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1.
Appl Radiat Isot ; 61(4): 609-16, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15246407

ABSTRACT

A few nondestructive examination (NDE) techniques are extensively being used worldwide to investigate aircraft structures for all types of defects. The detection of corrosion and delaminations, which are believed to be the major initiators of defects leading to aircraft structural failures, are addressed by various NDE techniques. In a combined investigation by means of visual inspection, X-ray radiography and shearography on helicopter main rotor blades, neutron radiography (NRad) at SAFARI-1 research reactor operated by Necsa, was performed to introduce this form of NDE testing to the South African aviation industry to be evaluated for applicability. The results of the shearography, visual inspection and NRad techniques are compared in this paper. The main features and advantages of neutron radiography, within the framework of these investigations, will be highlighted.

2.
Appl Radiat Isot ; 61(4): 617-23, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15246408

ABSTRACT

The natural drying process of concrete, which has a significant effect on its characteristics, for example durability, was studied at the neutron radiography facility at SAFARI-1 nuclear research reactor, operated by Necsa. Monitoring of the movement of the water in concrete samples, which were wet cured for one day and covered on all the sides but one, was done by means of a CCD camera system. In this paper the methodology in observing the drying process will be described together with results obtained from this investigation. The measured water content and porosity results were quantified and compared reasonably well with conventional gravimetrical measurements.

3.
J Lipid Res ; 42(8): 1231-8, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483624

ABSTRACT

CD36 and scavenger receptor class B, type I (SR-BI) are both class B scavenger receptors that recognize a broad variety of ligands, including oxidized low density lipoprotein (oxLDL), HDL, anionic phospholipids, and apoptotic cells. In this study we investigated the role of mouse CD36 (mCD36) as a physiological lipoprotein receptor. We compared the association of various lipoprotein particles with mCD36 and mSR-BI expressed in COS cells by adenovirus-mediated gene transfer. mCD36 bound human oxLDL and mouse HDL with high affinity. Human LDL bound poorly to mCD36, indicating that mCD36 is unlikely to play a significant role in LDL metabolism. The ability of mCD36 to mediate the selective uptake of cholesteryl esters (CE) from receptor-bound HDL was assessed. In comparison with mSR-BI, mCD36 inefficiently mediated the selective uptake of CE. Hepatic overexpression of mCD36 in C57BL/6 mice by adenovirus-mediated gene transfer did not result in significant alterations in plasma LDL and HDL levels. We conclude that mCD36, while able to bind HDL with high affinity, does not contribute significantly to HDL or LDL metabolism.


Subject(s)
CD36 Antigens/physiology , Lipoproteins, HDL/metabolism , Lipoproteins, LDL/metabolism , Membrane Proteins , Receptors, Immunologic , Receptors, Lipoprotein , Adenoviridae/genetics , Animals , CD36 Antigens/genetics , COS Cells , Cholesterol Esters/metabolism , Gene Expression , Genetic Vectors , Humans , Lipoproteins/blood , Liver/metabolism , Mice , Mice, Inbred C57BL , Rats , Rats, Inbred SHR , Receptors, Scavenger , Scavenger Receptors, Class B , Transfection
4.
J Biol Chem ; 276(19): 15832-9, 2001 May 11.
Article in English | MEDLINE | ID: mdl-11279034

ABSTRACT

High density lipoprotein (HDL) represents a mixture of particles containing either apoA-I and apoA-II (LpA-I/A-II) or apoA-I without apoA-II (LpA-I). Differences in the function and metabolism of LpA-I and LpA-I/A-II have been reported, and studies in transgenic mice have suggested that apoA-II is pro-atherogenic in contrast to anti-atherogenic apoA-I. The molecular basis for these observations is unclear. The scavenger receptor BI (SR-BI) is an HDL receptor that plays a key role in HDL metabolism. In this study we investigated the abilities of apoA-I and apoA-II to mediate SR-BI-specific binding and selective uptake of cholesterol ester using reconstituted HDLs (rHDLs) that were homogeneous in size and apolipoprotein content. Particles were labeled in the protein (with (125)I) and in the lipid (with [(3)H]cholesterol ether) components and SR-BI-specific events were analyzed in SR-BI-transfected Chinese hamster ovary cells. At 1 microg/ml apolipoprotein, SR-BI-mediated cell association of palmitoyloleoylphosphatidylcholine-containing AI-rHDL was significantly greater (3-fold) than that of AI/AII-rHDL, with a lower K(d) and a higher B(max) for AI-rHDL as compared with AI/AII-rHDL. Unexpectedly, selective cholesterol ester uptake from AI/AII-rHDL was not compromised compared with AI-rHDL, despite decreased binding. The efficiency of selective cholesterol ester uptake in terms of SR-BI-associated rHDL was 4-5-fold greater for AI/AII-rHDL than AI-rHDL. These results are consistent with a two-step mechanism in which SR-BI binds ligand and then mediates selective cholesterol ester uptake with an efficiency dependent on the composition of the ligand. ApoA-II decreases binding but increases selective uptake. These findings show that apoA-II can exert a significant influence on selective cholesterol ester uptake by SR-BI and may consequently influence the metabolism and function of HDL, as well as the pathway of reverse cholesterol transport.


Subject(s)
Apolipoprotein A-II/metabolism , CD36 Antigens/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/metabolism , Membrane Proteins , Receptors, Immunologic , Animals , Apolipoprotein A-I/blood , Apolipoprotein A-I/metabolism , Apolipoprotein A-II/blood , Binding, Competitive , CHO Cells , Cell Line , Cholesterol Esters/metabolism , Cricetinae , Humans , Kinetics , Phosphatidylcholines/metabolism , Phospholipids/metabolism , Receptors, Lipoprotein/metabolism , Receptors, Scavenger , Recombinant Proteins/metabolism , Scavenger Receptors, Class B
5.
J Lipid Res ; 42(2): 309-13, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11181762

ABSTRACT

Apolipoprotein A-I (apoA-I) is an important ligand for the high density lipoprotein (HDL) scavenger receptor class B type I (SR-BI). SR-BI binds both free and lipoprotein-associated apoA-I, but the effects of particle size, composition, and apolipoprotein conformation on HDL binding to SR-BI are not understood. We have studied the effect of apoA-I conformation on particle binding using native HDL and reconstituted HDL particles of defined composition and size. SR-BI expressed in transfected Chinese hamster ovary cells was shown to bind human HDL(2) with greater affinity than HDL(3), suggesting that HDL size, composition, and possibly apolipoprotein conformation influence HDL binding to SR-BI. To discriminate between these factors, SR-BI binding was studied further using reconstituted l-alpha-palmitoyloleoyl-phosphatidylcholine-containing HDL particles having identical components and equal amounts of apoA-I, but differing in size (7.8 vs. 9.6 nm in diameter) and apoA-I conformation. The affinity of binding to SR-BI was significantly greater (50-fold) for the larger (9.6-nm) particle than for the 7.8-nm particle. We conclude that differences in apoA-I conformation in different-sized particles markedly influence apoA-I recognition by SR-BI. Preferential binding of larger HDL particles to SR-BI would promote productive selective cholesteryl ester uptake from larger cholesteryl ester-rich HDL over lipid-poor HDL.


Subject(s)
Apolipoprotein A-I/chemistry , CD36 Antigens/metabolism , Lipoproteins, HDL/metabolism , Membrane Proteins , Receptors, Immunologic , Receptors, Lipoprotein , Electrophoresis, Polyacrylamide Gel , Humans , Particle Size , Protein Binding , Protein Conformation , Receptors, Scavenger , Recombinant Proteins/chemistry , Scavenger Receptors, Class B
6.
J Lipid Res ; 41(11): 1849-57, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11060355

ABSTRACT

During inflammatory states plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are reduced. Secretory group IIa phospholipase A(2) (sPLA(2)) is a cytokine-induced acute-phase enzyme associated with HDL. Transgenic mice overexpressing sPLA(2) have reduced HDL levels. Studies were performed to define the mechanism for the HDL reduction in these mice. HDL isolated from sPLA(2) transgenic mice have a significantly lower phospholipid content and greater triglyceride content. In autologous clearance studies, (125)I-labeled HDL from sPLA(2) transgenic mice was catabolized significantly faster than HDL from control mice (4.24 +/- 1.16 vs. 2.84 +/- 0.1 pools per day, P < 0.008). In both sPLA(2) transgenic and control mice, the cholesteryl ester component of HDL was more rapidly catabolized than the protein component, indicating a selective uptake mechanism. In vitro studies using CHO cells transfected with scavenger receptor class B type I (SR-BI) showed that sPLA(2)-modified HDL was nearly twice as efficient as a substrate for cholesteryl ester transfer. These data were confirmed in in vivo selective uptake experiments using adenoviral vector overexpression of SR-BI. In these studies, increased hepatic selective uptake was associated with increased (125)I-labeled apolipoprotein uptake in the kidney. We conclude that during inflammation sPLA(2) hydrolysis of HDL phospholipids alters the lipid composition of the particle, allowing for more efficient SR-BI-mediated selective cholesteryl ester uptake. This enhanced SR-BI activity generates HDL remnants that are preferentially catabolized in the kidney.


Subject(s)
Lipoproteins, HDL/blood , Membrane Proteins , Phospholipases A/metabolism , Receptors, Immunologic/metabolism , Receptors, Lipoprotein , Tyramine/analogs & derivatives , Animals , CD36 Antigens , CHO Cells , Cholesterol Esters/metabolism , Cricetinae , Gene Expression , Kinetics , Lipoproteins, HDL/analysis , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phospholipases A/genetics , Phospholipids/analysis , Receptors, Immunologic/genetics , Receptors, Scavenger , Scavenger Receptors, Class B , Transfection , Triglycerides/analysis , Tritium , Tyramine/metabolism
7.
Cytokine ; 12(9): 1337-47, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10975993

ABSTRACT

The lack of sensitive and relatively non-invasive measures has hampered monitoring the clinical course of spontaneously developing colitis in IL-2-deficient (-/-) mice. We selected (i) to study the correlation of the acute phase plasma proteins serum amyloid A (SAA) and serum amyloid P component (SAP) levels with colonic disease and (ii) to characterize the amyloidosis in the IL-2(-/-)animals. IL-2(-/-)mice exhibited increasing severity of gross intestinal inflammation with age, confined to the distal colon. Histologically, the colonic disease score increased serially in IL-2(-/-)animals. Wild-type mice showed no activity, while 16-week-old IL-2(+/-)animals had minimal colitis with small ulcers and lamina propria inflammatory infiltrate. Periportal hepatitis was present and positive Congo red staining indicated amyloidosis of the liver and spleen in 16 week IL-2(-/-)mice. SAA immunostaining in the liver and spleen was increased in the 8 week and 16 week IL-2(-/-)and 16 week IL-2(+/-)animals indicating AA amyloid deposits. Plasma SAA and SAP levels were markedly elevated, and generally preceded the onset of colitis and reflected its severity. Northern analysis showed markedly increased SAA expression in the liver and intestine of IL-2(-/-)and intestine of IL-2(+/-)16-week-old animals. Increased intestinal expression of SAA3 (lamina propria macrophages) indicates local inflammation in IL-2(+/-)animals at 16 weeks. Treatment of 3-week-old animals with systemic IL-2 or IL-1 receptor antagonist (IL-1ra) delayed inflammation, postponed the increase in SAA levels and minimized disease onset. These results further demonstrate that IL-2 plays a significant role in normal immune responses in the body and that plasma SAA levels both reflect colonic disease severity and may indicate subclinical disease in both IL-2(-/-)and IL-2(+/-)mice. Furthermore. The mechanism of IL-2-deficient induced colitis appears to be mediated in part through the increase in IL-1. In addition, the IL-2(-/-)mouse of spontaneous enterocolitis may provide a unique system for studying spontaneously developing AA amyloidosis.


Subject(s)
Colitis/blood , Colitis/diagnosis , Interleukin-2/genetics , Serum Amyloid A Protein/metabolism , Serum Amyloid P-Component/metabolism , Amyloidosis/blood , Amyloidosis/pathology , Animals , Blotting, Northern , Colitis/pathology , Coloring Agents/metabolism , Congo Red/metabolism , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Interleukin-2/pharmacology , Intestinal Mucosa/metabolism , Intestines/immunology , Intestines/pathology , Liver/metabolism , Liver/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA/metabolism , Receptors, Interleukin-1/antagonists & inhibitors , Spleen/metabolism , Spleen/pathology , Time Factors , Tissue Distribution
8.
J Lipid Res ; 41(7): 1035-47, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10884283

ABSTRACT

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.


Subject(s)
Acute-Phase Proteins/chemistry , Acute-Phase Reaction/blood , Lipids/chemistry , Lipoproteins, HDL/chemistry , Apolipoprotein A-I/analysis , Apolipoproteins/analysis , Arteriosclerosis/etiology , Fatty Acids/analysis , Humans , Inflammation/etiology , Particle Size , Phospholipids/analysis , Serum Amyloid A Protein/analysis , Triglycerides/analysis
9.
Arterioscler Thromb Vasc Biol ; 20(6): 1543-50, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10845870

ABSTRACT

The serum amyloid A (SAA) family of proteins consists of inducible acute-phase members and a constitutive member that are minor apolipoproteins of normal high density lipoprotein (HDL). During inflammation, HDL cholesterol and apolipoprotein A-I (apoA-I) protein are decreased, and these changes are thought to be partly related to the increase in acute-phase SAA proteins that associate with the HDL particle to become the major apolipoprotein species. To determine the specific role of SAA in the alteration of HDL in the absence of a generalized acute-phase response, acute-phase Saa1.1 transgene expression was directed via an inducible mouse metallothionein promoter. Elevated levels of SAA1.1 (28+/-9 mg/dL) comparable to a moderate acute-phase response were achieved over a 5-day period. SAA association with the HDL particles at this concentration did not significantly alter the apoA-I or HDL cholesterol levels or change the lipoprotein profiles in the transgenic mice compared with wild-type mice. In addition, we used adenoviral vectors to increase the SAA expression to levels seen in a major acute-phase response. Injection of adenovirus expressing the mouse SAA1.1 protein resulted in high-level expression (72+/-8 mg/dL) but did not alter apoA-I levels. However, the SAA associated with the HDL particle gave rise to significantly larger HDL particles ( approximately 10%). Adenoviral expression of the constitutive SAA4 protein resulted in an increase in HDL size ( approximately 10%) and an increase in very low density lipoprotein levels (20-fold) and triglyceride levels (1.7-fold). These studies suggest that increases in acute-phase SAA proteins alone are insufficient to alter HDL cholesterol or apoA-I levels during inflammation. A role for constitutive SAA4 in HDL-very low density lipoprotein interactions should be considered.


Subject(s)
Gene Expression , Lipoproteins/blood , Serum Amyloid A Protein/genetics , Acute-Phase Reaction , Adenoviridae/genetics , Animals , Apolipoprotein A-I/metabolism , Cholesterol, HDL/blood , Genetic Vectors , Lipoproteins, HDL/blood , Metallothionein/genetics , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Serum Amyloid A Protein/physiology , Zinc Sulfate/pharmacology
10.
J Biol Chem ; 275(14): 10077-84, 2000 Apr 07.
Article in English | MEDLINE | ID: mdl-10744687

ABSTRACT

Plasma levels of high density lipoprotein (HDL) cholesterol and its major protein component apolipoprotein (apo) A-I are significantly reduced in both acute and chronic inflammatory conditions, but the basis for this phenomenon is not well understood. We hypothesized that secretory phospholipase A(2) (sPLA(2)), an acute phase protein that has been found in association with HDL, promotes HDL catabolism. A series of HDL metabolic studies were performed in transgenic mice that specifically overexpress human sPLA(2) but have no evidence of local or systemic inflammation. We found that HDL isolated from these mice have a significantly lower phospholipid and cholesteryl ester and significantly greater triglyceride content. The fractional catabolic rate (FCR) of (125)I-HDL was significantly faster in sPLA(2) transgenic mice (4.08 +/- 0.01 pools/day) compared with control wild-type littermates (2.16 +/- 0.48 pools/day). (125)I-HDL isolated from sPLA(2) transgenic mice was catabolized significantly faster than (131)I-HDL isolated from wild-type mice after injection in wild-type mice (p < 0.001). Injection of (125)I-tyramine-cellobiose-HDL demonstrated significantly greater degradation of HDL apolipoproteins in the kidneys of sPLA(2) transgenic mice compared with control mice (p < 0.05). The fractional catabolic rate of [(3)H]cholesteryl ether HDL was significantly faster in sPLA(2)-overexpressing mice (6.48 +/- 0.24 pools/day) compared with controls (4.80 +/- 0.72 pools/day). Uptake of [(3)H] cholesteryl ether into the livers and adrenals of sPLA(2) transgenic mice was significantly enhanced compared with control mice. In summary, these data demonstrate that overexpression of sPLA(2) alone in the absence of inflammation causes profound alterations of HDL metabolism in vivo and are consistent with the hypothesis that sPLA(2) may promote HDL catabolism in acute and chronic inflammatory conditions.


Subject(s)
Apolipoprotein A-I/blood , Cholesterol, HDL/metabolism , Lipoproteins, HDL/metabolism , Phospholipases A/metabolism , Adrenal Glands/metabolism , Animals , Cholesterol/blood , Cholesterol Esters/blood , Group II Phospholipases A2 , Humans , Kidney/metabolism , Kinetics , Lipoproteins, HDL/blood , Lipoproteins, HDL/chemistry , Liver/metabolism , Mice , Mice, Transgenic , Phospholipases A/genetics , Phospholipids/blood , Spleen/metabolism , Triglycerides/blood
11.
Lab Invest ; 80(12): 1797-806, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11140693

ABSTRACT

Amyloid A (AA) amyloid deposition in mice is dependent upon isoform-specific effects of the serum amyloid A (SAA) protein. In type A mice, SAA1.1 and SAA2.1 are the major apolipoprotein-SAA isoforms found on high-density lipoproteins. During inflammation, both isoforms are increased 1000-fold, but only SAA1.1 is selectively deposited into amyloid fibrils. Previous studies showed that the CE/J mouse strain is resistant to amyloid induction. This resistance is not due to a deficiency in SAA synthesis, but is probably related to the unusual SAA isoform present. The CE/J mouse has a single acute-phase SAA protein (SAA2.2), which is a composite of the SAA1.1 and SAA2.1, with an amino terminus similar to the nonamyloidogenic SAA2.1. Recently, genetic experiments suggested that the SAA2.2 isoform might provide protection from amyloid deposition. To determine the amyloidogenic potential of the CE/J mouse, we generated SAA adenoviral vectors to express the various isoforms in vitro and in vivo. Purified recombinant SAA proteins demonstrated that SAA1.1 was fibrillogenic in vitro, whereas SAA2.2 was unable to form fibrils. Incubation of increasing concentrations of the nonamyloidogenic SAA2.2 protein with the amyloidogenic SAA1.1 did not inhibit the fibrillogenic nature of SAA1.1, or alter its ability to form extensive fibrils. Injection of the mouse SAA1.1 or SAA2.2 adenoviral vectors into mice resulted in isoform-specific expression of the SAA proteins. Amyloid induction after viral expression of the SAA1.1 protein resulted in the deposition of amyloid fibrils in the CE/J mouse, whereas SAA2.2 expression had no effect. Similar expression of the SAA2.2 protein in C57BL/6 mice did not alter amyloid deposition. These data demonstrate that the failure of the CE/J mouse to deposit amyloid is due to the structural inability of the SAA2.2 to form amyloid fibrils. This mouse provides a unique system to test the amyloidogenic potential of altered SAA proteins and to determine the important structural features of the protein.


Subject(s)
Apolipoproteins/genetics , Liver/metabolism , Adenoviridae , Amino Acid Sequence , Amyloid/analysis , Amyloid/biosynthesis , Animals , Apolipoproteins/chemistry , Genetic Vectors , Inflammation/physiopathology , Lipoproteins, HDL/blood , Liver/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Molecular Sequence Data , Protein Isoforms/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Serum Amyloid A Protein/chemistry
13.
Arterioscler Thromb Vasc Biol ; 19(5): 1291-8, 1999 May.
Article in English | MEDLINE | ID: mdl-10323782

ABSTRACT

Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.


Subject(s)
Arteriosclerosis/enzymology , Fatty Acids, Unsaturated/metabolism , Phospholipases A/physiology , Phospholipids/metabolism , Animals , Aorta/cytology , Arteriosclerosis/etiology , Aryldialkylphosphatase , Cells, Cultured , Cholesterol, Dietary/toxicity , Diet, Atherogenic , Dietary Fats/toxicity , Elapid Venoms/enzymology , Endothelium, Vascular/cytology , Esterases/deficiency , Female , Genetic Predisposition to Disease , Group II Phospholipases A2 , Humans , Male , Mass Spectrometry , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Oxidation-Reduction , Oxidative Stress , Phospholipases A/genetics , Phospholipases A/pharmacology , Phospholipases A2 , Rabbits , Recombinant Fusion Proteins/pharmacology
14.
J Lipid Res ; 40(4): 648-53, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10191288

ABSTRACT

Plasma concentrations of high density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo)A-I are significantly decreased in inflammatory states. Plasma levels of the serum amyloid A (SAA) protein increase markedly during the acute phase response and are elevated in many chronic inflammatory states. Because SAA is associated with HDL and has been shown to be capable of displacing apoA-I from HDL in vitro, it is believed that expression of SAA is the primary cause of the reduced HDL cholesterol and apoA-I in inflammatory states. In order to directly test this hypothesis, we constructed recombinant adenoviruses expressing the murine SAA and human SAA1 genes (the major acute phase SAA proteins in both species). These recombinant adenoviruses were injected intravenously into wild-type and human apoA-I transgenic mice and the effects of SAA expression on HDL cholesterol and apoA-I were compared with mice injected with a control adenovirus. Plasma levels of SAA were comparable to those seen in the acute phase response in mice and humans. However, despite high plasma levels of murine or human SAA, no significant changes in HDL cholesterol or apoA-I levels were observed. SAA was found associated with HDL but did not specifically alter the cholesterol or human apoA-I distribution among lipoproteins. In summary, high plasma levels of SAA in the absence of a generalized acute phase response did not result in reduction of HDL cholesterol or apoA-I in mice, suggesting that there are components of the acute phase response other than SAA expression that may directly influence HDL metabolism.


Subject(s)
Acute-Phase Reaction , Apolipoprotein A-I/metabolism , Cholesterol, HDL/blood , Gene Expression , Serum Amyloid A Protein/genetics , Adenoviridae/genetics , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Female , Gene Transfer Techniques , Humans , Kinetics , Lipopolysaccharides/pharmacology , Mice , Mice, Transgenic , Recombinant Proteins , Serum Amyloid A Protein/metabolism
15.
J Lipid Res ; 39(11): 2150-60, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9799801

ABSTRACT

Group IIA secretory phospholipase A2 is an acute phase enzyme, co-expressed with serum amyloid A protein. Both are present in atherosclerotic lesions. We report that human normal and acute phase high density lipoproteins and low density lipoprotein are effective substrates for human group IIA phospholipase A2. The enzyme hydrolyzed choline and ethanolamine glycerophospholipids at the sn -2 position resulting in an accumulation of the corresponding lysophospholipids, including the unhydrolyzed alkyl and alkenyl ether derivatives. The hydrolysis of acute phase high density lipoprotein was 2- to 3-fold more rapid and intensive than of normal high density lipoprotein. The hydrolysis of lipoproteins was noted at enzyme concentration as low as 0.05 microgram/mg protein, which was within the range observed in the circulation in acute and chronic inflammatory diseases. The enzyme hydrolyzed the different molecular species of the residual glycerophospholipids in proportion to their mass, showing no preference for the release of arachidonic acid. Group IIA phospholipase A2 preferentially attacked the hydroxy and hydroperoxy linoleates and possibly other oxygenated fatty acids, which were released from the glycerophospholipids at early times of incubation. There was no effect on the content or molecular species composition of the sphingomyelins or neutral lipids of the lipoproteins. In conclusion, human plasma lipoproteins are the first reported natural biological substrates for human group IIA phospholipase A2. The enhanced hydrolysis of acute phase high density lipoproteins is probably due to its association with serum amyloid A protein, which enhances the activity of the enzyme and may promote its penetration to the lipid monolayer. As sPLA2-induced hydrolysis of the lipoproteins leads to accumulation of lysophosphatidylcholine and potentially toxic oxygenated fatty acids, overexpression of this enzyme may be proatherogenic.


Subject(s)
Acute-Phase Reaction/blood , Lipoproteins, HDL/blood , Phospholipases A/metabolism , Aldehydes/metabolism , Chromatography, High Pressure Liquid , Fatty Acids, Nonesterified/metabolism , Group II Phospholipases A2 , Humans , Hydrolysis , Lipid Peroxides/metabolism , Mass Spectrometry , Phospholipases A2 , Phospholipids/metabolism , Ultracentrifugation
16.
Genomics ; 50(2): 199-205, 1998 Jun 01.
Article in English | MEDLINE | ID: mdl-9653646

ABSTRACT

Murine macrosialin and its human homologue CD68 are heavily glycosylated transmembrane proteins expressed specifically in macrophages and macrophage-related cells. Macrosialin is predominantly a late endosomal protein but is also found on the cell surface where it binds oxidized LDL, an important factor in atherogenesis. We have cloned and sequenced the murine macrosialin gene (Cd68) and localized it by linkage analysis to chromosome 11. The gene is 1908 nucleotides long from the start site of transcription to the end of the 3'UTR. It has six exons, which range in size from 79 to 434 nucleotides. The promoter lacks a classical TATA box but contains other protein binding sites consistent with preferential monocyte/macrophage gene expression. Although the function of macrosialin is unknown, it might play a role in lipoprotein regulation given its binding of oxidized LDL in vitro and its colocalization to a region on chromosome 11 involved in the control of HDL levels.


Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Chromosome Mapping , Macrophages, Peritoneal/metabolism , Animals , Antigens, CD/chemistry , Antigens, CD/physiology , Antigens, Differentiation, Myelomonocytic/chemistry , Antigens, Differentiation, Myelomonocytic/physiology , Base Sequence , Blotting, Southern , Cells, Cultured , Cloning, Molecular , DNA Probes , Exons , Genetic Linkage , Genomic Library , Humans , Introns , Mice , Molecular Sequence Data , Monocytes , Promoter Regions, Genetic , RNA/analysis , RNA/isolation & purification , Sequence Analysis, DNA , Transcriptional Activation
17.
J Biol Chem ; 273(24): 15241-8, 1998 Jun 12.
Article in English | MEDLINE | ID: mdl-9614139

ABSTRACT

The scavenger receptor class B, type I (SR-BI), binds high density lipoprotein (HDL) and mediates selective uptake of cholesteryl ester from HDL and HDL-dependent cholesterol efflux from cells. We recently identified a new mRNA variant that differs from the previously characterized form in that the encoded C-terminal cytoplasmic domain is almost completely different. In the present study, we demonstrate that the mRNAs for mouse SR-BI and SR-BII (previously termed SR-BI.2) are the alternatively spliced products of a single gene. The translation products predicted from human, bovine, mouse, hamster, and rat cDNAs exhibit a high degree of sequence similarity within the SR-BII C-terminal domain (62-67% identity when compared with the human sequence), suggesting that this variant is biologically important. SR-BII protein represents approximately 12% of the total immunodetectable SR-BI/II protein in mouse liver. Subcellular fractionation of transfected Chinese hamster ovary cells showed that SR-BII, like SR-BI, is enriched in caveolae, indicating that the altered cytoplasmic tail does not affect targeting of the receptor. SR-BII mediated both selective cellular uptake of cholesteryl ether from HDL as well as HDL-dependent cholesterol efflux from cells, although with approximately 4-fold lower efficiency than SR-BI. In vivo studies using adenoviral vectors showed that SR-BII was relatively less efficient than SR-BI in reducing plasma HDL cholesterol. These studies show that SR-BII, an HDL receptor isoform containing a distinctly different cytoplasmic tail, mediates selective lipid transfer between HDL and cells, but with a lower efficiency than the previously characterized variant.


Subject(s)
CD36 Antigens/chemistry , Carrier Proteins , Lipid Metabolism , Lipoproteins, HDL/metabolism , Membrane Proteins , RNA-Binding Proteins , Receptors, Lipoprotein/metabolism , Sialoglycoproteins , Adenoviridae/genetics , Alternative Splicing/genetics , Amino Acid Sequence , Animals , CHO Cells , Cholesterol/pharmacokinetics , Cholesterol Esters/metabolism , Cricetinae , Lysosomal Membrane Proteins , Mice , Molecular Sequence Data , RNA, Messenger/metabolism , Receptors, Scavenger , Scavenger Receptors, Class B , Transfection/genetics
18.
Biochem J ; 332 ( Pt 3): 721-8, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9620875

ABSTRACT

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.


Subject(s)
Adenoviridae/genetics , Genetic Vectors , Serum Amyloid A Protein/biosynthesis , Acute-Phase Proteins/metabolism , Amyloid Neuropathies/blood , Animals , Cell Line , Cell Line, Transformed , Haplorhini , Lipoproteins, HDL/blood , Lipoproteins, HDL/chemistry , Male , Mice , Mice, Inbred BALB C , Microscopy, Electron , Rats , Rats, Sprague-Dawley , Recombinant Proteins/biosynthesis , Recombinant Proteins/blood , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , Serum Amyloid A Protein/genetics , Serum Amyloid A Protein/ultrastructure
19.
J Lipid Res ; 38(11): 2232-9, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9392421

ABSTRACT

Lipoprotein metabolism is markedly altered during inflammation. The concentration of human secretory phospholipase A2 (sPLA2) can increase hundreds of fold in inflammatory fluids and in the circulation. It was detected in atherosclerotic lesions where many inflammatory genes are induced. As sPLA2 has been reported to act on lipoproteins as substrates, lipoprotein profiles in transgenic mice expressing sPLA2 were studied. HDL levels were markedly decreased in transgenic mice overexpressing sPLA2. HDL in the transgenics were smaller in size, with a significant decrease (11%) in phospholipid content compared to nontransgenic littermates. In sPLA2 transgenic mice and transgenic mice expressing both sPLA2 and human apolipoprotein B (apoB), the concentrations of apoB-containing lipoproteins were not altered. We conclude that sPLA2 alters HDL metabolism and could be responsible for the depressed levels of HDL that exist during chronic inflammatory diseases.


Subject(s)
Lipoproteins, HDL/metabolism , Lipoproteins/metabolism , Phospholipases A/metabolism , Animals , Apolipoproteins B/blood , Apolipoproteins B/metabolism , Cholesterol/blood , Chromatography, Liquid , Female , Fibroblasts , Humans , Inflammation/metabolism , Lipids/blood , Lipoproteins, LDL/metabolism , Male , Mice , Mice, Transgenic , Phospholipases A/genetics , Phospholipases A2 , Protein Binding
20.
J Lipid Res ; 38(8): 1583-90, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9300780

ABSTRACT

Serum amyloid A (SAA) is an acute phase reactant that can become the predominant apolipoprotein of high density lipoprotein (HDL) during severe inflammatory states. However, the function of SAA is unknown. To study the ability of SAA to form HDL in the absence of apolipoprotein A-I, we expressed the mouse SAA pI 6.15 (CE/J) isoform in apolipoprotein A-I knock-out (apoA-I (-/-)) mice using a recombinant adenovirus. As a control, apoA-I (-/-) mice were injected with an adenovirus expressing human apoA-I. High level expression of plasma SAA was obtained in the absence of any endogenous acute phase SAA production. SAA expression increased plasma HDL cholesterol levels about 2-fold, but to a lesser extent than the expression of apoA-I (about 10-fold). The HDL particles isolated by density ultracentrifugation from SAA-expressing mice were heterogeneous in size and composition and rich in free cholesterol as well as apoE and apoA-IV. Of the SAA expressed in the plasma, only a small fraction (4%) was associated with HDL particles in contrast to expressed apoA-I, of which 62% was associated with HDL. We conclude that SAA is unable to substitute for apoA-I in HDL particle formation.


Subject(s)
Apolipoprotein A-I/deficiency , Apolipoproteins/genetics , Serum Amyloid A Protein/genetics , Acute-Phase Reaction/blood , Adenoviridae/genetics , Animals , Apolipoprotein A-I/genetics , Apolipoproteins/metabolism , Apolipoproteins A/blood , Apolipoproteins E/blood , Cholesterol/blood , Cholesterol, HDL/blood , Cholesterol, HDL/chemistry , Gene Expression , Genetic Vectors , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Serum Amyloid A Protein/metabolism
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