Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.     

Lysosomal acid lipase deficiency in rats: lipid analyses and lipase activities in liver and spleen

We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for [14C]triolein, [14C]cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans.     

Secretory carcinoma of the endometrium

A case of secretory carcinoma of the endometrium in a 21-year-old woman is reported. Endometrial smears were interpreted as showing a differentiated adenocarcinoma. Smears of ascitic fluid obtained during subsequent surgery showed similar findings; periodic acid-Schiff staining of the smears revealed abundant positive material in the cytoplasm. These findings were interpreted as evidence of secretory carcinoma, which was confirmed by histopathologic study of the biopsy and surgical specimens.     

Genotypes of alcohol dehydrogenase and aldehyde dehydrogenase loci in Japanese alcohol flushers and nonflushers

Summary A much higher incidence of alcohol flushing among Orientals in comparison to Caucasians, i.e., >50% vs 5%–10%, has been attributed to racial differences in alcohol-metabolizing enzymes. A large majority of Orientals are atypical in alcohol dehydrogenase-2 locus (ADH ₂ ), and their livers exhibit significantly higher ADH activity than the livers of most Caucasians. Approximately 50% of Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH₂) activity, and elimination of acetaldehyde might be disturbed. We determined by means of hybridization of genomic DNA samples with allele specific oligonucleotide probes, genotypes of the ADH ₂ and ALDH ₂ loci in Japanese alcohol flushers and nonflushers. We found that all individuals with homozygous atypical ALDH ₂ ² /ALDH ₂ ² and most of those with heterozygous atypical ALDH ₁ ² /ALDH ₂ ² were alcohol flushers, while all subjects with homozygous usual ALDH ₁ ² /ALDH ₁ ² were nonflushers. Frequency of the atypical ADH ₂ ² was found to be higher in alcohol flushers than in nonflushers, but the statistical significance was not established in the sample size examined.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Endotoxic properties of chemically synthesized lipid A analogs. Studies on six inflammatory reactions in vivo, and one reaction in vitro

Biological activities of two groups of synthesized lipid A analogs, the counterpart of biosynthetic precursor, Lehmann's Ia type, 406, and E. coli lipid A type, 506, as well as their non-phosphorylated, and mono-phosphorylated analogs were investigated. The activities employed included four bone marrow cell reactions in mice, mice skin reaction, leukocytes migration in rabbits' cornea, and hemagglutination. Compound 406 and 506 elicited bone marrow reactions in mice and hemagglutination of mouse RBC, although 406 failed to elicit hemorrhage and necrosis also in mice skin. Compound 406 did not elicit corneal reaction in rabbits. The results suggest that for elicitation of this reaction and mice skin reaction, acyloxyacyl structure is required. Cytotoxicity and thromboplastin production of four bone marrow reactions had been reported by us to be endotoxic reactions, since these had not been elicited by peptidoglycan of Lactobacillus and Staphylococcus (1981) and 300 series synthesized analogs (1984) which did not have endotoxic structures. From these results, it seems that these two marrow reactions and hemagglutination require, as does the limulus test, the lipid A part structure as is present in 406.     

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.     

Reconstituted F1-ATPase complexes containing one impaired beta subunit are ATPase-active

Homogeneous populations of hybrid alpha 3 beta 3 gamma complexes of the thermostable F1-ATPase containing one, two, or three copies of the mutationally impaired beta subunits were produced using the solid phase reconstitution method. Two kinds of mutated beta subunits were used for the reconstitution, one of which lacked the ability to bind any adenine nucleotides. The complexes containing one impaired beta and two wild-type beta subunits retained a significant amount of ATPase activity with cooperative kinetics, whereas those containing two or three impaired beta subunits showed very little ATPase activity. These results imply that the catalysis of steady-state ATP hydrolysis can proceed even if one of the three beta subunits in F1-ATPase is not functional.     

Conformation of the fowl protamine, galline, and its binding properties to DNA

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25 mM EDTA/10 mM Tricine-HCl, pH 7.4, but hardly so in 30 mM NaCl/3 mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100 mM NaCl/50 mM Tricine-HCl, pH 7.4, at 37 C was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc) = 3.3 X 10(7)M-1, the co-operativity factor (q) = 800, and the number of nucleotides of DNA occupied by one galline molecule (n) = 28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.