Prenatal diagnosis of peroxisomal disorders. Biochemical and immunocytochemical studies on peroxisomes in human amniocytes.

Prenatal diagnoses of peroxisomal disorders, including peroxisome-deficient Zellweger syndrome, isolated deficiency of peroxisomal beta-oxidation enzyme and rhizomelic type chondrodysplasia punctata were investigated by means of the lignoceric acid oxidation assay, indirect immunofluorescence staining and pulse-chase experiments, using cultured amniocytes. Assessment of peroxisomal beta-oxidation activity by means of [1-14C]lignoceric acid oxidation is essential for the diagnosis of a single enzyme deficiency of peroxisomal beta-oxidation with detectable enzyme protein. For the diagnosis of Zellweger syndrome, the absence of peroxisomes was readily determined by immunofluorescence staining of only a few amniocytes. Evidence for abnormal processing of 3-ketoacyl-CoA thiolase leads to the diagnosis of rhizomelic chondrodysplasia punctata. All the fetuses were considered to be normal and the neonates were normal. Use of these methods requires only a small number of amniocytes and will facilitate the prenatal diagnosis of peroxisomal disorders.

[Two cases of early Acanthamoeba keratitis].

Early Acanthamoeba keratitis was diagnosed in two soft contact lens wearers. Both patients had initially been diagnosed as having herpes simplex keratitis and treated with antiherpes drugs. In one patient, slit-lamp examination disclosed dendritiform epithelial keratitis, subepithelial opacities, linear stromal infiltrate along the corneal nerve (radial keratoneuritis), and marked swelling and hyperemia of the limbal conjunctiva. Acanthamoeba castellanii was cultured from the corneal scrapings and contact lens case. The second patient also showed dendritiform keratitis and subepithelial opacities, with swelling of the limbal conjunctiva. Cultures were positive for A. polyphaga from the contact lens case, but negative from the corneal scrapings. The patients were cured of Acanthamoeba keratitis with medical therapy consisting of topical fluconazole and miconazole, systemic fluconazole, and topical corticosteroids. Recognition of distinctive characteristics of the clinical findings in early Acanthamoeba keratitis can lead to the early diagnosis of the disease.

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.