Tangier disease: isolation and characterization of LpA-I,LpA-II,LpA-I:A-II and LpA-IV particles from plasma

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 ± 0.14 and 0.14 ± 0.04 μmol of cholesterol esterified/h/μg of protein, and 7 ± 2.5 and 1.4 ± 0.3 μmol of cholesteryl ester transferred/h/μg of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2–1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 ± 0.16 and 0.01 ± 0.01 μmol of cholesterol esterified/h/μg of protein and, 41 ± 3.7 and 1 ± 0.4 μmol of cholesteryl ester transferred /h/μg of protein). The LpA-II particle in TD represented in fact in LpA-II: A-IV complex (75% mol apoA-II and 22% mol apoA-IV). This particle did not promote cholesterol efflux, but LCAT and CETP activity were present. LpA-IV particles had the capacity to promote cholesterol efflux and had both LCAT and CETP activity. LpA-IV may contribute to maintain the reverse cholesterol transport in TD. Our results indicate the potential importance of apoA-IV in maintaining reverse cholesterol transport in TD. In spite of the low steady state HDL-cholesterol levels in TD, LpA-I, LpA-I: A-II: A-IV complex and LpA-IV appear to be active in reverse cholesterol transport and may help to prevent premature CHD in TD.     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Thyroid hormone stimulates synthesis of a cardiac myosin isozyme. Comparison of the two-two-dimensional electrophoretic patterns of the cyanogen bromide peptides of cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits.

The CNBr peptides of [14C]carboxymethylated cardiac myosin heavy chains from euthyroid and thyrotoxic rabbits have been compared using a two-dimensional electrophoretic system. The results indicated that there were extensive differences in the peptide "maps" of these heavy chains, which included differences in the distribution of radiolabeled thiol peptides. Also, the patterns of heavy chain peptides from the cardiac myosins have been compared with those produced by the heavy chain myosin isozymes from skeletal muscles. Peptide maps of heavy chains from red skeletal muscle myosin closely resembled the pattern of peptides found with cardiac myosin heavy chains from euthyroid rabbits. However, peptide maps of heavy chains from white skeletal muscle myosin were dissimilar to those of the cardiac myosin isozymes. We conclude that thyroxine administration stimulates the synthesis of a cardiac myosin isozyme with a heavy chain primary structure which is different from either of the skeletal muscle myosin isozymes.     

Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylase in the uptake and the base-mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles.

Although uridine and adenosine are converted by membrane-associated nucleoside phosphorylases to ribose-1-phosphate (ribose-1-P) and the corresponding bases (uracil and adenine), only ribose -1-P is accumulated within Salmonella typhimurium LT2 membrane vesicles. In accordance with these observations, no uptake is observed when the vesicles are incubated with the bases or nucleosides labeled in their base moieties. The vesicles lack a transport system for ribos-1-P, since excess ribose-1-P does not inhibit the uptake of the ribose moiety of uridine. In addition, there is no exchange with preaccumulatedribose-1-P. Thus, uridine, rather than ribose-1-P, must serve as the initially transported substrate. The uptake of the ribose portion of uridine is coupled to electron transport, and the levels to which ribose-1-P are accumulated may be reduced by adding various bases to the reaction mixtures. The bases appear to inhibit the uridine phosphorylase reaction and/or cause an efflux of ribose-1-P from the vesicles. This loss of ribose-1-P reflects the accumulation of nucleosides in the external medium after being synthesized within the membranes. Synthesis of the nucleosides from intravesicular ribose-1-P and exogenous base proceeds even though the bases are not accumulated by the vesicles. Furthermore, ribose-1-P cannot significantly inhibit uridine phosphorylase activity unless the membranes are disrupted. These observations indicate that the membrane-associated nucleoside phosphorylases may have a transmembranal orientation with their base and ribose-1-P binding sites on opposite sides of the membranes. Such an asymmetric arrangement of these enzymes may facilitate the uptake of the ribosyl moiety of nucleosides by a group translocation mechanism. Thus, nucleosides may be cleaved during the membrane transport process, with the resultant bases delivered to the external environment while ribose-1-P is shunted to the intravesicular space.     

New chemical adaptor unit designed to release a drug from a tumor targeting device by enzymatic triggering

A new controlled drug delivery system for selective chemotherapy was developed. It is based on a chemical adaptor unit, that releases a drug by a spontaneous cyclization mechanism after cleavage of an enzymatic substrate. It also provides a generic linkage of a drug with a targeting device in a manner set to be triggered by defined enzymatic activity. The system is generic and allows using a variety of drugs, targeting devices, and enzymes by introducing the corresponding substrate as a trigger for drug release in the chemical adaptor.     

In vivo modulation of HDL phospholipid has opposing effects on SR-BI- and ABCA1-mediated cholesterol efflux

The effects of in vivo modulation of HDL phospholipid (PL) on scavenger receptor class BI (SR-BI)- and ATP binding cassette transporter 1 (ABCA1)-mediated efflux were examined by overexpressing either endothelial lipase (EL) or phosphatidylserine phospholipase (PS-PLA1) in human apolipoprotein A-I (apoA-I) transgenic mice. Overexpression of EL led to large reductions in the serum PL/apoA-I ratio (-60%), total cholesterol (TC; -89%), and HDL cholesterol (-91%). Relative to the serum before overexpression of EL, the efflux potential of the serum via SR-BI decreased by 90% and ABCA1-mediated efflux increased by 63%. In contrast to overexpression of EL, overexpression of PS-PLA1 led to increases in the PL/apoA-I ratio (88%), TC (78%), HDL cholesterol (57%), and HDL size. The efflux potential of the serum increased by 60% via SR-BI and decreased by 57% via ABCA1. There were significant positive correlations between SR-BI-mediated efflux and a number of serum parameters, including PL/apoA-I ratio, PL, TC, free cholesterol (FC), and HDL cholesterol. In striking contrast, the same correlations were seen with ABCA1-mediated efflux, but the relationships were inverse. In summary, in vivo modulation of HDL PL content affects ABCA1- and SR-BI-mediated efflux in a reciprocal manner. These findings indicate that the type of lipase acting on HDL in vivo will determine which FC efflux pathway the HDL serves. Additionally, the extent of lipolysis will determine the efficiency of FC removal via this pathway.     

Role of N-linked glycosylation in the secretion and activity of endothelial lipase

Human endothelial lipase (EL), a member of the triglyceride lipase gene family, has five potential N-linked glycosylation sites, two of which are conserved in both lipoprotein lipase and hepatic lipase. Reduction in molecular mass of EL after treatment with glycosidases and after treatment of EL-expressing cells with the glycosylation inhibitor tunicamycin demonstrated that EL is a glycosylated protein. Each putative glycosylation site was examined by site-directed mutagenesis of the asparagine (Asn). Mutation of Asn-60 markedly reduced secretion and slightly increased specific activity. Mutation of Asn-116 did not influence secretion but increased specific activity. In both cases, this resulted from decreased apparent K(m) and increased apparent V(max). Mutation of Asn-373 did not influence secretion but significantly reduced specific activity, as a result of a decrease in apparent V(max). Mutation of Asn-471 resulted in no reduction in secretion or specific activity. Mutation of Asn-449 resulted in no change in secretion, activity, or molecular mass, indicating that the site is not utilized. The ability of mutants secreted at normal levels to mediate bridging between LDL and cell surfaces was examined. The Asn-373 mutant demonstrated a 3-fold decrease in bridging compared with wild-type EL, whereas Asn-116 and Asn-471 were similar to wild-type EL.     

The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies

The rabbit antibody repertoire, which in the form of polyclonal antibodies has been used in diagnostic applications for decades, would be an attractive source for the generation of therapeutic human antibodies. The humanization of rabbit antibodies, however, has not been reported. Here we use phage display technology to select and humanize antibodies from rabbits that were immunized with human A33 antigen which is a target antigen for the immunotherapy of colon cancer. We first selected rabbit antibodies that bind to a cell surface epitope of human A33 antigen with an affinity in the 1 nm range. For rabbit antibody humanization, we then used a selection strategy that combines grafting of the complementarity determining regions with framework fine tuning. The resulting humanized antibodies were found to retain both high specificity and affinity for human A33 antigen.     

Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice

In addition to its role in the uptake of apolipoprotein B (apoB)-containing lipoproteins, apoE promotes hepatic very low density lipoprotein-triglyceride (VLDL-TG) production in animal models. However, it is not known if apoE increases the amount of TG per VLDL particle or the number of VLDL particles secreted. VLDL-apoB production is a measure of the rate of VLDL particle secretion. We determined the effects of apoE deficiency and apoE overexpression on VLDL-apoB production in mice. [(35)S]methionine was injected into endogenously label VLDL-apoB and Triton WR-1339 was simultaneously injected to block the catabolism of VLDL. Compared with wild-type mice, the VLDL-apoB production rate was decreased by 33% in apoE-deficient mice. Conversely, VLDL-apoB production was increased by 48% in mice overexpressing apoE compared with controls. Nascent VLDL, obtained from post-Triton plasma, had a decreased, not increased, content of TG per apoB in the apoE-overexpressing group compared with the control group. This study demonstrates that hepatic apoE expression increases the output of VLDL triglyceride by increasing the production rate of VLDL-apoB, suggesting that hepatic apoE influences the number of VLDL particles secreted by the liver.     

Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339

Triglyceride (TG), a water-insoluble energy-rich lipid, is secreted by the liver as part of very low density lipoproteins (VLDLs) to supply energy to extrahepatic tissues. Overproduction of VLDL is associated with increased risk of cardiovascular heart disease; this has renewed an interest in factors that affect hepatic TG production. The TG production rate is determined by measuring temporal increases in plasma TG under conditions in which TG hydrolysis by lipoprotein lipase (LPL) is inhibited. The nonionic detergent, Triton WR-1339 (Triton), has commonly been used to inhibit LPL for this purpose. Triton, in addition to inhibition of TG hydrolysis, has properties that have the potential to adversely influence lipoprotein metabolism. Another nonionic detergent, poloxamer 407 (P-407), also inhibits LPL. In these studies, we demonstrate that P-407 is comparable to Triton in the determination of TG production but without the unwanted side effects of Triton.