibility for acts of criminal violence. Evidence concerning genes associated with violent and antisocial behavior has been introduced in criminal courts in the USA and Italy, either alone or with associated environmental factors. One example of a “genetic defense” is based on low levels of monoamine oxidase A (MAOA) activity, with a prevalence of around 30% in Caucasian males. In countries with trial by jury it is particularly relevant to consider the views of publics on criminal liability and the significance they assign to evidence citing genetic influences on behavior. This article draws on largely qualitative research looking at participants' explanations of, and assigning of responsibility for, violent and antisocial behavior where environmental or genetic influences are claimed. Genetic factors were not viewed deterministically by participants but were considered by most to be irrelevant to personal responsibility. Notions of human agency, free will and choice were crucial to explanations of problem behaviors and ensured that offenders could be held responsible despite evidence on environmental and genetic factors.     

Cholesterol dynamics in membranes of raft composition: a molecular point of view from 2H and 31P solid-state NMR

Lipidic membrane systems that have been reported to be composed of sphingomyelin (SM)-cholesterol (Chol) microdomains or "rafts" by Dietrich et al. [palmitoyloleoyl-phosphatidylcholine(POPC)/SM/Chol, 1/1/1; Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson, K., and Gratton, E. (2001) Biophys. J. 80, 1417-1428] and by Schroeder et al. [SCRL: Liver-PC/Liver-phosphatidylethanolamine/SM/Cerebrosides/Chol, 1/1/1/1/2; Schroeder, R., London, E., and Brown, D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134] were investigated under the form of fully hydrated liposomes by the noninvasive solid-state (31)P and (2)H NMR method. Liposomes of binary lipid composition POPC/Chol and SM/Chol were also studied as boundary/control systems. All systems are found to be in the liquid-ordered phase (Lo) at physiological temperatures. Use of deuterium-labeled cholesterol afforded finding both the position of the sterol motional axis and its molecular order parameter. The axis of anisotropic rotation of cholesterol is such that the molecule is, on average, quasiperpendicular to the membrane plane, in all of the four systems investigated. Cholesterol order parameters greater than 0.8 are observed, indicating that the sterol is in a very motionally restricted environment in the temperature range 0-60 degrees C. The binary mixtures present "boundary" situations with the lowest values for POPC/Chol and the highest for SM/Chol. The SCRL raft mixture has the same ordering as the SM/Chol, i.e., the highest order parameter values over the temperature range. It demonstrates that in the SCRL mixture cholesterol dynamics is as in the binary system SM/Chol, therefore, suggesting that it might be depleted from the rest of the membrane to form complexes as if it were alone with SM. On the other hand, the mixture POPC/SM/Chol exhibits an intermediate ordering situation between those of SM/Chol and POPC/Chol. This strongly suggests that cholesterol could be in fast exchange, at the NMR time scale (milli- to microseconds), between two or more membrane regions of different dynamics and questions the statement of "rigid domains" made of SM and cholesterol in the model "raft" system POPC/SM/Chol.     

Effect of reconstituted discoidal high-density lipoproteins on lipid mobilization in RAW 264.7 and CHOK1 cells

Reconstituted discoidal high‐density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid‐free apolipoprotein A‐I (apoA‐I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol‐free rHDL were more effective than Chol‐containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA‐I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline‐containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol‐free 96 Å discs, the other rHDL as well as apoA‐I promoted PC efflux from RAW cells. Chol‐containing rHDL were more active than Chol‐free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA‐I and Chol‐free 78 Å rHDL were active enough to remove PC. J. Cell. Biochem. 113: 1208–1216, 2012. © 2011 Wiley Periodicals, Inc.     

Continuity and transformation of body logic

This article is concerned with two distinct corporeal logics. In the first, corporeality is founded on joints, tendons, and mobility; in the second, the envelope and its apertures are considered primordial. The first logic is extant in very few works. Although these texts (e.g. The Iliad, Beowulf) clearly share the same, very specific, conception of the body, they belong to different histories. The corporeal logic of the 'jointed body' (corps articulaire) cannot, therefore, be appraised in terms of longue durée. The texts represent, instead, a moment of transition between the psychodynamics of orality and literacy. A problem correlated to this fact is that readers (ancient and modern) no longer think using the same logic as that pertaining to the jointed body. They tend to translate information regarding the logic of the jointed body into data meaningful in their own logic.     

The roles of cholesterol in pulmonary surfactant: insights from comparative and evolutionary studies

In most eutherian mammals, cholesterol (Chol) comprises approximately 8-10 wt.% or 14-20 mol.% of both alveolar and lamellar body surfactant. It is regarded as an integral component of pulmonary surfactant, yet few studies have concentrated on its function or control. Throughout the evolution of the vertebrates, the contribution of cholesterol relative to surfactant phospholipids decreases, while that of the disaturated phospholipids (DSP) increases. Chol generally appears to dominate in animals with primitive bag-like lungs that lack septation, in the saccular lung of snakes or swimbladders which are not used predominantly for respiration, and also in immature lungs. It is possible that in these systems, cholesterol represents a protosurfactant. Cholesterol is controlled separately from the phospholipid (PL) component in surfactant. For example, in heterothermic mammals such as the fat-tailed dunnart, Sminthopsis crassicaudata, and the microchiropteran bat, Chalinolobus gouldii, and also in the lizard, Ctenophorus nuchalis, the relative amount of Chol increases in cold animals. During the late stages of embryonic development in chickens and lizards, the Chol to PL and Chol to DSP ratios decrease dramatically. While in isolated lizard lungs, adrenaline and acetylcholine stimulate the secretion of surfactant PL, Chol secretion remains unaffected. This is also supported in isolated cell studies of lizards and dunnarts. The rapid changes in the Chol to PL ratio in response to various physiological stimuli suggest that these two components have different turnover rates and may be packaged and processed differently. Infusion of [3H]cholesterol into the rat tail vein resulted in a large increase in Chol specific activity within 30 min in the lamellar body (LB) fraction, but over a 48-h period, failed to appear in the alveolar surfactant fraction. Analysis of the limiting membrane of the lamellar bodies revealed a high (76%) concentration of LB cholesterol. The majority of lamellar body Chol is, therefore, not released into the alveolar compartment, as the limiting membrane fuses with the cell membrane upon exocytosis. It appears unlikely, therefore, that lamellar bodies are the major source of alveolar Chol. It is possible that the majority of alveolar Chol is synthesised endogenously within the lung and stored independently from surfactant phospholipids. The role of cholesterol in the limiting membrane of the lamellar body may be to enable fast and easy processing by maintaining the membrane in a relatively fluid state.     

Insight into the putative specific interactions between cholesterol, sphingomyelin, and palmitoyl-oleoyl phosphatidylcholine

The effects of cholesterol (Chol) on phospholipid bilayers include ordering of the fatty acyl chains, condensing of the lipids in the bilayer plane, and promotion of the liquid-ordered phase. These effects depend on the type of phospholipids in the bilayer and are determined by the nature of the underlying molecular interactions. As for Chol, it has been shown to interact more favorably with sphingomyelin than with most phosphatidylcholines, which in given circumstances leads to formation of lateral domains. However, the exact origin and nature of Chol-phospholipid interactions have recently been subjects of speculation. We examine interactions between Chol, palmitoylsphingomyelin (PSM) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in hydrated lipid bilayers by extensive atom-scale molecular dynamics simulations. We employ a tailored lipid configuration: Individual PSM and Chol monomers, as well as PSM-Chol dimers, are embedded in a POPC lipid bilayer in the liquid crystalline phase. Such a setup allows direct comparison of dimeric and monomeric PSMs and Chol, which ultimately shows how the small differences in PSM and POPC structure can lead to profoundly different interactions with Chol. Our analysis shows that direct hydrogen bonding between PSM and Chol does not provide an adequate explanation for their putative specific interaction. Rather, a combination of charge-pairing, hydrophobic, and van der Waals interactions leads to a lower tilt in PSM neighboring Chol than in Chol with only POPC neighbors. This implies improved Chol-induced ordering of PSM's chains over POPC's chains. These findings are discussed in the context of the hydrophobic mismatch concept suggested recently.     

Temporary membrane distortion of vascular smooth muscle cells is responsible for their apoptosis induced by platelet-activating factor-like oxidized phospholipids and their degradation product, lysophosphatidylcholine

To obtain information about the mechanism of apoptosis induced by oxidized low density lipoproteins (oxLDL) in atherosclerotic plaques, we examined the effects of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF)-like lipids (PAF-LL), which can be derived from oxLDL, on rat vascular smooth muscle cells (VSMC). All the lipids with different structures examined induced apoptosis of VSMC, so we studied the mechanism of induction of apoptosis by LPC. LPC-induced apoptosis was inhibited by alpha-tocopherol (alpha-T) and cholesterol (Chol), but not by other antioxidants such as palmitoyl ascorbic acid and PAF receptor antagonist. The cells temporarily became spherical and highly permeable before induction of apoptosis, and their change in shape was prevented by alpha-T and Chol. From these results, we suggest that the apoptosis induced by oxLDL-derived phospholipids in VSMC is caused by temporary membrane distortion, not through specific receptors.     

Reconsidering Witchcraft: Postcolonial Africa and Analytic (Un)Certainties

African notions of witchcraft are neither archaic nor static but are highly flexible and deeply attuned to the conundrums of our contemporary world. Many anthropologists have recently argued that notions of the African witch provide commentaries on the meaning and merit of modernity as experienced in different historical and cultural settings. By exploring one particular type of witchcraft —that involving rain—amongst the lhanzu of Tanzania, this article suggests instead that some forms of witchcraft may be more pertinent to understanding local notions of "tradition" than "modernity." It is argued that the process of identifying rain witches provides lhanzu men and women with a way to circumscribe, contemplate, and, ultimately, reassert the veracity and significance of a conceptual category they call "tradition." The article concludes by critiquing the homogenizing effects of terms like the African witch and African witchcraft, compelling us to think in terms of pluralities rather than singulars. [Keywords: witchcraft, modernity, tradition, rainmaking, anthropological theory]     

Serum Cholesterol-Decreasing Effect of Heat-Moisture-Treated High-Amylose Cornstarch in Cholesterol-Loaded Rats

Rats were fed on a diet containing cholesterol (Chol) at a level corresponding to the standard Chol intake in humans, and the influence of heat-moisture-treated high-amylose cornstarch (HHA) on their serum Chol level was investigated. HHA decreased the serum level of Chol in rats fed on the diet containing 0.1% Chol, which corresponds to a Chol intake in humans of 800 mg/d, although the liver levels of Chol increased in these rats. HHA did not influence the fecal excretion of Chol/bile acids. It is possible that the decrease in serum Chol level in the rats fed on the high-Chol diet can be attributed to the promotion of Chol uptake in the liver.     

Serum cholesterol-decreasing effect of heat-moisture-treated high-amylose cornstarch in cholesterol-loaded rats

Rats were fed on a diet containing cholesterol (Chol) at a level corresponding to the standard Chol intake in humans, and the influence of heat-moisture-treated high-amylose cornstarch (HHA) on their serum Chol level was investigated. HHA decreased the serum level of Chol in rats fed on the diet containing 0.1% Chol, which corresponds to a Chol intake in humans of 800 mg/d, although the liver levels of Chol increased in these rats. HHA did not influence the fecal excretion of Chol/bile acids. It is possible that the decrease in serum Chol level in the rats fed on the high-Chol diet can be attributed to the promotion of Chol uptake in the liver.     

Sanguma and scepticism: questioning witchcraft in the highlands of Papua New Guinea

Some anthropologists prefer clear-cut depictions of the societies they study, preferring to ignore differences of opinion, failures of belief, and expressions of scepticism and doubt. This tendency is increasingly being challenged, and an emerging literature now explores scepticism. Contributing to this literature, I discuss the scepticism towards witchcraft expressed by my interlocutors in the Papua New Guinea highlands province of Chimbu. I do not juxtapose witchcraft to science, for witchcraft must be taken seriously – especially since accusations often have severe consequences. Expressions of scepticism bring into doubt the absolute certainty of witchcraft as the explanation for misfortunes such as illness and death, and its study provides a more nuanced picture of a society, showing how cultural norms can be transformed and the foundations of violence can be disrupted. This applies to the scepticism I heard expressed, because the challenge it poses to witchcraft accusations may help prevent further violence. An anthropology of scepticism could usefully be applied more broadly to many aspects of culture, since study of scepticism is one way of reaching an understanding of how social change occurs.     

Hypothesis test for causal explanations in human pathology: evaluation of pulmonary edema in 181 autopsied patients with leukemia

Symbolic logic, as used in the formal theory of scientific explanation proposed by Hempel and Oppenheim, has been suggested as the basis for automated medical diagnosis. In human autopsy pathology the determination of cause-and-effect relationships is a major area subject to logical analysis. We propose a modification of the Hempel-Oppenheim schema in which the logical relationships must only be satisfied “much” of the time, as determined by binomial significance tests. The analysis employs “certainty levels” logic with a more limited consistency requirement than classical logic. The analysis is applied to a series of 181 autopsied patients with leukemia in an attempt to determine a possible role of chemotherapeutic agents in the etiology of pulmonary edema. Among 51 patients who had received cytosine arabinoside (Ara-C) within 30 days of death, there was significantly more unexplained moderate or massive pulmonary edema than among patients with no or remote therapy (p<0.001). The results suggest that a symbolic logical analysis combined with a binomial significance test can elucidate cause-and-effect relationships observed at autopsy, especially when there are multiple possible explanations for the same effect.