Hereditary spherocytosis and elliptocytosis erythrocytes show a normal transbilayer phospholipid distribution. (33/2206)

Phosphatidylserine (PS) asymmetry was determined in red blood cells from patients with hereditary spherocytosis and elliptocytosis. No PS-exposing subpopulations were detected using the very sensitive method with fluorescently labeled annexin V. Treatment with N-ethylmaleimide or adenosine triphosphate (ATP) depletion to inactivate the flipase did not lead to formation of PS-exposing subpopulations in these cells, but elevated intracellular calcium levels did lead to extensive scrambling of the PS asymmetry. Although interactions of the membrane skeleton with the phospholipid bilayer have been suggested to stabilize the asymmetric distribution of PS across the bilayer, our data show that red blood cells with a severely damaged membrane skeleton are able to preserve asymmetry, even under conditions in which restoration of the asymmetric distribution is excluded. Moreover, the loss of membrane asymmetry in these cells requires active scrambling involving high levels of intracellular calcium as in normal cells. Our data show that the severe disorder of the membrane skeleton found in these cells does not affect the activity of flipase or scramblase, indicating that these proteins are not regulated by, nor coupled to the membrane skeleton assembly, and that possible thrombotic events in spherocytosis patients are not likely associated with altered PS topology of the red blood cells.  (+info)

CD14 is a component of multiple recognition systems used by macrophages to phagocytose apoptotic lymphocytes. (34/2206)

Expression of the aminophospholipid phosphatidylserine (PS) on the surface of apoptotic lymphocytes and lipid-symmetric erythrocytes triggers their phagocytosis by macrophages. Phagocytosis by both activated and unactivated macrophages, which utilize different recognition systems, can be blocked by certain monoclonal antibodies directed against the LPS receptor, CD14. Here we investigate the requirement for CD14 in the phagocytosis of both apoptotic thymocytes and lipid-symmetric erythrocytes by both activated and unactivated macrophages. We show that phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages is completely abolished when CD14 is removed from macrophages by cleaving its glycosylphosphatidylinositol tether with phospholipase C. This treatment also substantially reduces phagocytosis of apoptotic lymphocytes by both types of macrophages. Unactivated LR-9 mouse macrophages which are deficient in CD14 expression are completely unable to phagocytose either apoptotic thymocytes or lipid-symmetric erythrocytes. These results argue that CD14 is an absolute requirement for the phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages, despite their different recognition systems, that CD14 contributes at least substantially to the phagocytosis of apoptotic lymphocytes by both activated and unactivated macrophages, and that activated macrophages may also possess an alternate, CD14-independent mechanism for phagocytosis of apoptotic lymphocytes.  (+info)

A review of nutrients and botanicals in the integrative management of cognitive dysfunction. (35/2206)

Dementias and other severe cognitive dysfunction states pose a daunting challenge to existing medical management strategies. An integrative, early intervention approach seems warranted. Whereas, allopathic treatment options are highly limited, nutritional and botanical therapies are available which have proven degrees of efficacy and generally favorable benefit-to-risk profiles. This review covers five such therapies: phosphatidylserine (PS), acetyl-l-carnitine (ALC), vinpocetine, Ginkgo biloba extract (GbE), and Bacopa monniera (Bacopa). PS is a phospholipid enriched in the brain, validated through double-blind trials for improving memory, learning, concentration, word recall, and mood in middle-aged and elderly subjects with dementia or age-related cognitive decline. PS has an excellent benefit-to-risk profile. ALC is an energizer and metabolic cofactor which also benefits various cognitive functions in the middle-aged and elderly, but with a slightly less favorable benefit-to-risk profile. Vinpocetine, found in the lesser periwinkle Vinca minor, is an excellent vasodilator and cerebral metabolic enhancer with proven benefits for vascular-based cognitive dysfunction. Two meta-analyses of GbE demonstrate the best preparations offer limited benefits for vascular insufficiencies and even more limited benefits for Alzheimer's, while "commodity" GbE products offer little benefit, if any at all. GbE (and probably also vinpocetine) is incompatible with blood-thinning drugs. Bacopa is an Ayurvedic botanical with apparent anti-anxiety, anti-fatigue, and memory-strengthening effects. These five substances offer interesting contributions to a personalized approach for restoring cognitive function, perhaps eventually in conjunction with the judicious application of growth factors.  (+info)

Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation. (36/2206)

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca2+-dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-alpha and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-alpha is responsible for the initial Ca2+- and phosphatidylserine-dependent electrostatic membrane binding of PKC-alpha, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp58 and Phe60) and C1b (Tyr123 and Leu125) domains of the native PKC-alpha molecule and measured the effects of mutations on vesicle binding, enzyme activity and monolayer penetration. Results show that the hydrophobic residues in the C1a domain are essential for the membrane penetration and activation of PKC-alpha, whereas those in the C1b domain are not directly involved in these processes. Based on these results in conjunction with our previous structure-function studies of the C2 domain (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), we propose a mechanism for the in vitro membrane binding and activation of conventional PKC that accounts for the temporal and spatial sequences of PKC activation.  (+info)

Inhibition of NF-kappa B activity in human T lymphocytes induces caspase-dependent apoptosis without detectable activation of caspase-1 and -3. (37/2206)

NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL.  (+info)

Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex. (38/2206)

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.  (+info)

Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein. (39/2206)

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.  (+info)

Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2. (40/2206)

Phosphatidylserine (PtdSer) is synthesized in mammalian cells by two base-exchange enzymes: PtdSer synthase (PSS)-1 primarily uses phosphatidylcholine as a substrate for exchange with serine, whereas PSS2 uses phosphatidylethanolamine (PtdEtn). We previously expressed murine PSS1 in McArdle hepatoma cells. The activity of PSS1 in vitro and the synthesis of PtdSer and PtdSer-derived PtdEtn were increased, whereas PtdEtn synthesis from the CDP-ethanolamine pathway was inhibited [Stone, Cui and Vance (1998) J. Biol. Chem. 273, 7293-7302]. We have now cloned and stably expressed a murine PSS2 cDNA in McArdle cells and M.9.1.1 cells [which are ethanolamine-requiring mutant Chinese hamster ovary (CHO) cells defective in PSS1]. Expression of the PSS2 in M.9.1.1 cells reversed the ethanolamine auxotrophy. However, the PtdEtn content was not normalized unless the culture medium was supplemented with ethanolamine. In both M.9.1.1 and hepatoma cells transfected with PSS2 cDNA the rate of synthesis of PtdSer and PtdSer-derived PtdEtn did not exceed that in parental CHO cells or control McArdle cells respectively, in contrast to cells expressing similar levels of murine PSS1. These observations suggest that PtdSer synthesis via murine PSS2, but not PSS1, is regulated by end-product inhibition. Moreover, expression of murine PSS2 in McArdle cells did not inhibit PtdEtn synthesis via the CDP-ethanolamine pathway, whereas expression of similar levels of PSS1 activity inhibited this pathway by approx. 50%. We conclude that murine PSS1 and PSS2, which are apparently derived from different genes, independently modulate phospholipid metabolism. In addition, mRNAs encoding the two synthases are differentially expressed in several murine tissues, supporting the idea that PSS1 and PSS2 might perform unique functions.  (+info)