Phospholipid composition of the mammalian red cell membrane can be rationalized by a superlattice model

Although the phospholipid composition of the erythrocyte membrane has been studied extensively, it remains an enigma as to how the observed composition arises and is maintained. We show here that the phospholipid composition of the human erythrocyte membrane as a whole, as well as the composition of its individual leaflets, is closely predicted by a model proposing that phospholipid head groups tend to adopt regular, superlattice-like lateral distributions. The phospholipid composition of the erythrocyte membrane from most other mammalian species, as well as of the platelet plasma membrane, also agrees closely with the predictions of the superlattice model. Statistical analyses indicate that the agreement between the observed and predicted compositions is highly significant, thus suggesting that head group superlattices may indeed play a central role in the maintenance of the phospholipid composition of the erythrocyte membrane.     

Intracellular delivery of lipopolysaccharide during DNA transfection activates a lipid A-dependent cell death response that can be prevented by polymyxin B

The presence of lipopolysaccharide (LPS) as a contaminant in plasmid DNA prepared from Escherichia coli is well documented, and we have previously demonstrated that LPS internalization during adenovirus-mediated gene transfer can generate a toxicity in some primary cell types. We demonstrate here that in addition to adenoviral systems, several commonly used nonviral methods of gene transfer also activate this cell death response in the presence of LPS. Subcomponents of LPS were analyzed and the toxicity was found to be due to the lipid A component of LPS. The LPS-chelating antibiotic polymyxin B, when present at concentration of 10-30 micrograms/ml, can block this toxicity.     

Crosslinking of apolipoprotein E by products of lipid peroxidation

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.     

Voltage-induce capacitance relaxation of lipid bilayer membranes. Effects of membrane composition

The specific capacity of black lipid membranes of different phospholipids dissolved in n-alkanes was investigated. The hydrocarbon thickness of these membranes, as calculated from the electric capacity with a dielectric constant of 2.1, was in most cases close to 5 nm. It was found that the specific capacity is not constant with time after blackening. It shows a linear time dependence characteristic for each lipid/solvent system. The influence of a transmembrane potential on the capacity of the membranes was measured. It was shown that the extent of the capacity change, obtained 10 s after applying a voltage, was strongly dependent on the lipid composition as well as the solvent content of the membranes. The capacity change of the membranes seems to be caused mainly by a thickness change and not by an area increase of the membranes.     

Occult B cell malignancies can be detected by three-color flow cytometry in patients with cytopenias

Patients with unexplained cytopenias often present a diagnostic dilemma with minimal morphologic or cytogenetic changes to identify the underlying disease process. We have used multidimensional flow cytometry in a study of patients with cytopenias and found that this technology established, changed, or refined the diagnosis in 17/121 patients. Using the flow cytometric technique of CD45 and right angle light scatter (SSC) gating with two additional markers in a three-color analysis, eight of 121 patients were found to have hairy cell leukemia (HCL), in the absence of definitive morphologic findings of HCL. Two additional patients were found to have non-Hodgkin's lymphoma (NHL). Myeloid abnormalities, myelodysplasia (MDS) or acute leukemia was detected in seven of 56 patients with unexplained pancytopenia. Six of 65 patients identified with cytopenias resulting from lymphoid neoplasms had been referred for bone marrow transplantation (BMT) with a presumptive diagnosis of MDS, with subsequent deferral of BMT upon correct diagnosis. The screening technique is incorporated into an extensive immunophenotyping scheme to identify hematopoietic abnormalities using multidimensional flow cytometry (MDF). HCL cells (detected as low as 1.3%) reside in the same position as normal monocytes in the CD45 and SSC plots but could be distinguished from monocytes based on the expression of HLA-DR without CD11b, and expression of CD19. Further phenotyping of the abnormal population confirmed immunoglobulin light chain restriction, CD11c, and CD25 expression. Non-Hodgkin's lymphoma was detected as aberrant mature lymphocytes expressing B lymphoid markers, CD5 and light chain restriction. Myeloid abnormalities were identified in the myeloblast or maturing myeloid cell fractions. The flow cytometric scheme described can be used in primary diagnosis. The technique is definitive, sensitive, and stresses the importance of distinguishing lymphoid from myeloid etiology of cytopenias.     

Role of apolipoprotein B and lipoprotein lipase gene polymorphism in variability of serum lipid levels

The serum lipid level is determined by the complex interaction of genetic and environmental factors. The genetic component of this system includes apolipoprotein B (apoB) and lipoprotein lipase (LPL) genes. Three RFLP markers (XbaI, MspI, EcoRI) in the apoB gene and PvuII-RFLP in the LPL gene were genotypes and five lipid traits (fasting plasma levels of total cholesterol, triglycerides, HDL-, LDL-, and VLDL- cholesterol) were measured in 122 unrelated individuals without clinical signs of cardiovascular disease. Several alleles and genotypes which were associated with significant effects on cholesterol, triglycerides, and LDL-cholesterol levels were identified. Analysis of intragenotypic variance and multivariate measures of the mean values of lipid concentrations indicate that apoB gene variation may contribute both to the level and variability of plasma lipids.     

Epileptic seizures can be anticipated by non-linear analysis

Epileptic seizures are a principal brain dysfunction with important public health implications, as they affect 0.8% of humans. Many of these patients (20%) are resistant to treatment with drugs. The ability to anticipate the onset of seizures in such cases would permit clinical interventions. The view of chronic focal epilepsy now is that abnormally discharging neurons act as pacemakers to recruit and entrain other normal neurons by loss of inhibition and synchronization into a critical mass. Thus, preictal changes should be detectable during the stages of recruitment. Traditional signal analyses, such as the count of focal spike density, the frequency coherence or spectral analyses are not reliable predictors. Non-linear indicators may undergo consistent changes around seizure onset. Our objective was to follow the transition into seizure by reconstructing intracranial recordings in implanted patients as trajectories in a phase space and then introduce non-linear indicators to characterize them. These indicators take into account the extended spatio-temporal nature of the epileptic recruitment processes and the corresponding physiological events governed by short-term causalities in the time series. We demonstrate that in most cases (17 of 19), seizure onset could be anticipated well in advance (between 2-6 minutes beforehand), and that all subjects seemed to share a similar 'route' towards seizure.     

Platelet membrane actin may be partially embedded in lipid bilayer and disulfide linked

Three genes coding for the endonuclease, methylase, and specificity subunits of a type I restriction-modification (R-M) system in the Lactococcus lactis plasmid pIL2614 have been characterized. Plasmid location, sequence homologies, and inactivation studies indicated that this R-M system is most probably of type IC.     

Secondary pneumonia. It can be prevented by care

An oncology nursing team at the Freiburg University Hospital have developed a detection and prevention plan for respiratory complications. The plan was originally applied only to oncology patients, but due to its positive results it has since been put in practice in other parts of the hospital. Each patient that enters the hospital fills out a medical survey that is used as a baseline to decide what level of risk the patient presents in acquiring a respiratory infection during their hospitalization. A point system obtained from the survey is evaluated on a scale that allows the nursing care to be adjusted accordingly.     

Extinction can be enhanced by a concurrent excitor.

Five experiments used rat subjects to investigate the impact on extinction of the presence of other conditioned stimuli. In Experiments 1 (Pavlovian magazine approach) and 2 (instrumental discriminative training), an excitatory stimulus (X) was extinguished alone, in conjunction with a previously reinforced other stimulus (A), in conjunction with previously nonreinforced other stimulus (B), or it was spared extinction. Responding during extinction was greatest to AX, and subsequent testing of X alone showed AX extinction to have produced the most decrement to X. Experiment 3 found similar results using a within-subject design. Experiment 5 continued separate reinforced presentations of A during extinction. This procedure not only promoted extinction of X but also converted it into a conditioned inhibitor. These experiments bear on the mechanisms of overshadowing and stimulus processing, as well as provide information on the determinants of extinction. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Studies on receptor binding site of insulin: the hydrophobic B12Val can be substituted by hydrophilic thr

L. Benjamin's (1984) structural analysis of social behavior (SASB) system was used as the organizing framework within which to characterize the phenomenology of self and other relationship experience among subtypes of alcoholic men. Within the context of a community-based study of psychopathology, groups of antisocial alcoholic (AAL), nonantisocial alcoholic (NAAL), and nonalcoholic (control) men completed ratings of their introject (self-concept) and spousal experience. Group differences in demography and psychopathology provided strong support for subtype variations among alcoholic men that could not be attributed to global differences in adaptive functioning. SASB data showed consistency in circumplex ordering across the groups in ratings of self-experience and in ratings of the spousal relationship. AAL men were the most self-neglecting, blaming, and least trusting, and control men were the most relationally connected, with NAAL men falling in between. Despite the importance of the subtyping distinction, in some areas, alcoholism, regardless of subtype, was the core differentiating factor.     

Role of cholesterol in regulating apolipoprotein B secretion by the liver

The review examines the evidence that the supply of cholesterol available for incorporation into nascent lipoprotein particles exerts a regulatory influence on apolipoprotein (apo) B secretion by the liver. Support for this hypothesis comes both from in vitro experiments and from recent observations in normal subjects and patients with dyslipidemia associated with familial hypercholesterolemia, obesity, noninsulin dependent diabetes mellitus, growth hormone deficiency and cholesteryl ester storage disease. The findings do not negate a role for triglyceride synthesis in determining apoB secretion in very low density lipoprotein, but the inhibitory effects on the latter process of pharmacological blockade of cholesterol synthesis or esterification suggest that it is conditional upon an adequate supply of cholesteryl ester.     

Homozygous protein C deficiency can be detected by prenatal diagnosis

Homozygous protein C deficiency (HPCD) with purpura fulminans is a rare condition with an estimated incidence of 1-2 per 400,000 births. About 20 case reports have appeared since the first one was published in 1983. HPCD provides an excellent illustration of the fundamental importance of the protein C anticoagulant pathway. This severe coagulopathy results in serious organ damage, often already in utero, and without treatment it is incompatible with life. The treatment options include fresh frozen plasma and protein C concentrate in the acute phase, followed by oral anticoagulant therapy. Over 160 different point mutations in the protein C gene have been identified in recent years, offering new possibilities for prenatal diagnosis. The article describes the case of a family who lost two children with congenital HPCD. But where the specific point mutation was identified thus enabling prenatal diagnosis to be performed in a subsequent pregnancy.     

Natural ligands of the B cell adhesion molecule CD22 beta can be masked by 9-O-acetylation of sialic acids

CD22 beta is a B cell-restricted phosphoprotein expressed on the surface of mature resting B cells. It mediates interactions with other cells partly or exclusively via recognition of alpha 2-6-linked sialic acids on glycoconjugates. The sialylated N-linked oligosaccharides recognized best by CD22 beta are common to many glycoproteins, suggesting that additional regulatory mechanisms may exist. Since the exocyclic side chain of sialic acid is required for recognition, we explored the effects of a naturally occurring modification of the side chain, 9-O-acetylation. Semisynthetic N-linked oligosaccharides terminating with 9-O-acetylated, alpha 2-6-linked sialic acids showed markedly reduced binding to CD22 beta relative to their non-O-acetylated counterparts. Murine lymphoid cells were probed for natural CD22 beta ligands that might be O-acetylated using recombinant soluble forms of CD22 beta (CD22 beta Rg) and influenza C esterase (CHE-Fc, which specifically removes 9-O-acetyl esters from sialic acids). By flow cytometry analysis, CD22 beta Rg binding to splenic B cells and a subset of T cells was increased by pretreatment with CHE-Fc, indicating that some potential CD22 beta ligands are naturally "masked" by 9-O-acetylation. Unmasking of these CD22 beta ligands by removal of 9-O-acetyl esters from intact splenocytes substantially increases their CD22 beta-dependent adhesion in an in vitro adhesion assay. Probing of murine lymphoid tissue sections by CD22 beta Rg and CHE-Fc treatment demonstrates regionally restricted and differentially expressed patterns of distribution between masked and unmasked ligands. For example, lymph node-associated follicular B cells express high levels of CD22 beta ligands, none of which are masked by 9-O-acetylation. In contrast, the ligands on lymph node-associated dendritic cells are almost completely masked by 9-O-acetylation, suggesting that masking may regulate interactions between CD22 beta-positive B cells and dendritic cells. In the thymus, only medullary cells express CD22 beta ligands, and a significant portion of these are masked by 9-O-acetylation, particularly at the cortical-medullary junction. Thus, 9-O-acetylation of sialic acids on immune cells is in a position to negatively regulate CD22 beta adhesion events in a manner depending on both cell type and tissue localization.     

Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments

Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.     

Translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apolipoprotein B are determined by the presence of beta sheet domains

Apolipoprotein (apo) B100 is an atypical secretory protein in that its translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal alpha globular domains but no beta sheet domains, and apoB13,16,beta, which has an amphipathic beta sheet domain of apoB100 inserted into apoB13,16. These constructs, along with carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induced proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,beta, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined by the presence of a lipid binding beta sheet domain. It is possible that beta sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel.