Organization and chromosomal localization of the human ECEL1 (XCE) gene encoding a zinc metallopeptidase involved in the nervous control of respiration.

ECEL1 (endothelin-converting enzyme-like 1; previously known as XCE) is a putative zinc metalloprotease that was identified recently on the basis of its strong identity with endothelin-converting enzyme. Although the physiological function of ECEL1 is unknown, inactivation of the corresponding gene in mice points to a critical role of this protein in the nervous control of respiration. In the present study we have characterized the human ECEL1 gene. It was located to region q36-q37 of chromosome 2 and shown to be composed of 18 exons spanning approx. 8 kb. The structure of the ECEL1 gene displays some striking similarities with those of genes of related metallopeptidases, supporting the hypothesis that they are all derived from a common ancestor. A short phylogenetic study describing the relationship between the various members of this gene family is also presented.

Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms.

Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization.

Organization of the gene encoding the human endothelin-converting enzyme (ECE-1).

The two human endothelin-converting enzyme (ECE-1) isoforms, which differ by their N-terminal region, are encoded by a single gene. The gene is composed of 19 exons that span more than 68 kilobases and has been mapped to the 1p36 band of the human genome. The two isoform mRNAs display different tissue distributions. Their precursors are transcribed from two distinct start sites, upstream from exon 1 and exon 3, respectively. Sequence analysis of the two putative promoters revealed the presence of motifs characteristic for several transcription factors. Comparison of the ECE-1 gene structure with those of other zinc metalloproteases, as well as a phylogenetic study, confirm the existence of a metalloprotease subfamily composed of ECE-1, ECE-2, neutral endopeptidase, Kell blood group protein, and two bacterial enzymes.

Genetic toxicology studies with a tumor promoter.

A diuretic antihypertensive agent, SC-33643 (8-[2-ethoxyethyl]-7-phenyl-[1,2,4]triazolo[4,3-c]pyrimidine-5- amine, also known as bemitradine), was tested in the Ames test, in the mouse lymphoma TK +/- mutation assay, in the Chinese hamster ovary cell hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) mutation test and in the CHO chromosome aberration assay with and without metabolic activation. Additionally, the compound was tested in the rat primary hepatocyte unscheduled DNA synthesis (UDS) assay and in the mouse bone marrow micronucleus assay. The results were uniformly negative. Contrary to expectations based on the results of the battery of genetic toxicology tests, the compound produced liver, thyroid and mammary tumors in the rat (reported separately). Subsequently, SC-36741 (5-amino-7-phenyl-[1,2,4]triazolo-[1,5-c] pyrimidine-8-ethanol, also known as desethylbemitradine), a major metabolite of SC-33643, was tested in the Ames test, in the CHO/HGPRT mutation test and in the CHO chromosome aberration assay with and without metabolic activation, and was also tested in the rat primary hepatocyte/UDS assay and in the mouse bone marrow micronucleus assay. This metabolite also produced negative results in these tests. Therefore, SC-33643 is a non-genotoxic carcinogen producing tumors in rats without altering DNA or chromosomes.

Genetic toxicology studies of an anti-AIDS drug.

SC-48334 (N-butyldeoxynojirimycin) is an experimental anti-AIDS drug which is currently in clinical trials. This drug is an aminosugar derivative. Its biological properties have been previously published [1]. Since many antiviral agents which are nucleic acid analogs exhibit mutagenic and/or clastogenic properties, the genotoxic potential of SC-48334 was examined in the Ames Salmonella/microsome assay, the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay and the mouse bone marrow micronucleus assay. No toxic or mutagenic effects were observed in either the bacterial or mammalian in vitro mutation assays. Likewise, no clastogenic activity was observed in the in vivo micronucleus assay. Therefore, the administration of this drug in humans is not likely to have mutagenic effects and would probably not have a carcinogenic effect.

Spontaneous osteogenic sarcoma in the rat.

A spontaneous osteogenic sarcoma of the spinal column (tail, sacrum and lumbar regions) with metastases to the lungs, kidneys and right ovary were found in a 15-month-old BHE rat. The literature on spontaneous osteogenic sarcoma in rats indicates that this tumour is rare (0.22 per cent), occurs in several strains and has no sex, age or site predilections; non-metastatic cases are more common than metastatic ones.

3-Alkyl-3-hydroxyglutaric acids: a new class of hypocholesterolemic HMG CoA reductase inhibitors. 1.

Derivatives of 3-hydroxy-3-methylglutaric acid (HMG), a portion of the substrate for HMG CoA reductase, were prepared and tested for their inhibitory action against rat liver HMG CoA reductase and for their hypocholesterolemic activity. Structure-dependent competitive inhibition was observed. Optimal structures had a free dicarboxylic acid with an alkyl group of 13-16 carbons at position 3. 3-n-Pentadecyl-3-hydroxyglutaric acid (3j) (IC50 = 50 microM) reduced serum cholesterol in the Triton-treated rat and HMG CoA reductase activity in the 20,25-diazacholesterol-treated rat.

22-Hydroxycholesterol derivatives as HMG CoA reductase suppressors and serum cholesterol lowering agents.

A series of 22-hydroxycholesterol derivatives with a modified side chain terminus was prepared. These agents were evaluated in vitro and in vivo for their ability to suppress HMG CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. In tissue culture assays, 22-hydroxycholesterol as well as the side chain modified analogues were potent inhibitors of HMG CoA reductase. However, only those sterols with a modified side chain terminus were effective suppressors of liver reductase when administered ig to rats. 22-Hydroxy-25-methylcholesterol (4a) and 25-fluoro-22-hydroxycholesterol (15a) significantly lowered serum cholesterol levels when administered ig to primates; 25-chloro-22-hydroxycholesterol (15b) and the analogue with a cyclopropyl terminus, 20b, were ineffective. The cholesterol-lowering sterols did not significantly alter lipoprotein levels; however, the two compounds have been shown to inhibit acyl-coenzyme A:cholesterol acyl-transferase (ACAT) in tissue culture studies.

Esophageal impaction in BHE rats.

Spontaneous esophageal impaction was observed in 33 BHE rats, aged 6 to 26 months, from 157 rats which died or were killed when moribund due to spontaneous conditions. The clinical course lasted less than 48 hours and included weakness, excessive salivation, forward head motions, matting of chin and perioral hair with bedding material, followed by recovery or death. In some animals, the clinical episodes reoccurred. The esophagus was dilated (up to 7.0 mm) and impacted with bedding or food. The caudal half of the esophagus was affected more than the cranial half. The esophageal muscle was thin and pale at the site of impaction. Histologically, there was esophageal myodegeneration.