Bovine Lipoprotein Research Articles

Functionalisation of brush polyethylene glycol polymers with specific lipids extends their elimination half-life through association with natural lipid trafficking pathways

Polymeric prodrugs have been applied to control the delivery of various types of therapeutics. Similarly, conjugation of peptide therapeutics to lipids has been used to prolong systemic exposure. Here, we extend on these two approaches by conjugating brush polyethylene glycol (PEG) polymers with different lipid components including short-chain (1C2) or medium-chain (1C12) monoalkyl hydrocarbon tails, cholesterol (Cho), and diacylglycerols composed of two medium-chain (2C12) or long-chain (2C18) fatty acids. We uniquely evaluate the integration of these lipid-polymers into endogenous lipid trafficking pathways (albumin and lipoproteins) and the impact of lipid conjugation on plasma pharmacokinetics after intravenous (IV) and subcutaneous (SC) dosing to cannulated rats. The IV and SC elimination half-lives of Cho-PEG (13 and 22 h, respectively), 2C12-PEG (11 and 17 h, respectively) and 2C18-PEG (12 h for both) were prolonged compared to 1C2-PEG (3 h for both) and 1C12-PEG (4 h for both). Interestingly, 1C2-PEG and 1C12-PEG had higher SC bioavailability (40 % and 52 %, respectively) compared to Cho-PEG, 2C12-PEG and 2C18-PEG (25 %, 24 % and 23 %, respectively). These differences in pharmacokinetics may be explained by the different association patterns of the polymers with rat serum albumin (RSA), bovine serum albumin (BSA) and lipoproteins. For example, in pooled plasma (from IV pharmacokinetic studies), 2C18-PEG had the highest recovery in the high-density lipoprotein (HDL) fraction. In conclusion, the pharmacokinetics of brush PEG polymers can be tuned via conjugation with different lipids, which can be utilised to tune the elimination half-life, biodistribution and effect of therapeutics for a range of medical applications. Statement of significanceLipidation of therapeutics such as peptides has been employed to extend their plasma half-life by promoting binding to serum albumin, providing protection against rapid clearance. Here we design and evaluate innovative biomaterials consisting of brush polyethylene glycol polymers conjugated with different lipids. Importantly, we show for the first time that lipidated polymeric materials associate with endogenous lipoprotein trafficking pathways and this, in addition to albumin binding, controls their plasma pharmacokinetics. We find that conjugation to dialkyl lipids and cholesterol leads to higher association with lipid trafficking pathways, and more sustained plasma exposure, compared to conjugation to short and monoalkyl lipids. Our lipidated polymers can thus be utilised as delivery platforms to tune the plasma half-life of various pharmaceuticals.

Agarose gel electrophoresis determination of bovine lipoproteins compared with a wet chemistry method

Quantification of serum lipoproteins provides information relative to the overall metabolic health, degree of lipid mobilization, and hepatic function of dairy cattle. Automated assays performed on benchtop chemistry analyzers and commercially available kits use reagents developed for human lipoproteins. The substantial physical and chemical differences between bovine and human lipoproteins potentially confounds the use of these assays in evaluating bovine lipoproteins. In this study, we prospectively analyzed serum lipoproteins from 56 Holstein cows using horizontal slab agarose gel electrophoresis to semi-quantify the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions by optical densitometry. Ultracentrifugation was used to confirm the electrophoretic separation pattern of the lipoproteins. The values obtained using the electrophoretic method were compared with values obtained by direct measure of HDL cholesterol, total cholesterol, and triglyceride (TG) concentrations on a Roche chemistry analyzer, and calculated LDL cholesterol. Correlation between these methods was poor for HDL (Passing-Bablok regression line: y = 30.31 + 0.853x) and could not be calculated for LDL. Automated HDL values were equal to, or higher than, the total cholesterol concentration in 25 of the 56 samples. The TG concentrations were above the reference interval in 18 samples, and these samples had an average of 96% of the cholesterol measured as HDL by the automated method, and 78% HDL by electrophoresis. Given that it is physiologically impossible to have more cholesterol within the HDL fraction than in the total serum fraction, and the increased proportion of TG found in LDL and very-low-density lipoprotein, our results draw into question the accuracy of the Roche automated assay in quantifying bovine lipoprotein fractions.

Technical note: Characterization of ceramide in bovine lipoproteins

The hepatic synthesis and export of ceramide is enhanced in diabetic monogastrics. Moreover, ceramide in lipoproteins can mediate the development of insulin resistance. We have previously demonstrated that circulating ceramide increases during the progression of insulin resistance in postpartum dairy cows. Considering that the origins of circulating ceramide required investigation, our objective was to develop a method to characterize the ceramide profile of lipoprotein fractions collected from dairy cows. Serum was collected from 4 nonpregnant and nonlactating Holstein dairy cows. Serum lipoproteins were isolated using size exclusion chromatography by fast protein liquid chromatography (SEC-FPLC). Measurement of triacylglycerol (TAG), phospholipid, total cholesterol, and protein was performed using standard colorimetry practices. Following lipid extraction, fractions were analyzed using electrospray ionization tandem mass spectrometry. Data were analyzed as repeated measures using a mixed model. Lipoprotein isolation using SEC-FPLC and subsequent colorimetric analyses confirmed the presence of 4 distinct fractions: TAG-rich, low density (LDL), and large (buoyant) and small (dense) high density lipoprotein (HDL) subclasses. As expected, the fraction representing mixed very low density lipoproteins and chylomicrons primarily contained TAG. Low density lipoprotein fractions were equally enriched with cholesterol and phospholipid. Buoyant HDL contained elevated levels of cholesterol, phospholipid, and protein. In contrast, the fraction containing dense HDL primarily contained protein. Our method revealed that LDL are enriched with ceramides. Ceramides were also compartmentalized to a lesser extent within both HDL subclasses and TAG-rich lipoproteins. Comparable to whole serum, C16:0-ceramide was the predominant ceramide quantified in all lipoprotein subclasses. Interestingly, the proportion of C24:0-ceramide to total ceramide was elevated in TAG-rich lipoproteins, relative to all other lipoprotein subclasses. We conclude that SEC-FPLC coupled with mass spectrometry is a means to quantify ceramides in lipoprotein fractions. Moreover, ceramides are enriched within bovine LDL, and lipoprotein ceramide profiles reflect levels observed in whole serum. Future investigation will need to determine the biological importance of lipoprotein ceramides with distinct C-chains at amide residues.

Effect of dietary vegetable oils on the fatty acid profile of plasma lipoproteins in dairy cows

ABSTRACTThe aim of this study was to elucidate the effect of dietary supplementation of soybean oil (SO) and hydrogenated palm oil (HPO) on the transport of fatty acids (FA) within plasma lipoproteins in lactating and non-lactating cows. Three lactating and three non-lactating Holstein cows were used in two different 3 × 3 Latin square experiments that included three periods of 21 d. Dietary treatments for lactating cows consisted of a basal diet (control; no fat supplement) and fat-supplemented diets containing SO (500 g/d per cow) or HPO (500 g/d per cow). For non-lactating cows, dietary treatments consisted of a basal diet (control; no fat supplement) and fat-supplemented diets containing SO (170 g/d per cow) or HPO (170 g/d per cow). Compared with the control and SO diet, HPO addition increased (p < 0.05) the concentration of C16:0, C18:0, C18:2cis-9,12, C18:3cis-9,12,15 and total saturated and polyunsaturated FA in the plasma of lactating cows. In non-lactating cows, the SO addition increased the plasma concentration of C18:1trans-11. In lactating cows, concentrations of C16:0, C18:0 and total saturated FA were increased (p < 0.05) by HPO addition in the high-density lipoprotein (HDL). Total saturated FA were increased (p < 0.05) by HPO in very-low-density lipoprotein (VLDL). In non-lactating cows, the concentration of C18:0 was increased (p < 0.05) by HPO in HDL, whereas C18:1trans-11 was increased (p < 0.05) by SO in the low-density lipoprotein. Overall, it was found that distribution and transport of FA within the bovine plasma lipoproteins may be influenced by chain length and degree of unsaturation of dietary lipids. Also, the distribution of individual FA isomers such as C18:1trans-11 and C18:2cis-9,trans-11 may vary depending on the physiological state of the cow (lactating or non-lactating), and are increased in plasma (lactating cows) and the HDL (non-lactating cows) when cows are fed SO.

Association studies on the bovine lipoprotein lipase gene polymorphism with growth and carcass quality traits in Qinchuan cattle

Lipoprotein lipase (LPL) is considered as an essential enzyme in lipid deposition and tissue metabolism. It has been proposed to be a lead candidate gene for genetic markers of lipid deposition and energy balance. In this paper, polymorphisms in the LPL gene were investigated in 554 Chinese Qinchuan cattle by PCR-RFLP and DNA sequencing. Seven single nucleotide polymorphisms (SNPs) were identified, which included one mutation (g.91C > T) in the 5′untranslated region (UTR), four synonymous mutations (g.17015A > G, g.18362G > A, g.18377T > C and g.19873T > C) and two mutations (g.25225A > G and g.25316T > G) in the 3′UTR. The frequencies of SNP g.18377T > C and g.25316T > G were skewed from Hardy–Weinberg equilibrium in all the samples (chi-square test, P < 0.05). An association analysis showed that five loci (except for g.91C > T and g.18377T > C) were significantly correlated with some growth and carcass quality traits. These results demonstrate that LPL might be a potential candidate gene for marker-assisted selection (MAS).

Resorufin Butyrate as a Soluble and Monomeric High-Throughput Substrate for a Triglyceride Lipase

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z′ > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.

Inclusion of bovine lipoproteins and the vitamin E analogue, Trolox, during in vitro culture of bovine embryos changes both embryo and fetal development

This experiment investigated effects of lipoproteins and Trolox (vitamin E analogue) on bovine embryo and fetal development. The treatments were: in vitro culture (IVC) in synthetic oviducal fluid alone (SOF); with bovine lipoproteins (2% v/v; SOFLP); with Trolox (100μM; SOFT); and with lipoproteins and Trolox (SOFLPT). In vitro culture with lipoproteins increased fatty acid content of blastocysts (P<0.001) whereas inclusion of Trolox had no effect (P>0.05). Whereas lipoproteins reduced zygote development to blastocysts (P=0.03), Trolox facilitated increased development (P<0.001) and counteracted the reduction observed with lipoproteins (interaction, P=0.009). Lipoproteins also compromised (P<0.001) but presence of Trolox (P>0.05) had no effect on blastocyst morphological grade. Pregnancy rates resulting from synchronous transfer of IVP embryos were not affected by IVC treatment. At Day 70 of pregnancy, compared with SOF, fetal weight was lower in SOFLP but not SOFLPT (interaction, P<0.001). Liver weight (g kg(-1) fetal weight) was greater (P=0.03) in treatments containing Trolox. Placentome numbers were greater in SOF and SOFLPT compared with SOFLP and SOFT (interaction, P=0.002); superior embryo grades were also associated with increased numbers of placentomes (P=0.024). In conclusion, the interactive effects of lipoprotein and Trolox inclusion on in vitro embryo development were also evident in fetal development at Day 70.

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Differential transport of trans fatty acids by bovine plasma lipoprotein fractions: 1. Soya oil and partially hydrogenated vegetable oil

Differential transport of trans fatty acids by bovine plasma lipoprotein fractions: 1. Soya oil and partially hydrogenated vegetable oil

Differential transport of trans fatty acids by bovine plasma lipoprotein fractions: 2. Fish oil and partially hydrogenated vegetable oil

Differential transport of trans fatty acids by bovine plasma lipoprotein fractions: 2. Fish oil and partially hydrogenated vegetable oil

Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

Application of Proteomic Tools To Detect the Nonspecificity of a Polyclonal Antibody against Lipoprotein Lipase

Specific antibodies are essential tools for studying proteins. P66 is a chicken polyclonal antibody raised against bovine lipoprotein lipase (LPL) that has been used in earlier studies. Here, we developed a two-dimensional (2D) Western blot with reducing gels, using commercial bovine LPL (53 kDa) as a standard and P66 for detection. Our results revealed incomplete purification of commercial LPL and nonspecificity of P66, both undetectable in one-dimensional analysis. Antithrombin III (ATIII) was identified as both a major contaminant in commercial LPL and a cross-reacting protein with P66. Although LPL purification methods were presumably designed to eliminate ATIII, here we demonstrate that some procedures fell short of this objective and thus led to the production of a nonspecific antibody. Our results define 2D electrophoresis/Western blot and mass spectrometric protein identification as the most reliable procedure for validating LPL purity and the specificity of antibodies against this enzyme.

Conversion of Low Density Lipoprotein-associated Phosphatidylcholine to Triacylglycerol by Primary Hepatocytes

We have studied the uptake and metabolism of phosphatidylcholine (PC), the major phospholipid of low density lipoproteins (LDL), by cultures of primary hepatocytes. Strikingly, in the absence of the LDL receptor, PC incorporation into hepatocytes was inhibited by only 30%, whereas cholesteryl ether uptake was inhibited by 60-70%. On the other hand, scavenger receptor class B, type I, the other important receptor for LDL in the liver, was found to be responsible for the uptake of the remaining 30-40% of LDL-cholesteryl ether. PC uptake was, however, only partially inhibited (30%) in scavenger receptor class B, type I, knock-out hepatocytes. Once LDL-PC was taken up by hepatocytes, approximately 50% of LDL-[(3)H]oleate-PC was converted to triacylglycerol rather than degraded in lysosomes as occurs for LDL-derived cholesteryl esters. The remainder of the LDL-derived PC was not significantly metabolized to other products. Triacylglycerol synthesis from LDL-PC requires a PC-phospholipase C activity as demonstrated by inhibition with the phospholipase C inhibitor D609 or activation with rattlesnake venom. Small interfering RNA-mediated suppression of acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), but not DGAT1, decreased the acylation of the LDL-derived diacylglycerol. These findings show that PC in LDL particles is taken up not only by the classical receptors but also by additional mechanism(s) followed by metabolism that is completely different from the cholesteryl esters or apoB100, the other main components of LDL.

Embryonic stem cells cultured in serum free medium acquire bovine apolipoprotein B‐100 from feeder cell layers and serum replacement medium

Previous studies have demonstrated that cell populations that are cultured with heterologous animal products can acquire xenoantigens, potentially limiting their clinical utility because of immune responses. Embryonic stem cells (ESCs) are an attractive source of multiple potential cellular therapies and are typically derived and routinely cultured on murine embryonic fibroblast (MEFs) feeder cell layers in commercially available serum replacement (SR) medium or fetal calf serum (FCS) containing medium. Recently we found that a strong antibody response was generated in human subjects after the second infusion of therapeutic cells cultured in FCS containing medium. This response was specific for bovine apolipoprotein B‐100 (apoB‐100) which is the major protein component of LDL and targets its binding to abundant low density lipoprotein receptors on the cell surface from which it is internalized. Here we have shown that ESCs cultured on MEFs in SR medium acquired bovine apoB‐100 from MEFs and from the SR medium as well. Our findings also suggest that bovine low density lipoproteins (LDL) are used as critical nutrients for ESC propagation.

120 EFFECTS OF BOVINE PLASMA LIPOPROTEINS ON ACCUMULATION OF CYTOPLASMIC LIPID DROPLETS AND MITOCHONDRIAL MORPHOLOGY IN BOVINE EMBRYOS

The excessive accumulation of cytoplasmic lipid droplets (LD) may affect the embryo quality such as the sensitivity of embryos to chilling and freezing. Previous studies demonstrated that the presence of serum in embryo culture medium caused the abnormal accumulation of cytoplasmic LD. However, the mechanism and source of cytoplasmic LD accumulation in bovine embryos cultured in serum-containing medium is unclear. In this study, the effects of bovine plasma lipoproteins (BPL) on the accumulation of cytoplasmic LD were investigated in bovine embryos. Bovine oocytes were matured in IVMD101 serum-free medium and inseminated in BO-based medium. After insemination, embryos were cultured in IVMD101 supplemented BPL (Sigma) at various concentrations (60, 150, 300, 600 μg mL–1) or 5% calf serum (CS) in a humidified atmosphere of 5% CO2 in air at 38.5°C. The embryos of 2-, 8-cell, morula, and blastocyst stages were used for experiments. Embryos were fixed and stained with Sudan black B (SBB) for histochemical evaluation. Some embryos were prepared for transmission electron microscopy. The effect of media supplemented with BPL on in vitro development of bovine embryos is shown in Table 1. The proportion of embryos developing to morula and blastocyst stages decreased with increased levels of BPL (Table 1). Histochemical examinations demonstrated that embryos cultured in BPLcontaining media had numerous lipid droplets in their cytoplasm as observed in the embryos developed in CS-supplemented medium. The increase in number of LD was dose-dependent. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of morulae and blastocysts cultured in medium supplemented with BPL (300 μg mL–1). Many elongated mitochondria were found in embryos developed in IVMD101 medium at the morula and blastocyst stages, whereas many of the mitochondria in the morulae and blastocysts developed in IVMD101 medium supplemented with BPL or CS were of an immature form such as spherical or ovoid shape. These results indicate that bovine embryos cultured in BPL-containing media abnormally accumulated LD in their cytoplasm, suggesting that the BPL directly affects the accumulation of cytoplasmic LD. Furthermore, the excessive accumulation of cytoplasmic LD in embryos may affect the mitochondrial development of bovine embryos. Table 1. Effects of BPL on in vitro development of IVM/IVF bovine embryos This work was parially supported by Grant-in-Aid for Scientific Research (17380164), on Priority Areas ‘Lifesurveyor’(19021006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Special Coordination Funds for Promoting Science and Technology of Japan, and the Japan Livestock Technology Association.

In Vitro Interaction between Hepatitis C Virus (HCV) Envelope Glycoprotein E2 and Serum Lipoproteins (LPs) Results in Enhanced Cellular Binding of Both HCV E2 and LPs

Hepatitis C virus (HCV) particles in serum associate with lipoproteins (LPs), and the low-density lipoprotein receptor (LDLr) has been implicated in virus attachment and entry into cells. To clarify the basis of interactions between virus and LPs, we determined whether HCV interacts with human LPs via its envelope glycoprotein E2. The binding of serum-derived virus-like particles, HCV E2, and HCV E2-LP complexes to CD81 and LDLr was studied. Incubation of HCV E2 protein with human and bovine LPs (very low density, low density, and high density) enhanced the binding of both HCV E2 and LPs to CD4+ lymphoblastoid (MOLT-4) cells, foreskin fibroblasts, and hepatocytes. The binding of HCV E2 to MOLT-4 cells was not enhanced when it was preincubated with lipid-free apoprotein B, which suggests that E2 interacts with the lipid moiety of human lipoproteins. The LP interaction was specific for HCV E2--incubation of HIV gp120 with LPs did not enhance gp120 binding to MOLT-4 cells. The enhanced HCV E2 binding required expression of both human CD81 and LDLr. These data suggest that HCV E2 associates with LDL and that the resulting complex enhances binding of both ligands to cells, which may contribute to the finding that HCV-infected individuals have significantly lower levels of LDL than control subjects.

Determination of Bovine Serum Low-density Lipoprotein Cholesterol using the N-geneous Method

The N-geneous method is a recently developed method for determination of low-density lipoprotein cholesterol (LDL-C) in human serum. In the present study, we attempted to adapt this method to bovine serum. The values of LDL-C obtained using the N-geneous method were highly correlated with those from the method using ultracentrifugation and heparin sepharose affinity chromatography (r = 0.934, p < 0.001). The reproducibility of this method was acceptable (intra-assay CV 4.2%, inter-assay CV 7.6%) for clinical use. Using the N-geneous method, serum LDL-C was evaluated in cows around parturition, and in cows with fatty liver induced by fasting. The concentration of LDL-C decreased significantly in cows close to parturition. A reduced concentration of LDL-C was also observed in cows with fatty liver. In both cases, the changes of LDL-C were similar to those of apolipoprotein B (apoB)-100, and the values of LDL-C were highly correlated (r = 0.876, p < 0.001) with those of apoB-100. These results suggest that the concentration of LDL-C reflects the level of apoB-100. The N-geneous method is simple and rapid, and might to be a useful tool to elucidate the clinical significance of LDL-C in bovine serum.

Adsorption of Lipoproteins onto Mineral Dust Surfaces: A Possible Factor in the Pathogenesis of Particle-induced Pulmonary Fibrosis?

We compare the adsorption behavior of high density lipoproteins (HDL) and low density lipoproteins (LDL) on "fibrogenic" and "nonfibrogenic" mineral dusts. The adsorption tests with bovine lipoprotein concentrate and human serum produced the following results: 1) All seven examined fibrogenic dusts (SiO2 DQ12, SiO2 F600, silica, graphite, TiC, kaolin, talc) adsorbed significantly more high density lipoproteins (HDL), than the five examined nonfibrogenic (inert) dusts (TiO2, SnO2, Al2O3, Fe2O3, Fe3O4). This different behavior was particularly conspicuous in the presence of competing adsorbates (serum proteins). 2) In contrast, the adsorption of LDL did not correlate with the fibrogenicity of the mineral dusts. 3) The known silicosis-protective substance polyvinylpyridine-N-oxide inhibits the HDL adsorption of alpha-quartz. These results indicate that the adsorption of HDL could have a causal relationship with the triggering of a fibrotic reaction. The adsorption on the surface of fibrogenic dust particles provides an exceptional opportunity for the intake of HDL by macrophages. During the phagocytosis of the inhaled dust particles, the HDL adsorbed on the surface of the particles could be taken up by macrophages regardless of the receptor. There the HDL particles and/or compounds associated with them, such as lecithin-cholesterol-acyltransferase, could stimulate the macrophages to release fibrogenic mediators by some yet unknown mechanism.

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture.

There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serum-free media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animal-sourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1~ 3x10(6) viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.

Bovine Paraoxonase 1 Activities in Serum and Distribution in Lipoproteins

Paraoxonase 1 (PON1) is an enzyme associated with high-density lipoprotein (HDL) and has a protective effect against oxidation of lipoproteins in mammals. We investigated PON1 enzyme activities in bovine serum and its distribution among bovine serum lipoproteins. Paraoxonase activity and arylesterase activity in serum (152 Holstein cows and 42 Japanese Blacks) were 275 +/- 55 U/ml and 130 +/- 27 U/ml (mean +/- SD), respectively. There was a high correlation (r=0.962) between the two enzyme activities, and the activity ratio of paraoxonase/arylesterase did not exhibit individual variation. More than 85% of both paraoxonase and arylesterase activities were detected in the HDL fraction separated by ultracentrifugation. The 43-kDa protein in the HDL fraction was identified as bovine PON1 by N-terminal amino acid sequence analysis. Bovine PON1 was purified by ultracentrifugation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an anti-bovine PON1 antiserum was developed. The concentration of PON1 protein determined by immunoblotting was closely correlated (r=0.976) with paraoxonase activity in serum. Bovine HDL was further fractionated into subpopulations, and the distribution of PON1 was examined. Paraoxonase activity and PON1 protein increased with decreasing HDL size and approximately 60% of total paraoxonase activity was distributed in the heavy HDL fraction. The different distributions of PON1 among HDL subpopulations might be concerned to the function and metabolism of bovine HDL.