Eva Zsigmond

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute ≈60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, α-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Genetic Deficiency of Acylation Stimulating Protein (ASP(C3ades-Arg)) Does Not Cause Hyperapobetalipoproteinemia in Mice

The acylation stimulating protein (ASP) is a 76-amino acid peptide that has been proposed as a potent mediator of triglyceride synthesis and, when functionally impaired, as a major cause of hyperapobetalipoproteinemia (HyperapoB). Purification and sequence analysis of ASP from human sera have revealed that ASP is identical to the complement C3-derived activation peptide C3ades-Arg. Because C3 is the precursor for C3ades-Arg and therefore ASP, a deficiency in C3 would be predicted to result in a phenotype characteristic of HyperapoB. To test this hypothesis in vivo, the current study was undertaken in which ASP(C3ades-Arg)-deficient mice were used as a model system. No significant differences were found in the triglyceride, cholesterol, or free fatty acid concentrations in the plasma of fasted normal and ASP(C3ades-Arg)-deficient animals. In addition, plasma lipoprotein analyses indicated that the very low density lipoprotein, low density lipoprotein, and high density lipoprotein cholesterol and triglyceride concentrations as well as the apolipoprotein B-48 and B-100 levels were not significantly different in the plasma of ASP(C3ades-Arg)-deficient and wild type mice. Furthermore, when challenged with an oral fat load, the ASP(C3ades-Arg)-deficient mice showed no impaired ability to clear triglycerides and free fatty acids from their circulation when compared with their wild-type littermates. Collectively, these results indicate that ASP(C3ades-Arg) deficiency does not cause HyperapoB in mice and that the physiological importance of impaired ASP(C3ades-Arg) function as a cause of hyperapobetalipoproteinemia needs to be reevaluated.

A critical evaluation of the putative role of C3adesArg (ASP) in lipid metabolism and hyperapobetalipoproteinemia.

The acylation stimulating protein, ASP is a small, basic serum protein capable of stimulating triglyceride synthesis in cultured fibroblasts and adipocytes. Sequence analysis of ASP has shown that ASP is identical to C3adesArg the inactive fragment of the complement anaphylatoxin peptide, C3a. It has been proposed that C3adesArg (ASP) can be generated by mature adipocytes secreting the three complement proteins: complement protein C3, factor B and factor D (adipsin). There have also been indications that adipocytes may express a specific C3adesArg (ASP)-receptor that is distinct from the recently cloned C3a-receptor. This suggests that C3adesArg (ASP) acts as an adipocyte autocrine and that it plays a central role in the metabolism of adipose tissue. Based on these observations a hypothesis for the etiology of hyperapobetalipoproteinemia (hyperapoB) has been proposed. Hyperapobetalipoproteinemia (hyperapoB), is a familial lipoprotein disorder characterized by increased hepatic secretion of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) particles. If C3adesArg (ASP) function in the adipose tissue is impaired, a reduced rate of triglyceride synthesis will follow, generating an increased flux of fatty acids to the liver. In response to an increased flow of fatty acids, the liver will increase its production of VLDL particles yielding the phenotype of hyperapoB. This review critically assesses this hypothesis and the potential role of C3adesArg (ASP) as a major determinant for triglyceride synthesis in the light of data collected in vitro and in vivo.

Modulation of mitochondrial ATPase sensitivity to inhibitors and stimulators by diet-induced changes in membrane lipid

Weanling rats were fed diets differing in fatty acid composition to determine if changes induced in cardiac mitochondrial membrane structural components alter the sensitivity of mitochondrial ATPase to inhibition by oligomycin and stimulation by 2,4-dinitrophenol. Mitochondrial ATPase assayed in situ within the mitochodrial membrane isolated from animals fed diets higher in fatty acids of longer chain length, exhibited greater oligomycin sensitivity and lower 2,4-dinitrophenol-induced stimulation. Concomitant diet-induced changes occur in the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and cardiolipin, increasing overall length of fatty-acyl tails in the membrane phospholipids. Diet fat mediated alterations in oligomycin sensitivity of mitochondrial ATPase and membrane fatty acid chain length suggest that in vivo changes in thickness of the lipid bilayer may alter mitochondrial ATPase functions. The present study extends the concept that dietary fat affects mitochondrial membrane structure and function by demonstrating that the membrane-dependent sensitivity of mitochondrial ATPase to inhibitors and stimulators may be modulated by dietary fat.

Epididymal C4b-binding protein is processed and degraded during transit through the duct and is not essential for fertility.

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP-/-) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP-/- mice were fertile and no significant differences were observed between the C4BP-/- and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation.

IMMUNOCHEMICAL QUANTITATION OF LIPOPROTEIN LIPASE

Publisher Summary Lipoprotein lipase (LPL) has an important function in the regulation of plasma lipoprotein metabolism and is also involved in adipose tissue differentiation and in the prediction of adipocyte cell size. The enzyme hydrolyzes the triacylglycerol components of very-low-density lipoproteins (VLDL) and chylomicrons to glycerol and free fatty acids, and hence it regulates the supply of the latter to various tissues. In view of the critical role that LPL plays in normal metabolism, as well as in pathophysiological states, assays have been developed that measure LPL enzyme activity and immunoreactive mass. This chapter discusses the assessment of LPL immunoreactive material by immunological methods. Various immunoassays have previously been described here. This chapter describes two immunoassay protocols designed to quantitate human LPL (hLPL), making use of a monoclonal antibody (M40) raised against bovine milk LPL that cross-reacts with hLPL and a polyclonal anti-hLPL antibody (immunoglobulin Y, IgY) prepared in chickens. The antibody capture enzyme-linked immunosorbent assay (ELISA) is designed to detect LPL in samples with low-level contamination by other proteins, such as culture medium, whereas the two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) is more sensitive and is best suited for detecting LPL in more complex samples, such as cellular extracts or post-heparin plasma. It also presents a procedure for preparing LPL from post-heparin plasma and detecting the protein by immunoblot analysis. Immunoblotting provides a semiquantitative measure of LPL immunoreactive mass as well as the apparent molecular weight of the protein.

HUMAN LIPOPROTEIN LIPASE : PRODUCTION IN VITRO, PURIFICATION AND GENERATION OF POLYCLONAL ANTIBODY

Publisher Summary Lipoprotein lipase (LPL) is a hydrolytic enzyme, produced by adipose tissue, heart, skeletal muscle, as well as in small amounts by many other tissues. LPL is a homodimeric protein located on the luminal surface of the vascular endothelium, being anchored to the latter, by interaction with cell surface glycosaminoglycans. Lipoprotein lipase has been purified to apparent homogeneity from bovine milk, human milk, as well as post-heparin plasma. Many of the biochemical and enzymatic properties of LPL have been determined from the studies on LPL isolated from these sources. However, LPLs prepared from these sources are often relatively unstable due to the copurification of contaminating proteases. Because of the difficulty of obtaining a homogeneous LPL preparation from natural sources that is stable and free from protease contamination, this chapter turns to using purified recombinant LPL produced in vitro. It expresses human LPL (hLPL) in yeast and in baculovirus and has found that the immunoreactive LPL, produced by such systems, is enzymatically inactive. Enzymatically active recombinant LPL can be produced in mammalian cells,such as COS-1 cells, ATCC, Rockville, MD, by transient transfection. This chapter describes the production of hLPL by CHO-K1 ceils, transfected with hLPL complementary DNA (cDNA) in the expression vector pEEl4, and the use of heparin-Sepharose affinity chromatography to purify the enzyme secreted by CHO-K1 cells into the medium.

Derivation and characterization of the human embryonic stem cell line CR-4: Differentiation to human retinal pigment epithelial cells

The CR-4 human embryonic stem cell line was derived from the inner cell mass of a developing blastocyst. This cell line has been adapted to grow in feeder-free conditions and is especially well-suited for differentiation to retinal pigment epithelium. The line demonstrates a normal human 46,XX female karyotype. Pluripotency was assessed through qRT-PCR for expression of NANOG, OCT-4, and SOX-2. A teratoma assay was performed and results were positive for all three germ layers. Testing for Mycoplasma was negative.

Hepatic Triglyceride Lipase and Lipoprotein Lipase Action in Vitro and in Vivo

Hepatic triglyceride lipase (HTGL) and lipoprotein lipase (LPL) are two evolutionarily related enzymes that play key roles in lipoprotein metabolism. Hepatic triglyceride lipase appears to be involved in the hydrolysis of intermediate density lipoprotein (IDL) triglyceride to produce low density lipoprotein (LDL), and that of high density lipoprotein (HDL)-2 triglyceride and phospholipid to produce HDL-3 [1,2]. It may also be required for the uptake of HDL triglyceride and cholesteryl esters by the liver [3–5]. Lipoprotein lipase is essential for the metabolism of the triglyceride-rich lipoproteins, chylomicron and very low density lipoproteins (VLDL). The LPL-mediated hydrolysis of these lipoproteins produces chylomicron remnants and IDL, respectively, releasing necessary components for the production of HDL-2. Thus, both enzymes are involved in HDL metabolism, and HL activity is inversely, whereas LDL activity is directly correlated with plasma HDL levels [6].