Molecular characterization of human hepatic lipase deficiency. In vitro expression of two naturally occurring mutations.

Individuals with hepatic lipase (HL) deficiency are often characterized by elevated levels of triglycerides and cholesterol and may be subject to premature atherosclerosis. Missense mutations in the HL gene have been identified in two affected families: substitutions of serine for phenylalanine at amino acid 267 and threonine for methionine at amino acid 383 (S267F and T383M, respectively). To confirm the role of S267F and T383M, respectively). To confirm the role of mutations separately into human HL cDNA by site-directed mutagenesis, and the resulting constructs were independently expressed in COS cells. HL activity and mass were measured and compared with wild-type HL transfectants to determine the effect of these mutations on lipase activity and secretion. Although similar amounts of HL protein were detected intracellularly after transfection with the wild-type and mutant constructs, S267F and T383M HL activity levels were markedly decreased: in S267F, no HL activity was detected, and activity levels in T383M were 38% of wild-type HL. Heparin-induced secretion of the two HL mutants was also severely affected: no detectable activity could be measured in the media of S267F, although some inactive mass (12% of wild-type HL) was secreted; mutant T383M secreted 4% and 20% of wild-type activity and mass, respectively. These results indicate that the single amino acid substitution present in HL S267F is sufficient to render the enzyme completely nonfunctional; in contrast, the T383M mutant retains partial activity but is poorly secreted. Thus, these defects appear capable of accounting for the HL-deficient phenotypes exhibited by individuals carrying the T383M and S267F mutations.

Genetic variation in mouse apolipoprotein A-IV expression is determined pre- and post-transcriptionally.

Among inbred mouse strains there is a striking genetic variation in the levels of apolipoprotein A-IV (apoA-IV) mRNA in the liver, although intestinal mRNA levels vary only twofold in these strains. In the present study we have characterized the apoA-IV expression phenotypes in strains C57BL/6J and 129/J, and investigated the molecular basis for the genetic variation. We report that the two strains differ eight- to tenfold both in the levels of apoA-IV mRNA and in the rate of apoA-IV protein synthesis in liver. Presumably due to the increased synthetic rate, strain 129 exhibits a threefold higher concentration of apoA-IV protein in the circulation. mRNA synthesis and turnover studies indicate that both transcriptional and post-transcriptional events contribute to the genetic variation in steady state apoA-IV mRNA levels. An analysis of the levels of apoA-IV mRNA derived from 129 and C57BL/6 alleles in F1 mice indicates that the genetic control of apoA-IV mRNA levels involves both cis-acting elements linked to the apoA-IV gene, and genetically distinct trans-acting factors.

The surface structure of photoreceptor outer segment disks.

The double-membrane disks in the outer segment of photoreceptors in the rabbit retina were isolated structurally intact after crosslinking with glutaraldehyde. The following criteria were used to determine that intact whole disks were isolated: (1) the disk diameter, 1.4 microns; (2) the presence of one incision extending toward the center of the disk; (3) the edge structure; and (4) the presence of particulate material with the dimensions of the particles corresponding to those present in the particulate fracture face of freeze-fractured outer segment disks. The disks were analyzed after negative staining or after freeze-drying and surface replication. The surface of the disks appeared smooth. In damaged disks negative staining revealed the presence of particulate material located below the disk surface. The observations are interpreted to reveal that the lipid bilayers of the disks are located at the disk surfaces shielding the rhodopsin molecules from contact with the cytoplasm present between the disks.

Identification and characterization of liver nonparenchymal cells by flow cytometry.

Our objective was to correlate biochemical or functional properties that could be measured by fluorescence to individual nonparenchymal cell types by the simultaneous detection of forward angle light scatter and fluorescence. Cells were released from liver following selective digestion of hepatocytes by Pronase perfusion. To fractionate the cells according to size, isolates were subjected to unit gravity sedimentation on a 1 to 5% sucrose gradient. Flow cytometry was used to analyze the resulting fractions as well as the original unfractionated cells. This reaffirmed that forward angle light scatter is linearly related to the sedimentation velocity (size) of the cells and that it can be used to assess cell sizes in an unfractionated population. Endocytosis of fluorescently tagged particulates was studied by injecting either fluorescein isothiocyanate-tagged colloidal albumin or Coumarin-tagged latex beads (0.57 micron diameter) prior to Pronase perfusion. Beads were found only in the largest cells (Kupffer cells). Since endothelial cells could endocytose only colloidal albumin, they were readily differentiated from nonendocytosing, nonsinusoidal cells ("lymphocytes"). Staining cell protein with fluorescein isothiocyanate made possible the determination of relative protein content per cell ("lymphocyte": endothelial:Kupffer cell was 0.57:1.0:2.0). To determine relative esterase activities, mixed cell isolates were incubated in fluorescein diacetate ("lymphocyte":endothelial:Kupffer cells was 0.13:1.0:2.4). Uptake of rhodamine 123 was used to assess mitochondrial function ("lymphocyte":endothelial:Kupffer cells was 0.45:1.0:14.6). Mitochondrial volume per cell is known to be 1:2 for endothelial:Kupffer cells. Therefore, the large difference in rhodamine uptake is not related to volume.