Biomedical subjects

J Y Lehtonen

Publications and source records attributed to J Y Lehtonen.

At least 19 recordsLinked to original sources

PubMed2006

Results and future of carotid endarterectomy in a medium-sized Finnish central hospital.

BACKGROUND AND AIMS: Carotid endarterectomies (CEA) should, by general agreement, be performed only at specialised institutions and on patients who are expected to have a low rate of complications, i.e., a 6% surgical risk for patients with symptomatic and a 3% risk for patients with asymptomatic carotid artery disease. We have reviewed the midterm results after CEA in a medium-sized Finnish central hospital to audit whether our results fulfil current quality standards. PATIENTS, MATERIALS AND METHODS: There were 116 consecutively operated patients at the Päijät-Häme Central Hospital during the years 1999-2003 on whom 122 CEAs were made. All CEAs were performed by conventional methods and shunts and patches were used when necessary. RESULTS: The overall survival rate was 81.0% during the 3.6 +/- 1.5 years [range 1.3-6.3 years] follow-up. The incidence of early major stroke was 2.5% and of late major stroke 1.6%. Other complications included cranial nerve deficit (2.5%), wound haematoma (3.3%) and postoperative hypertension (3.3%) or hypotension (2.5%). Significant changes in medication were needed after surgery in several patient subgroups. CONCLUSIONS: The results of CEA at the Päijät-Häme Central Hospital are at least acceptable. The primary prevention of strokes with pharmacotherapy needs aggressive improvement.

Adult↗

PubMed2001

Intracellular third loops in AT1 and AT2 receptors determine subtype specificity.

The recently cloned angiotensin II type 2 (AT2) receptor is a member of the seven transmembrane G-protein coupled receptor superfamily with a relatively low sequence homology with the angiotensin II type 1 (AT1) receptor subtype and counteracts the growth action of AT1 receptor. Intracellular third loops are known to be involved in interactions with various G proteins. We hypothesized that the intracellular third loop plays critical roles in determining the specificity of opposite functions of AT1 and AT2 receptor subtypes and examined this possibility using chimeric AT1 receptor, of which intracellular third loop is replaced with that of AT2 receptor. We transfected this chimeric receptor into PC 12 cells and observed that stimulation of this receptor inhibited extracellular signal-regulated kinase (ERK) activation and induces apoptosis, whereas the binding characteristics of this receptor remained those of ATI receptor. Taken together, these results support the notion that intracellular third loop is the critical determinant for mutually antagonistic AT1 and AT2 receptors' signaling pathways.

Animals↗

PubMed2000

Interferon-gamma induces AT(2) receptor expression in fibroblasts by Jak/STAT pathway and interferon regulatory factor-1.

The expression of angiotensin II type 2 (AT(2)) receptor is closely associated with cell growth, differentiation, and/or injury. We examined the effect of interferon (IFN)-gamma on AT(2) receptor expression in mouse fibroblast R3T3 cells and demonstrated that IFN-gamma treatment increased the expression of AT(2) receptor mRNA as well as its binding. Interferon regulatory factor (IRF)-1 was induced in mouse fibroblast R3T3 cells after IFN-gamma stimulation, and electrophoretic mobility shift assay showed an increase in IRF-1 binding with the IRF-specific binding sequence in the AT(2) receptor gene promoter region after IFN-gamma stimulation. The IRF-1 gene promoter contains an IFN-gamma-activated sequence (GAS) motif for possible binding of signal transducer(s) and activator(s) of transcription (STAT). Indeed, in R3T3 cells, IFN-gamma treatment resulted in rapid activation of Janus kinase (Jak) 1, Jak2, and STAT1 via tyrosine phosphorylation. Electrophoretic mobility shift assay with the GAS probe revealed increased STAT1 binding to the IRF-1 gene promoter in response to IFN-gamma stimulation. Transfection of GAS-binding oligonucleotides inhibited the effect of IFN-gamma on IRF-1 production, resulting in the AT(2) receptor trans-activation. Taken together, our data show that IFN-gamma upregulates AT(2) receptor expression in R3T3 cells via the activation of the intracellular Jak/STAT pathway and production of IRF-1.

3T3 Cells↗

PubMed1999

Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis.

This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta-chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta-chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.

Acyltransferases↗

PubMed1999

Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor.

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1999

Stimulation of different subtypes of angiotensin II receptors, AT1 and AT2 receptors, regulates STAT activation by negative crosstalk.

Angiotensin II type 2 (AT2) receptor exerts an inhibitory action on cell growth. In the present study, we report that the stimulation of AT2 receptor in AT2 receptor cDNA-transfected rat adult vascular smooth muscle cells (VSMCs) inhibited angiotensin II type 1 (AT1) receptor-mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) 1alpha/beta, STAT2, and STAT3 without influence on Janus kinase. AT2 receptor activation also inhibited the tyrosine phosphorylation of STAT1alpha/beta induced by interferon-gamma, epidermal growth factor, and platelet-derived growth factor. Similar effects of AT2 receptor were observed in R3T3 fibroblast and mouse fetal VSMCs, which express endogenous AT2 receptor. Moreover, AT2 receptor inhibited serine phosphorylation of STAT1alpha and STAT3 via the inhibition of extracellular signal-regulated kinase (ERK) activation. Stimulation of AT2 receptor inhibited the binding of STATs with sis-inducing element in c-fos promoter, resulting in decreased c-fos expression. Taken together, our results suggest that AT2 receptor can crosstalk negatively with multiple families of growth receptors by inhibiting ERK and STAT activation.

Animals↗

PubMed1999

Evidence for the extended phospholipid conformation in membrane fusion and hemifusion.

Molecular-level mechanisms of fusion and hemifusion of large unilamellar dioleoyl phosphatidic acid/phosphocholine (DOPA/DOPC, 1:1 molar ratio) vesicles induced by millimolar Ca2+ and Mg2+, respectively, were investigated using fluorescence spectroscopy. In keeping with reduction of membrane free volume Vf, both divalent cations increased the emission polarization for 1,6-diphenyl-1,3, 5-hexatriene (DPH). An important finding was a decrease in excimer/monomer emission intensity ratio (Ie/Im) for the intramolecular excimer-forming probe 1, 2-bis[(pyren-1-)yl]decanoyl-sn-glycero-3-phosphocholine (bis-PDPC) in the course of fusion and hemifusion. Comparison with another intramolecular excimer-forming probe, namely, 1-[(pyren-1)-yl]decanoyl-2-[(pyren-1)-yl]tetradecanoyl-sn-gl ycero-3-p hosphocholine (PDPTPC), allowed us to exclude changes in acyl chain alignment to be causing the decrement in Ie/Im. As a decrease in Vf should increase Ie/Im for bis-PDPC and because contact site between adhering liposomes was required we conclude the most feasible explanation to be the adoption of the extended conformation (P.K.J., Chem. Phys. Lipids 63:251-258) by bis-PDPC. In this conformation the two acyl chains are splaying so as to become embedded in the opposing leaflets of the two adhered bilayers, with the headgroup remaining between the adjacent surfaces. Our data provide evidence for a novel mechanism of fusion of the lipid bilayers.

Biophysical Phenomena↗

PubMed1999

Interactions of cyclosporin A with phospholipid membranes: effect of cholesterol.

Cyclosporin A (CsA) is a highly hydrophobic drug used to prevent graft rejection after organ transplantation. Interactions of CsA with phosphatidylcholine as well as with binary mixtures containing phosphatidylcholine and cholesterol were investigated by measuring the penetration of CsA into lipid monolayers at an air/water interface, by differential scanning calorimetry, and by imaging with fluorescence microscopy the effects of CsA on the lateral distribution of a fluorescent probe, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1, 3-diazol)aminocaproyl-phosphocholine, in monolayers. Film penetration studies revealed the association of CsA with lipids to be a biphasic process. Cholesterol diminished the intercalation of CsA into the monolayer at surface pressures of >19 mN/m. CsA broadened the main transition of dimyristoylphosphatidylcholine (DMPC)/beta-cholesterol (10:1, mol/mol) multilamellar vesicles. The behavior of the transition enthalpy was more complex; the behavior of DMPC/beta-cholesterol multilamellar vesicles in the XCsA of 0 to 0.1 showed at most ratios a increase, but several well distinct dips were observed. The results are interpreted in terms of regular structures in tertiary alloy. Influence of CsA on lateral organization could be verified for lipid domains observed by fluorescence microscopy of lipid monolayers. More specifically, CsA altered the distribution of 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1, 3-diazol)aminocaproyl-phosphocholine in a dipalmitoylphosphatidylcholine film and in DPPC/beta-cholesterol (88:10, mol/mol) mixtures in a manner that suggests that CsA partitions into the boundaries between fluid and gel domains. To our knowledge, this constitutes the first demonstration of a change in lipid domain morphology to be induced by a drug molecule.

Calorimetry, Differential Scanning↗

PubMed1999

AT2 receptor and vascular smooth muscle cell differentiation in vascular development.

The angiotensin II type 2 (AT2) receptor is transiently expressed at late gestation in the fetal vasculature, but its expression rapidly declines after birth. We have previously demonstrated that the expression of this receptor mediates decline in vascular DNA synthesis that occurs at this stage of vascular development. To examine further the role of the AT2 receptor in vasculogenesis, we have focused on the effect of the AT2 receptor on vascular smooth muscle cell (VSMC) differentiation. In this study, we examined the time-dependent expression of differentiation markers for VSMCs in the aorta of wild-type and AT2 receptor-null mice. alpha-Smooth muscle actin was expressed at the early stage of differentiation and exhibited unchanged expression before and after the peak of AT2 receptor expression, which was observed at embryonic day 20, neonatal day 1, and thereafter. No difference in alpha-smooth muscle actin expression was observed between the wild-type and AT2 receptor-null mice. In contrast, the mRNA levels for calponin, expressed in the late stage of VSMC differentiation, were significantly higher in the wild-type mouse aorta as compared with the AT2 receptor-null mice, which correlates with expression of the AT2 receptor. Moreover, the protein levels of calponin and high-molecular-weight caldesmon (h-caldesmon) showed lower expression in the aorta of AT2 receptor knockout mice at 2 and 4 weeks after birth. Taken together, our results suggest that the AT2 receptor promotes vascular differentiation and contributes to vasculogenesis.

Aging↗

PubMed1999

Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth.

Angiotensin II type 2 (AT2) receptor is abundantly expressed in vascular smooth muscle cells (VSMC) of the fetal vasculature during late gestation (embryonic day 15-20), during which the blood vessels undergo remodeling. To examine directly the influence of AT2 receptor expression in the developmental biology of VSMC, we studied cultures of VSMC from fetal and postnatal wild-type (Agtr2(+)) and AT2 receptor null (Agtr2(-)) mice. Consistent with in vivo data, AT2 receptor binding in cultured Agtr2(+) VSMC increased by age, peaking at embryonic day 20, and decreased dramatically after birth. Angiotensin II-induced growth in Agtr2(+) VSMC (embryonic day 20) was increased by the AT2 receptor blocker PD123319, indicating that the AT2 receptors are functional and exert an antigrowth effect in Agtr2(+) VSMC. Growth of VSMC in response to serum decreased age dependently and was higher in Agtr2(-) than in Agtr2(+), inversely correlating with AT2 receptor expression. However, serum-induced growth in Agtr2(+) and Agtr2(-) VSMC and the exaggerated Agtr2(-) VSMC growth was maintained even in the presence of PD123319 or losartan, an AT1 receptor blocker. Moreover, Agtr2(-) VSMC showed greater growth responses to platelet-derived growth factor and basic fibroblast growth factor, indicating that Agtr2(-) cells exhibit a generalized exaggerated growth phenotype. We studied the mechanism responsible for this phenotype and observed that extracellular signal-regulated kinase (ERK) activity was higher in Agtr2(-) VSMC at baseline and also in response to serum. ERK kinase inhibitor PD98059 inhibited both growth and ERK phosphorylation dose-dependently, while the regression lines between growth and ERK phosphorylation were identical in Agtr2(+) and Agtr2(-) VSMC, suggesting that increased ERK activity in Agtr2(-) VSMC is pivotal in the growth enhancement. Furthermore, the difference in ERK phosphorylation between Agtr2(+) and Agtr2(-) was abolished by vanadate but not by okadaic acid, implicating tyrosine phosphatase in the difference in ERK activity. These results suggest that the AT2 receptor expression during the fetal vasculogenesis influences the growth phenotype of VSMC via the modulation of ERK cascade.

Angiotensin II↗

PubMed1999

Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis.

We previously demonstrated that the intracellular third loop (i3 loop) of angiotensin II type 2 receptor (AT2) plays a key role in mediating the biological functions of this receptor. To determine which residues are important for AT2 signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 within the intermediate portion of the i3 loop resulted in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases (ERK), and SHP-1 activation. In contrast to well characterized heptahelical receptors, the AT2 functions were not affected by deletions of the amino- or carboxyl-terminal portions of the i3 loop. Alanine substitutions further demonstrated that lysine 240, asparagine 242, and serine 243 are key residues for AT2-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT2-induced ERK inhibition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT2 function and that SHP-1 is a proximal effector of the AT2 receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.

Alanine↗

PubMed1998

Binding of quinacrine to acidic phospholipids and pancreatic phospholipase A2. Effects on the catalytic activity of the enzyme.

Binding of quinacrine to phospholipids and porcine pancreatic phospholipase A2 (PLA2) was investigated using fluorescence resonance energy transfer, Langmuir films, assay for the enzymatic activity, and molecular modeling. No significant binding of this drug to the zwitterionic phosphatidylcholine was observed whereas a high affinity for acidic phospholipids was revealed by quenching of pyrene-labeled phospholipid analogues. Partial reversal of this binding was observed due to the addition of 4 mM CaCl2. Quinacrine efficiently and independently of the lipid surface pressure penetrated into monolayers of phosphatidylglycerol while only a weak penetration into phosphatidylcholine films was evident. Quinacrine also bound to eosin-labeled PLA2, and the addition of 4 mM CaCl2 reversed this interaction almost completely. In the presence of acidic phospholipids both the drug and the enzyme were attached to the lipid surface. Studies on the influence of quinacrine on the activity of PLA2 toward pyrene-labeled phospholipid analogues revealed that the hydrolysis of phosphatidylcholine was progressively reduced as a function of increasing [quinacrine]. At low [CaCl2] and low quinacrine:lipid molar ratios (<1:5) quinacrine enhanced slightly the rate of hydrolysis of acidic phospholipids whereas at higher drug:lipid molar ratios (>1:2) an inhibition was observed. In the presence of 1 mM CaCl2 quinacrine inhibited PLA2-catalyzed hydrolysis of phosphatidylglycerol only when the drug:lipid molar ratio exceeded 1:1. The presence of 4 mM CaCl2 abolished nearly completely the inhibition with all the substrate analogues used. Our data suggest that the inhibition of PLA2 by quinacrine is due to its binding to the enzyme. This is supported also by molecular modeling which suggested a binding site for quinacrine close to the active site and Ca2+ binding site of the enzyme. Importantly, our data indicate that quinacrine binds avidly to acidic phospholipids and their presence may influence the drug-enzyme interaction and the inhibition of the enzyme action. Accordingly, presence of quinacrine may interfere also with other processes that require the presence of acidic lipids and/or Ca2+, such as the function of the nicotinic acetylcholine receptor.

Animals↗

PubMed1997

Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes.

Binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and natural ceramide (up to a mole fraction Xcer = 0.25) were investigated by measuring the excimer:monomer fluorescence emission intensity ratio IE:IM for the pyrene labeled phospholipid probe 1-palmitoyl-2[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC), by monitoring fluorescence polarization of diphenylhexatriene (DPH), as well as using differential scanning calorimetry (DSC). Increasing Xcer > 0.10 both below and above the main transition temperature Tm increased IE:IM for PPDPC maximally approximately 1.6-fold at Xcer = 0.25. Above Tm, and when Xcer approaches 0.10, fluorescence polarization P for DPH increases steeply, reflecting an overall decrease in acyl chain motions. At Xcer = 0.10 there is a discontinuity in P and upon further increase in the content of ceramide a smaller, yet significant increase in P is evident. DSC revealed ceramide to increase in the pretransition temperature until at Xcer exceeding 0.07 this transition was no longer evident. Simultaneously, increasing Xcer up to 0.05 increased Tm from 23.9 to 24.6 degrees C. Total enthalpy delta Hm of the main transition diminished progressively upon increase in Xcer up to approximately 0.10. Above this concentration of ceramide a new endotherm became evident at 22.5 degrees C, and exceeding Xcer = 0.14 this endotherm became dominant. Our results indicate an enrichment of the pyrene labeled phospholipid analog into microdomains concomitant with the formation of a distinct ceramide-enriched phase at Xcer > 0.10.

Calorimetry, Differential Scanning↗

PubMed1997

Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease.

The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes. Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM. Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold. Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively. An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles. This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000. The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody. Interestingly, excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease. Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved. Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident. The addition of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased IE/IM for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated. More specifically for protein-free bilayers at XDPPG = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in IE/IM, under these conditions the increase in IE/IM due to lac permease was strongly reduced, by approximately 84%. The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity. In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 A and thus exceed the hydrophobic thickness of POPG of approximately 25.8 A. Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers.

Binding Sites↗

PubMed1996

Binding of daidzein to liposomes.

Turbidity and differential scanning calorimetry measurements revealed the plant derived antineoplastic isoflavone, daidzein, to bind to large unilamellar liposomes. Comparing different unsaturated phospholipids most pronounced aggregation due to daidzein was observed for phosphatidylinositol (PI) while the inclusion of cholesterol strongly attenuated the aggregation. Interestingly, aggregation was not observed for the structurally very closely related isoflavone, genistein. The extent of aggregation was nonlinearly dependent on the content of PI in egg phosphatidylcholine (eggPC) vesicles. The saturated dimyristoyl phospholipids, phosphatidylserine, phosphatidylcholine, phosphatidic acid, as well as phophatidylglycerol were also extensively aggregated by daidzein at 10 degrees C, i.e., below their main phase transition temperature whereas their aggregation at 35 degrees C in the fluid phase was strongly reduced. Vesicle aggregation could be accompanied by membrane fusion, however, neither contents mixing nor lipid mixing of the LUVs (large unilamellar vesicles) was observed in the presence of daidzein. Strong perturbation of the thermal phase behaviour of both dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) multilamellar vesicles by daidzein was revealed by differential scanning calorimetry. More specifically, for DMPC increasing quantities of daidzein progressively decreased both the main transition temperature Tm and its enthalpy whereas for DMPS a decrease in delta H was not observed, thus indicating the modes of interaction of daidzein with these phospholipids to differ. Our results indicate daidzein to reside in the polar headgroup/interfacial region of PI and PS membranes. The interactions of daidzein with phospholipids could represent an additional contributor to the growing list of effects of this isoflavone on cellular functions.

Antineoplastic Agents↗

PubMed1996

Activation of phospholipase A2 by amyloid beta-peptides in vitro.

Amyloid beta-peptides (A beta) are centrally involved in the pathogenesis of Alzheimer's disease. Using secretory phospholipase A2 (PLA2) from porcine pancreas as a model and in the presence of a limiting Ca2+ concentration of approximately 50 nM, the synthetic peptide A beta 1-42 activates the hydrolysis of the pyrene-labeled acidic phospholipid analog 1-palmitoyl-2-[(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) maximally 2.3-fold, whereas an inhibition of PLA2 action by 50% on the corresponding phosphatidylcholine derivative (PPHPC) was observed. The above effects were evident at 0.24 nM A beta 1-42 corresponding to A beta 1-42:phospholipid and A beta 1-42:PLA2 molar ratios of 1:10 650 and 1:7.6, respectively. The presence of 10 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in PPHPC reversed the inhibitory effect of A beta 1-42 peptide and for these vesicles the hydrolytic activity of PLA2 toward the fluorescent phosphatidylcholine was enhanced approximately 1.8-fold by A beta 1-42. In contrast, inclusion of 10 mol % POPG into PPHPG did not influence either the hydrolytic rate toward the latter lipid or the activating effect of A beta 1-42. Ca2+ concentrations exceeding 15 microM abolished the enhancing effect of A beta 1-42 on the hydrolysis of PPHPG whereas a slight activation of PPHPC hydrolysis now became evident. With limiting [Ca2+] preaggregated A beta 1-42 enhanced the hydrolysis of both PPHPG as well as PPHPC but the peptide concentrations required were higher by 3-4 orders of magnitude. The synthetic peptide A beta 25-35 corresponding to the hydrophobic membrane-spanning segment of the beta amyloid precursor protein activated PLA2 when using PPHPG as a substrate; however, compared to A beta 1-42 the extent of activation was less (approximately 2-fold) and required higher (1 nM) peptide. A beta 25-35 did not affect the hydrolysis of the phosphatidylcholine derivative. The hydrophilic peptide A beta 1-28 had no effect on PLA2-catalyzed hydrolysis of either PPHPG or PPHPC under the conditions used in the present study. Interestingly, the above activating effects of A beta 1-42 and A beta 25-35 on PLA2-catalyzed hydrolysis of the acidic phospholipid substrate parallel their toxicity on cultured neurons whereas A beta 1-28 had no influence either on cultured cells or on PLA2 activity.

Alzheimer Disease↗

PubMed1996

Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids.

The excimer-to-monomer fluorescence emission intensity ratio (IE/IM) of the fluorescent probe 1-palmitoyl-2-[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC, 1 mol%) was measured at 30 degrees C as a function of the thickness of fluid liposomal membranes composed of phosphatidylcholines (PCs) with homologous monounsaturated acyl chains of varying lengths N (= number of carbon atoms). Upon decreasing N from di-24:1 PC to di-14:1 PC, the rate of excimer formation was sigmoidally augmented from 0.02 to 0.06. This increase in IE/IM can arise either from enhanced lateral mobility or from the lateral enrichment of PPDPC into domains, or both. Direct evidence for partial lateral segregation of PPDPC being involved is provided by experiments where 1.6 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N- (5-fluoresceinthiocarbamoyl) (DPPF) was included together with PPDPC into the bilayers. Notably, because of spectral overlap DPPF can function as a resonance energy transfer acceptor for pyrene excimer. Fluorescence intensity ratio (F/Fo) measured at 480 nm for PPDPC/DPPF (yielding F) and PPDPC (yielding Fo) containing membranes as a function of N reveals a sharp maximum for di-20:1 PC, i.e., the quenching of pyrene excimer fluorescence by DPPF is least efficient in this lipid and is enhanced upon either decrease or increase in N. This is compatible with colocalization of DPPF in PPDPC enriched domains when N not equal to 20, whereas in di-20:1 PC these probes appear to be effectively dispersed. The driving force for the enrichment of PPDPC in thin (N < 20) and thick (N > 20) PC matrices is likely to be hydrophobic mismatch of the effective lengths of the matrix phospholipids and the fluorescent probes. We also measured fluorescence polarization (P) for 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as the IE/IM for the intramolecular excimer forming probe 1,2-bis[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a function of N. In brief, neither the fluorescence polarization data and nor the measurements of intramolecular chain dynamics using bisPDPC concur with enhanced lateral diffusion as the sole cause for the increase in the IE/IM for PPDPC in thin membranes. Our findings suggest hydrophobic mismatch as the cause of microdomain formation of lipids in fluid, liquid crystalline bilayers, while simultaneously allowing for a high rates of lateral diffusion. Such hydrophobic mismatch-induced compositional fluctuations would also offer one plausible explanation for the chain length diversity observed for biological membranes.

Biophysical Phenomena↗

PubMed1996

Characteristics of the binding of tacrine to acidic phospholipids.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrate) is an inhibitor of acetylcholinesterase currently used in the treatment of the symptoms of Alzheimer's disease. The present study demonstrates preferential binding of this drug to acidic phospholipids, as revealed by fluorescence polarization, penetration into lipid monolayers, and effects on the thermal phase behavior of dimyristoyl phosphatidic acid (DMPA). A fivefold enhancement in the polarization of tacrine emission is evident above the main phase transition temperature (T(m)) of DMPA vesicles, whereas below T(m) only a 0.75-fold increase is observed. In contrast, the binding of tacrine to another acidic phospholipid, dimyristoylphosphatidylglycerol, did not exhibit strong dependence on T(m). In accordance with the electrostatic nature of the membrane association of tacrine, the extent of binding was augmented with increasing contents of egg PG in phosphatidylcholine liposomes. Furthermore, [NaCl] > 50 mM dissociates tacrine (albeit incompletely) from the liposomes composed of acidic phospholipids. Inclusion of the cationic amphiphile sphingosine in egg PG vesicles decreased the membrane association of tacrine until at 1:1 sphingosine: egg PG stoichiometry binding was no longer evident. Tacrine also penetrated into egg PG but not into egg PC monolayers. Together with broadening of the main transition and causing a shoulder on its high temperature side, the binding of tacrine to DMPA liposomes results in a concentration-dependent reduction both in the combined enthalpy delta H of the above overlapping endotherms and the main transition temperature T(m). Interestingly, these changes in the thermal phase behavior of DMPA as a function of the content of the drug in vesicles were strongly nonlinear. More specifically, upon increasing [tacrine], T(m) exhibited stepwise decrements. Simultaneously, sharp minima in delta H were observed at drug:lipid stoichiometries of approximately 2:100 and 25:100, whereas a sharp maximum in delta H was evident at 18:100. The above results are in keeping with tacrine causing phase separation processes in the bilayer and may also relate to microscopic drug-induced ordering processes within the membrane.

Cholinesterase Inhibitors↗