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Purpose: Matrix regenerating agents (RGTAs) emerged as promising in vivowound-healing agents. These agents could prove beneficial for the treatment of dry eye disease-associated corneal micro-erosions; therefore, we aimed to evaluate the wound healing efficacy of regenerative agents (RGTAs or serum) in an in vitromodel of hyperosmolarity (HO) stressed and non-stressed human corneal epithelial cells. Patients and methods: The migration and proliferation induced by the regenerative agents was evaluated using an in vitroscratch wound assay and brome-deoxy-uridine incorporation. The inflammatory profile and effects of osmoregulators were also investigated. The two-tailed paired t-test calculated the statistical significance, with P-value<0.05 considered significant. Results: The most efficient inducer of re-epithelization was 2% serum, followed closely by 2% RGTA with an average improvement in cell migration of 1.8- and 1.4-fold, respectively, when compared with the non-treated control. Hyperosmolar stress significantly reduced the restorative effects of both serum and RGTAs; these effects were, however, neutralized by the osmoregulator betaine. Conclusion: These findings suggest that RGTAs could provide efficient treatment for dry-eye associated corneal micro-lesions if ocular surface HO is neutralized.

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an inherent protease activity. After incubation of HDL3in the presence of purified plasma PLTP, the d < 1.25 g/ml particles (fusion particles) contained intact 28.2 kDa apoA-I while the d > 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleavage of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typically an 8–10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lagphase was necessary for the development of the cleavage activity during incubation at 37°C. The specific apoA-I cleavage activity of different PLTP preparations varied between 0.4–0.8 μg apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the Cterminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleavage occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical N-terminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Recombinant PLTP produced in CHO cells or using the baculovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated proteolytic cleavage of apoA-I might affect plasma HDL metabolism by generating a novel kinetic compartment of apoA-I with an increased turnover rate.—Jauhiainen, M., J. Huuskonen, M. Baumann, J. Metso, T. Oka, T. Egashira, H. Hattori, V. M. Olkkonen, and C. Ehnholm. Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I. J. Lipid Res.1999. 40: 654–664.

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. We proposed a covalent catalytic mechanism of action for LCAT (Jauhiainen, M., and Dolphin, P. J. (1986) J. Biol. Chem. 261, 7032-7034) in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols we have probed the geometry of the catalytic site. p-Aminophenylarsendichloride noncompetitively inactivates cholesterol esterification (Ki = 0.23 mM) by LCAT via alkylation of both catalytic cysteine residues. This reagent does not significantly inactivate lecithin cleavage by LCAT. Full enzyme activity is restored by treatment with 2,3-dimercapto-1-propanesulfonic acid. Treatment of LCAT with p-bromoacetylaminophenylarsenoxide blocks the subsequent incorporation of diisopropyl fluorophosphate and iodoacetamide and inactivates both cholesterol esterification and lecithin cleavage. These activities are not restored following 2,3-dimercapto-1-propanesulfonic acid treatment. However, the reduced cysteine thiols are regenerated and can catalyze cholesteryl arachidonate formation from arachidonyl-CoA. The control reagent, bromoacetylaniline, which lacks the sulfhydryl-reactive arsenical moiety, does not inactivate LCAT nor is this reagent incorporated into the LCAT protein. We conclude that the two catalytic cysteine residues of LCAT (Cys31 and Cys184) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by the bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.D., and Dolphin, P.J. (1987) Biochim. Biophys. Acta918, 175–188). This proposal is evaluated here by use of nonhydrolyzable sn-2-difluoroketone phosphatidylcholine analogues, known to inhibit calcium-dependent phospholipase A2. These compounds inhibited the calcium-independent phospholipase A2activity of LCAT in a time and concentration dependent manner. The most potent analogues had a 100-fold higher affinity for the enzyme than the substrate, lecithin, when present within lecithin/apoA-I proteoliposomes. The inhibition was dependent upon the presence of a difluoromethylene group α to the sn-2-carbonyl carbon of the analogues. The inhibition is attributed to the formation of a tetrahedral adduct between the catalytic serine residue of LCAT and the sn-2-carbonyl carbon atom of the analogues which is stabilized by the electronegative fluorine atoms present upon the carbon atom α to the carbonyl carbon. This adduct mimics that proposed by us to occur during lecithin cleavage by LCAT, and the data substantiate the existence of this transition state adduct prior to the release of lysolecithin and formation of a fatty acylserine oxyester of the enzyme.

Background: Serum has the ability to neutralize the procoagulant properties of anionic liposomes, with transfer of phospholipids (PLs) to both high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) particles. Phospholipid transfer protein (PLTP) mediates transfer of PLs between HDL and other lipoproteins and conversion of HDL into larger and smaller particles. Objectives: To examine the role of PLTP in the neutralization of procoagulant liposomes. Methods: Procoagulant liposomes were incubated with different lipoproteins in the presence or absence of PLTP, and then tested for their ability to stimulate thrombin formation. Results and Conclusions: In the absence of added PLTP, the lipoprotein‐enriched fraction, total HDL, HDL3and very high‐density lipoprotein (VHDL) were all able to neutralize the procoagulant properties of the liposomes. In these samples, endogenous PLTP was present, as judged by Western blotting. In contrast, no PLTP was present in LDL, HDL2and lipoprotein‐deficient serum, all of which displayed no ability to neutralize the procoagulant liposomes. The phospholipid (PL) transfer activity was dependent on both enzyme (PLTP) and PL acceptor (lipoproteins). After treatment of the VHDL fraction with antiserum against PLTP, the neutralization of procoagulant activity was reduced, but could be regained by the addition of active PLTP. The neutralizing activity was dependent on a catalytically active form of PLTP, and addition of a low activity form of PLTP had no effect. In conclusion, PLTP was found to mediate transfer of anionic PLs to HDL and LDL, thereby neutralizing the effect of procoagulant liposomes, resulting in a reduction of procoagulant activity.

To determine whether hydrogenated fat consumption alters triglyceride metabolism and cholesterol esterification rates, 14 women (65-71 years of age) were provided with each of four diets for 5-week periods according to a randomized cross-over design. The experimental diets contained either soybean oil (SO), low trans squeeze (SQM), medium trans tub (TM), or high trans stick (SM) margarines. Triglyceride uptake by adipose tissue was determined by measuring plasma acylation-stimulating protein (ASP), FFA, glucose, and insulin levels, while rates of transfer and esterification rate of newly synthesized cholesterol (ER) were derived by using plasma CETP levels and the deuterium incorporation methodology. Plasma ASP levels were lowest (P < 0.05) in subjects on the SM diet (33.4 +/- 12.7 nM) compared with the SO (48.7 +/- 17.0 nM) and SQM (50.7 +/- 15.7 nM) diets. Conversely, FFA were highest (P < 0.05) on the SM diet (0.86 +/- 0.45 mM) relative to all the other diets. No differences were observed in plasma glucose and insulin levels among diets. A trend toward higher CETP levels after consumption of the SM diet was observed. However, the ER was lowest (P < 0.05) after the SM (0.111 +/- 0.062 g x day(-1)) diet and highest after consumption of the SQM (0.216 +/- 0.123 g x day(-1)) diet. In addition, ASP levels were negatively correlated with FFA (r = -0.63, P < 0.05), LDL cholesterol (r = -0.56, P < 0.05), and TG (r = -0.41, P < 0.05), whereas FFA was positively correlated with apolipoprotein B-containing lipoproteins (r = 0.58 and 0.47, for VLDL and LDL cholesterol, P < 0.05), and negatively correlated with HDL cholesterol (r = -0.51, P < 0.05). The ER was found to positively correlate with HDL cholesterol and HDL2 subfraction (r = 0.53 and 0.45, respectively, P < 0.05). Taken together, these data demonstrate that the alterations in circulating lipid levels, commonly observed with consumption of hydrogenated fat-rich diets, can be explained in part by changes in ASP activity as well as newly synthesized cholesterol ER.

Relative to saturated fatty acids, trans-fatty acids/hydrogenated fat-enriched diets have been reported to increase low density lipoprotein (LDL) cholesterol levels and either decrease or have no effect on high density lipoprotein (HDL) cholesterol levels. To better understand the effect of trans-fatty acids/hydrogenated fat on HDL cholesterol levels and metabolism, 36 subjects (female, n = 18; male, n = 18) were provided with each of three diets containing, as the major sources of fat, vegetable oil-based semiliquid margarine, traditional stick margarine, or butter for 35-day periods. LDL cholesterol levels were 155 +/- 27, 168 +/- 30, and 177 +/- 32 mg/dl after subjects followed the semiliquid margarine, stick margarine, and butter-enriched diets, respectively. HDL cholesterol levels were 43 +/- 10, 42 +/- 9, and 45 +/- 10 mg/dl, respectively. Dietary response in apolipoprotein (apo) A-I levels was similar to that in HDL cholesterol levels. HDL(2) cholesterol levels were 12 +/- 7, 11 +/- 6, and 14 +/- 7 mg/dl, respectively. There was virtually no effect of dietary fat on HDL3 cholesterol levels. The dietary perturbations had a larger effect on particles containing apoA-I only (Lp A-I) than apoA-I and A-II (Lp A-I/A-II). Cholesterol ester transfer protein (CETP) activity was 13.28 +/- 5.76, 15.74 +/- 5.41, and 14.35 +/- 4.77 mmol x h(-1) x ml(-1), respectively. Differences in CETP, phospholipid transfer protein activity, or the fractional esterification rate of cholesterol in HDL did not account for the differences observed in HDL cholesterol levels.These data suggest that the saturated fatty acid component, rather than the trans- or polyunsaturated fatty acid component, of the diets was the putative factor in modulating HDL cholesterol response.

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP.In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.

The properties of the mature and pro-forms of recombinant apolipoprotein A-I (apoA-I) were compared with those of apoA-I isolated from human plasma. When the synthesis and secretion of pro- and mature forms of apoA-I from a baculovirus/insect cell expression system were compared in parallel experiments, the amount of the pro-form of apoA-I synthesized and secreted was severalfold higher than that of the mature form of apoA-I. A comparison of the properties of the pro- and mature forms of recombinant apoA-I and human plasma apoA-I showed no difference between all three in their secondary structure, their ability to self-associate, lipid-binding capacity, lecithin: cholesterol acyltransferase activation, and binding to the phospholipid transfer protein. The properties of reconstituted high density lipoprotein (HDL) particles formed from the proteins and their ability to promote cholesterol and phospholipid efflux from human skin fibroblasts were also similar. However, their ability to bind to plasma HDL subfractions differed, because twice as much proapoA-I associated with prebeta(1)-HDL and prebeta(2)-HDL subfractions compared with both mature recombinant and plasma apoA-I. Correspondingly, the amount of proapoA-I in alpha-HDL subfractions, especially in alpha(1)-HDL and alpha(2)-HDL, was decreased. We conclude that while the propeptide of apoA-I is required for the effective synthesis and secretion of apoA-I, cleavage of this peptide is a requisite for the effective interconversion of HDL subfractions.

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160+/-40 kDa and an inactive form with an average mass of 520+/-120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition.

The plasma phospholipid transfer protein (PLTP) is an important regulator of high density lipoprotein (HDL) metabolism. We have here, based on sequence alignments of the plasma LPS-binding/lipid transfer protein family and the X-ray structure of the bactericidal/permeability increasing protein (BPI), modeled the structure of PLTP. The model predicts a two-domain architecture with conserved lipid-binding pockets consisting of apolar residues in each domain. By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets are shown to be essential for PLTP-mediated phospholipid transfer. A solid phase ligand binding assay was used to determine the HDL-binding ability of the mutants. The results suggest that the observed decreases in phospholipid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDL-binding, but the C-terminal lipid-binding pocket may be involved in the association of PLTP with HDL. Further, the essential structural role of a disulfide bridge between cysteine residues 146 and 185 is demonstrated. The structural model and the mutants characterized here provide powerful tools for the detailed analysis of the mechanisms of PLTP function.

The plasma concentration of human lipoprotein(a) [Lp(a)] is correlated with the risk of heart disease. A distinct feature of the Lp(a) particle is the apolipoprotein (a) [apo(a)], which is associated with apoB‐100, the main protein component of low‐density lipoprotein. We now report that apo(a), which has extensive homology to plasminogen, binds to immobilized fibronectin. The binding of Lp(a) was localized to the C‐terminal heparin‐binding domain of fibronectin. Incubation of Lp(a) with fibronectin resulted in fragmentation of fibronectin. The cleavage pattern, as visualized by gel electrophoresis and immunoblotting, was reproducibly obtained with Lp(a) purified from five different individuals and was distinct from that obtained upon proteolysis of fibronectin by plasmin or kallikrein. The use of synthetic peptide substrates demonstrated that the amino acid specificity for Lp(a) was arginine rather than lysine. The proteolytic activity of Lp(a) was localized to apo(a) and experiments with inhibitors indicated that the proteolytic activity was of serine proteinase‐type.

Genetically hypercholesterolaemic RICO rats (male, 6 weeks old) were randomly distributed into 6 experimental groups. The zero-time basal group A was sacrificed at the start of the experiment while the other groups were fed for 6 weeks and then sacrificed. Group B was fed a stock diet. Control group C was fed a high-sucrose (45%) diet with 0.5% added cholesterol. In the diet of group D, only the magnesium (Mg) content was reduced from the level of group C (883 ppm) to 200 ppm. The diet of group E was the same as that of group D with the addition of 12 ppm of fluoride (F) and the diet of group G was the same as that of group E, but with its Mg content elevated from 200 ppm to 300 ppm. Analysis of aortic blood samples, taken before sacrifice, indicated significant increases in total serum cholesterol (p<0.01), very low density lipoprotein (VLDL) (p<0.001) and low density lipoprotein (LDL), (p<0.001) cholesterol, and a trend to lower high density lipoprotein (HDL) cholesterol in group C, as compared to group B. Significantly lower total (p<0.05), VLDL (p<0.01) and LDL (p<0.01) triglycerides were observed in group C when compared to group B. The LDL phospholipids were significantly higher in group C (p<0.001) than in group B. When cholesterol levels in groups D, E and G were compared with group C, the VLDL cholesterol in group E and the LDL cholesterol in group G were slightly but significantly (p<0.05) reduced, while total cholesterol and the other subtractions were unaltered. The LDL triglycerides of groups E and G were significantly smaller still than the already small fraction in group C. The VLDL triglyceride in group E was significantly lower than that of group C (35% reduction, p<0.001), D and G (p<0.05). Phospholipids were slightly but significantly reduced in the VLDL fraction of group E and in the LDL fraction of group G (p<0.05 and 0.01, respectively), as compared to those of group C.

In the present work we have tested a new immunoturbidometric (IT) lipoprotein(a) (Lp(a)) assay and compared it with two other Lp(a) assay systems (IRMA and ELISA) commonly used in clinical chemistry laboratories. In addition, we have examined the effects of long-term storage and plasminogen on the results of Lp(a). Intra-assay and inter-assay coefficients of variation of the IT method were from 1.6 to 5.5% and from 2.8 to 10.7% depending on the concentration of Lp(a). The correlations between methods were 0.957 (IRMA vs. IT), 0.969 (IRMA vs. ELISA) and 0.956 (IT vs. ELISA) and the regression curves were IRMA = 1.07*IT + 11, IRMA = 1.84*ELISA + 2 and IT = 1.62*ELISA + 17, respectively. Storage of the samples for 5 years at - 70°C did not affect serum Lp(a) levels. There was a slight increasing effect of high concentrations of plasminogen on the Lp(a) results, but on physiological serum levels of plasminogen the effect was not significant. We conclude that the IT method provides a simple way to screen serum Lp(a) levels.

alpha-Tocopherol (alpha-T), an important anti-oxidant of plasma lipoproteins and cell membranes, is secreted from liver together with very-low-density lipoproteins into the blood stream. Other serum lipoprotein classes gain alpha-T by exchange and transfer processes. We show here that the lipoprotein-free d > 1.22 g/ml fraction of human or pig serum increases the exchange rate of alpha-T by a factor of 2-4 as compared with spontaneous exchange/transfer. The alpha-T exchange/transfer (alpha-TET) activity was purified by multiple-step column chromatography. It gave a single band in PAGE with an apparent molecular mass of 75 kDa, and was found to be identical with the phospholipid transfer protein (PLTP). PLTP catalysed alpha-T exchange between different lipoprotein classes, as well as the transfer of alpha-T from artificial liposomes to high-density lipoproteins. The alpha-TET activity measured with a newly developed assay in ten healthy people was 2.45 +/- 0.88 nmol.ml-1.h-1.alpha-TET activity was negatively correlated with plasma low-density lipoprotein-cholesterol (r = -0.75; P < 0.01). It is concluded that human PLTP catalyses exchange/transfer processes of alpha-T between lipid compartments. This factor may be of relevance in atherogenesis and tumour initiation and growth.

Phospholipid transfer protein (PLTP) converts high density lipoproteins (HDL) to larger and smaller particles during incubation in vitro. The present study shows that these conversions are enhanced in triglyceride (TG)-enriched HDL. When pig HDL were enriched with TG by incubation with cholesteryl ester transfer protein (CETP) and very low density lipoproteins, the TG/apoA-I molar ratio increased from 0.7/1 to 11.4/1 and the diameter increased from 9.0 to 9.5 nm. When unmodified pig HDL were incubated with PLTP, the diameter increased from 9.0 to 9.5 nm. Incubation of the TG-enriched pig HDL with PLTP generated larger (13.0 nm) and smaller (7.5 nm) particles. The composition of the large particles was comparable to the non-incubated, TG-enriched HDL. The small particles contained only phospholipids and apolipoprotein (apo) A-I. Studies were also carried out with spherical, reconstituted HDL (rHDL) containing only cholesteryl esters in their core. When the rHDL were enriched with TG by incubation with Intralipid and CETP, the diameter increased from 9.0 to 9.2 nm and the TG/apoA-I molar ratio became 5.7/1. When unmodified rHDL were incubated with PLTP, the diameter of 15% of the particles increased to 11.0 nm, 18% decreased to 7.6 nm, and 67% were unchanged. When the TG-enriched rHDL were incubated with PLTP, the diameter of 62% of the particles increased to 11.4 nm, 26% decreased to 7.6 nm, and 12% did not change. Preincubation with Intralipid alone or CETP alone did not enrich the rHDL with TG or enhance the size changes. These results show that the TG content of HDL has a major influence on PLTP-mediated size changes.

Plasma phospholipid transfer protein (PLTP) plays a critical role in lipoprotein metabolism and reverse cholesterol transport. We have studied the biosynthesis and secretion of PLTP using a stably transfected inducible HeLa cell line. Pulse-chase analysis revealed that: i) the major secreted forms of PLTP carry complex N-glycans; ii) N-glycosylation is crucial for PLTP secretion; iii) Endo H-resistant forms of PLTP could not be enriched using a 20 degrees C temperature block, indicating that the transport of PLTP from the endoplasmic reticulum to the Golgi apparatus is exceptionally sensitive to low temperatures; and iv) treatment of the PLTP-producing cells with the reducing agent dithiothreitol caused a reversible secretion arrest, suggesting a role of disulfide bonds in the correct folding of PLTP. Transient expression of C-terminally truncated PLTP variants in COS cells demonstrated that: i) the 30 C-terminal amino acids are dispensable for PLTP secretion, whereas deletion of 35-50 residues results in a complete absence of secretion; and ii) the deletion of 30 C-terminal amino acid residues almost completely abolished the phospholipid transfer activity of PLTP. The present study describes for the first time the biosynthesis of phospholipid transfer protein and provides tools for detailed elucidation of the structure-function relationships in the protein.

The interaction of plasma phospholipid transfer protein (PLTP) with HDL has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-mediated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule. To study the PLTP/apolipoprotein interaction we used a solid phase ligand binding assay, the ELISA technique, and apoA-I and apoA-II affinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a concentration-dependent manner, and the binding could be displaced by preincubating the PLTP sample with purified apolipoproteins. To determine which portion of apoA-I is recognized by PLTP, we coated ELISA plates with either recombinant full-length apoA-I or three shortened apoA-I forms sequentially truncated from the C-terminus. To characterize the PLTP binding ability of the C-terminal region of apoA-I, we used both C-terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. To further confirm the identity of the binding region, we probed the interaction with a polyclonal and several monoclonal anti-apoA-I antibodies. The antibodies that inhibited the interaction between PLTP and apoA-I were directed towards apoA-I epitopes localized between amino acids 27-141. The polyclonal antibody, R33, and the monoclonal antibody A-I-1 (epitope between amino acids 27-48) were most effective and reduced PLTP binding by 70%. These results show that PLTP binds to both apoA-I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.

Low density lipoprotein (LDL) particles can undergo fusion in the arterial intima, where they are bound to proteoglycans. Here we studied the effect of human arterial proteoglycans on proteolytic fusion of LDL in vitro. For this purpose, an assay was devised based on fluorescence resonance energy transfer that allowed continuous monitoring of fusion of proteoglycan-bound LDL particles. We found that addition of human arterial proteoglycans markedly increased the rate of proteolytic fusion of LDL. The glycosaminoglycans isolated from the proteoglycans also increased the rate of fusion, demonstrating that this effect was produced by the negatively charged sulfated polysaccharides in the proteoglycans. Furthermore, heparin, chondroitin 6-sulfate, and dextran sulfate, three commercially available sulfated polysaccharides, also increased the rate of LDL fusion, with heparin and chondroitin 6-sulfate being as effective as and dextran sulfate more effective than human proteoglycans. The ability of the sulfated polysaccharides to increase the rate of proteolytic fusion of LDL depended critically on their ability to form insoluble complexes with LDL, which, in turn, resulted in an increased rate of LDL proteolysis and, in consequence, in an increased rate of LDL fusion. The results reveal a novel mechanism regulating LDL fusion and point to the potentially important role of arterial proteoglycans in the generation of LDL-derived lipid droplets in the arterial intima during atherogenesis.

To study the function of plasma phospholipid transfer protein (PLTP) in vivo, a liver directed adenoviral gene transfer system was used to overexpress human PLTP in mice. For the experiments, two strains of mice, wild type (C57/B1) and mice transgenic for the human apoA-I gene (HuApoA-ITg), were utilized. Five days after injection of the recombinant PLTP adenovirus, wild type mice showed a 4-fold increase in serum PLTP activity in (12.2+/-1.3 micromol/ml per h to 48.1+/-8.6 micromol/ml per h (+394%), P < 0.001). The PLTP overexpression induced significant reduction of serum cholesterol (2.46+/-0.08 to 0.69+/-0.42 mmol/l (-72%), P < 0.001), phospholipids (3.10+/-0.06 to 0.90+/-0.24 mmol/l (-71%), P < 0.01), and triglycerides (0.2+/-0.07 to 0.08+/-0.03 mmol/l (-69%), (P < 0.001). ApoA-I was hardly detectable in the serum. These lipid changes were due to a dramatic reduction of high density lipoprotein (HDL). The HuApoA-ITg mice displayed higher basal HDL level and PLTP activity. Adenovirus mediated PLTP overexpression in these mice resulted in a similar decrease of the lipid levels as that seen in the C57/B1 mice. However, the lipoprotein profile revealed a redistribution of HDL, with the appearance of larger buoyant HDL species. The results demonstrate that plasma phospholipid transfer protein in vivo causes high density lipoprotein (HDL) conversion and thereby plays a central role in HDL metabolism.

Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement of the phospholipase A2 and transacylase activities of the enzyme using proteoliposome substrates and following selective chemical modification of serine, histidine, and cysteine residues of pure homogeneous LCAT indicated the following catalytic mechanism: HS-Cys-E-Ser-OH + lecithin in equilibrium HS-Cys-E-Ser-O-FA + lysolecithin, HS-Cys-E-Ser-O-FA in equilibrium FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH + cholesterol-OH in equilibrium HS-Cys-E-Ser-OH + cholesterol-O-FA, where FA denotes fatty acid. Modification of 2 LCAT cysteine residues with 5,5‘-dithiobis-(2-nitrobenzoic acid) or treatment with ferricyanide inactivated the transacylase but not the phospholipase A2 activity. Modification of 1 serine residue with phenylmethanesulfonyl fluoride or 1 histidine residue with diethyl pyrocarbonate inhibited cholesteryl ester formation and phospholipase A2 activity. Proteoliposome substrates protected both activities against chemical inactivation. Lecithin alone protected the phospholipase A2 activity against phenylmethanesulfonyl fluoride inactivation but not the transacylase against 5,5‘-dithiobis-(2-nitrobenzoic acid) inactivation. Incubation of native LCAT with arachidonyl-CoA or the lecithin-apo-A-I proteoliposome resulted in acylation of three enzyme sites, only one of which was stable to neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- and thioesters were demonstrable in both cases. No transfer of arachidonic acid from iodoacetamide-modified LCAT to cholesterol occurred, indicating that the fatty-acylated serine residue cannot directly esterify cholesterol. Cholesterol arachidonate was formed upon incubation of phenylmethanesulfonyl fluoride-modified LCAT with arachidonyl-CoA.

The plasma concentration of lipoprotein (a) [Lp(a)] is correlated with the risk of atherosclerosis. It is a lipoprotein particle consisting of apoprotein (a) [Lp(a)] is correlated with the risk of atherosclerosis. It is a lipoprotein particle consisting of apoprotein (a) [apo(a)], a protein showing considerable amino acid sequence identity with plasminogen. bound to low-density lipoprotein. The apo(a) portion of Lp(a) was recently shown to have serine-proteinase-type amidolytic activity and to be able to degrade the adhesive glycoprotein fibronectin. To characterize this enzyme activity further, we used chromogenic peptide substrates and inhibitors. Of the substrates tested, those with arginine at the scissile bond [N-alpha-benzoyl-L-Arg p-nitroanilide (pNA), N-alpha-benzoyl-Ile-Glu-Gly-Arg-pNA, N-alpha-benzyloxycarbonyl-Arg-Gly-Arg-pNA] gave the highest hydrolysis rates. Synthetic substrates with plasmin specificity (Val-Leu-L-Lys-pNA and Val-Phe-L-Lys-pNA) were not hydrolysed by Lp(a). Neither tissue plasminogen activator nor urokinase had any effect on the enzyme activity. The addition of antibodies to these plasminogen activators did not inhibit the enzyme activity of Lp(a). Inhibition experiments with phenylmethanesulphonyl fluoride, carbodi-imide, dichloroisocoumarin and competitive peptide inhibitors demonstrated that Lp(a) has enzyme activity that closely resembles that of serine proteinases. Whether this serine-proteinase activity of Lp(a) plays any role in the genesis of atherosclerosis remains to be established.

Previous observations have suggested that low intakes of fluoride prevent pathological calcifications of internal organs, including the aortic wall, in experimental animals, fed a basically low magnesium diet. Our group found recently that fluoride has some potentially preventive effect against atherosclerotic serum lipid profiles in genetically hypercholesterolaemic rats. To study whether the apparently positive potential of fluoride against atherosclerosis is also reflected in aortic tissue, through its well known activation of adenylate cyclase, the aortic cAMP content of the rats used in our recent study was determined. Out of a total of 56 male RICO rats, mean weight 160 g, the control group C was fed an adequate diet, with 44% sucrose, a magnesium content of 883 p.p.m. and with 0.5% cholesterol. Group D had the same diet as group C except that the magnesium content was reduced to 200 p.p.m. Group E had the same diet as group D but with the fluoride content elevated from 1.9 to 12 p.p.m. Group G had the same diet as group E but with the magnesium content elevated from 200 to 300 p.p.m. After a feeding period of 6 weeks, the aortas of the animals were removed, cleaned and kept at -70°C until analysed. The mean cAMP content of the aortas, measured by radioimmunoassay, in groups C, D, E and G was 439, 546, 681, and 1394 μmol mg-1 protein, respectively. In group G only, the cAMP content was significantly higher than that of the other groups (p<0.001). The mean calcium and magnesium contents of the aortas of different groups did not significantly differ from each other. Thus in RICO rats, fed a high-sugar low-magnesium diet with cholesterol, supplementation of the diet with a small amount of fluoride elevates the cAMP content of the aorta, provided that the intake of Mg is not very low.

The object of this study was to compare the indirect diagnostic methods on the basis of urinary galactose determination in the diagnosis of lactose malabsorption with the actual lactase activities. One hundred and seven patients were studied. The specificity and sensitivity of the strip test were 97%. With 30% actual prevalence the positive predictive value was 94%, and the negative predictive value was 99%. In common prevalences of hypolactasia the strip test was reliable.

The aim of this study was to continue our previously published work and to compare the different indirect diagnostic methods for hypolactasia with the lactase to sucrase ratio obtained by jejunal biopsy. The following tests were performed in 63 adult patients: the breath hydrogen test, the lactose tolerance test with ethanol (serum galactose measurement after oral lactose load with ethanol), the urinary lactose tolerance test (urinary galactose measurement after oral lactose load with ethanol), and the strip test (like the former but using a special test strip for urinary galactose). Specificities of all these tests were good (96-98%). The 3-h breath hydrogen test was less sensitive (69%) than the other methods (81-94%). The strip test is recommended for the general practitioner for the diagnosis of this common cause of abdominal complaints.

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.