Molecular cloning and sequencing of a human cDNA encoding ornithine decarboxylase antizyme.

We report the cloning of a cDNA encoding the human homolog of ornithine decarboxylase antizyme from a human gingival fibroblast cDNA library. The human antizyme is 84% identical to the rat sequence and shows almost no homology to the E. coli antizyme. Northern analysis studies show that this gene is expressed in both human gingival and synovial fibroblasts.

Mechanistic features associated with induction of metalloproteinases in human gingival fibroblasts by interleukin-1.

Human gingival fibroblasts were treated with recombinant interleukin-1 (IL-1) to determine the effect of this stimulus on the relative expression of collagenase (MMP-1), stromelysin (MMP-3) and plasminogen activator (PA) mRNA. The steady-state mRNA levels for these genes were determined on Northern blots. IL-1 induced steady-state levels of these mRNAs to different extents. Nuclear run-on transcription studies showed that IL-1 induction of neutral metalloproteinase may be transcriptionally regulated. Actinomycin D and protein kinase inhibitors decreased the mRNA production for all three metalloproteinases, whereas cycloheximide decreased the production of collagenase and stromelysin mRNA. Protein kinase inhibitors (H7/H8) decreased production of the three mRNAs to different extents. This study demonstrates a potentially important role for IL-1 in the regulation of metalloproteinase expression in human gingival fibroblasts. The ability of IL-1 to induce the expression of stromelysin, collagenase and PA may define a pivotal role for this cytokine in the pathogenesis of periodontitis.

Regulation of dipeptidyl aminopeptidase I and angiotensin converting enzyme activities in cultured murine brain cells by cortisol and thyroid hormone.

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis in vitro of sialosylgalactosyldiacylglycerol by mouse brain microsomes.

A sialyltransferase enzyme, present in the microsomal fraction of mouse brain, catalyzes the synthesis in vitro of a lipid, characterized as 1,2-diacyl-3-beta-D-galactosyl (3 comes from 2 N-acetylneuraminosyl)-sn-glycerol, (sialosylgalactosyldiacylglycerol) from 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol (galactosyldiacylglycerol) and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc). The enzymatic activity increases proportionally, over a given range, with increasing concentrations of both substrates and of enzyme. The apparent Km of the enzyme for galactosyldiacylglycerol is 130 microM, and for CMP-NeuNAc, 780 microM. The reaction proceeds optimally at pH 6.2. The product of the enzymatic reaction was characterized as a lipid which contained galactosyldiacylglycerol and N-acetylneuraminic acid. 14C-labeled lipid, synthesized from [14C]N-acetylneuraminic acid, and 3H-labeled lipid, synthesized from [3H]galactosyldiacylglycerol, ran with identical RF values when chromatographed on thin layers of silica gel. The water-soluble products, obtained by mild alkaline deacylation of these two labeled lipids, migrated the same when electrophoresed on paper. The ratio 14C/3H was calculated as 0.83 for doubly labeled lipid, [14C]sialosyl-[3H]galactosyldiacylglycerol. Degradation of this doubly labeled lipid by mild alkaline deacylation, followed by mild acid hydrolysis, yielded products that cochromatographed with standards galactosylglycerol and N-acetylneuraminic acid. Analysis of the products resulting from periodate oxidation of the 3H-labeled lipid demonstrated that the N-acetylneuraminic acid is linked to carbon 3 of the galactose.

Positional distribution of fatty acids in the glycerophospholipids of Tetrahymena pyriformis.

The positional distributions of the fatty acids in the major glycerophospholipids of Tetrahymena pyriformis W were analyzed. A comparison was made of the acyl distributions in normal and ergosterol-grown cells. It was assumed that the positional arrangement of fatty acids would serve as an indicator of acyltransferase enzyme specificity. The acyltransferases in this protozoan have substrate specificities that direct unsaturated groups, particularly polyunsaturates, to the 2-carbon of the glycerophospholipids. An exception is gamma-linolenic acid, which represents a substantial proportion of the total acids at both carbons. Saturated and iso-acids are esterified primarily at the 1-carbon. The qualitative pattern of the fatty acyl distribution is the same in both normal and ergosterol-grown organisms. Sterol substitution produces quantitative differences in the acyl components at both the 1- and 2-carbons of the glycerophospholipids. These differences include a shortening of the average chain length and a decrease in total unsaturation at both the 1- and 2-positions. In addition, there is a modification at the 2-carbon in the relative amounts of the products of two pathways involved in the biosynthesis of fatty acids. The data are interpreted to indicate that the fatty acid transformations in the glycerophospholipids of organisms that contain ergosterol are not the result of altered acyltransferase specificities.

The association of the sulphogalactosylglycerolipid of rat brain with myelination.

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4mumol per brain) thereafter into adult life. 2. The activity of adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H(2) (35)SO(4) at 20 days of age lost (35)S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro.

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.