Common genetic variation in a basal promoter element alters DDAH2 expression in endothelial cells.

Synthesis of the vasodilator nitric oxide (NO) can be inhibited by the endogenous methylarginines L-NMMA and ADMA. ADMA is elevated in a number of cardiovascular disorders in which NO availability is reduced. Elimination of ADMA from the body occurs primarily by enzymatic breakdown through the action of DDAH, of which two isoforms exist, DDAH1 and DDAH2. In this study we have identified a core promoter region of the DDAH2 gene, and transcription factor sites that play an important role in the regulation of DDAH2 expression. Using PCR-SSCP analysis we also identified six common polymorphisms. One of these polymorphisms (an insertion/deletion at position -871) within the core promoter element influenced basal transcription. The discovery of a functional polymorphism within the DDAH2 promoter suggests that there may be common, individual differences in the ability to metabolise ADMA in vivo, that in turn, might underlie susceptibility to cardiovascular disease.

Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins.

Endogenously produced asymmetrically methylated arginine residues are competitive inhibitors of all three isoforms of nitric oxide synthase (NOS). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) specifically hydrolyzes these asymmetrically methylated arginine residues to citrulline and methylamines. Previously we have proposed that regulation of asymmetric methylarginine concentration by DDAH may provide a novel mechanism for the regulation of NOS activity in vivo. Recently we reported the cloning of human DDAH and identified a novel human DDAH isoform (DDAH I and DDAH II, respectively). Here we report that the DDAH1 gene maps to chromosome 1p22 and confirm that DDAH2 maps to the MHC III region of chromosome 6p21.3. Extensive analysis of the distribution of DDAH1 and DDAH2 mRNA in 50 human tissues indicates differential expression of DDAH isoforms in brain regions, in immune cells, and during development. DDAH2 expression predominates in highly vascularized tissues that express the endothelial NOS isoform and in immune tissues that can express iNOS. Whereas DDAH2 is expressed at relatively high levels in all fetal tissues examined, DDAH1 expression varies little between fetal and adult tissues. The chromosomal localization of the DDAHs is consistent with gene duplication, and consistent with this, comparison of the gene structures indicates that the intron/exon organization is highly conserved. Phylogenetic analysis of DDAH sequences from diverse species suggests that DDAH gene duplication occurred prior to the emergence of bony fish some 400 million years ago. Overall the data suggest that DDAH2 may be the more ancient of the two genes.

Intracellular localization of dimethylarginine dimethylaminohydrolase overexpressed in an endothelial cell line.

Methylarginines are endogenous inhibitors of nitric oxide synthase (NOS) and have been implicated in the regulation of the nitric oxide pathway in health and disease. Cellular concentrations of free methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH). There are two isoforms of DDAH which have distinct tissue distributions with some relationship to NOS isoforms. We have determined the intracellular localization of both DDAH isoforms by overexpression of epitope-tagged DDAH in an immortalized endothelial cell line. Immunofluorescence confocal microscopy and immunoblotting indicate that both isoforms are predominantly cytosolic with no specific association with organelles or the plasma membrane. These data suggest that the key role for DDAH may be to ensure that under normal conditions the levels of methylarginines are kept low throughout the whole cell.

Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases.

Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3. 18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.

A unique molecular basis for enzyme mistargeting in primary hyperoxaluria type 1.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is normally targeted to the peroxisomes in human liver cells. However, in a third of patients suffering from the autosomal recessive disease primary hyperoxaluria type 1 (PH1), AGT is mistargeted to the mitochondria. Such organelle-to-organelle mistargeting is without parallel in human genetic disease. AGT mistargeting results from the combination of a common Pro11-->Leu polymorphism and a rare Gly170-->Arg mutation. The former generates a functionally weak mitochondrial targeting sequence (MTS) while the latter, in combination with the former, increases the efficiency of this MTS by slowing the rate at which AGT dimerises. The fact that the intracellular compartmentation of AGT can be determined, at least in part, by its oligomeric status highlights the fundamental differences in the molecular requirements for protein import into two intracellular organelles--the peroxisomes and mitochondria.

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1.

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.

Systematic expression of the complete coding sequence of apoB-100 does not reveal transmembrane determinants.

We have investigated the hypothesis that apolipoprotein B undergoes a regulated process of translocation into the endoplasmic reticulum (ER) which causes the protein to adopt a transmembrane configuration. Protein segments representing the complete coding sequence of apolipoprotein B were first expressed by in vitro translation of transcripts from seven overlapping transcripts. Two regions were identified (located at residue 2425 and between residues 4149 and 4348) that can undergo incomplete translocation into pancreatic microsomes. Ribosome pausing at these sites uncoupled translation from translocation, leading to the synthesis of large cytoplasmically oriented segments of protein. In contrast, when these two regions were expressed by transfection in cultured cells, transmembrane structures were not detected. Endogenous apolipoprotein B-100 synthesis in HepG2 cells generates a spectrum of nascent chains, indicating that ribosome pausing can also occur in intact cells. However, the cellular pause products were cotranslationally translocated. While endogenous apolipoprotein B-100 in HepG2 cells was fully translocated, discrete proteolytic fragments were generated from the amino terminus of the protein when proteases gained access to the lumen of permeabilized microsomes. These products were similar in size and sequence to apoliprotein B proteolytic fragments previously ascribed as the luminal domains of transmembrane apoB-100 molecules (Du, E. Z., Kurth, J., Wang, S. L., Humiston, P., and Davis, R. A. 1994. J. Biol. Chem. 269: 24169-24176).

Regulation of hepatic apolipoprotein-B-containing lipoprotein secretion.

The overall secretion of hepatic lipid as VLDL may be regulated by (i) changing the number of particles secreted as a result of altering apolipoprotein B output and (ii) changing the degree of lipidation of the particles with triacylglycerol so that their diameters vary over a threefold range. Substantial progress has been made in understanding the pathway of triacylglycerol incorporation into VLDL. Less is understood about the processes that commit apolipoprotein B either to secretion or to presecretory degradation, although both regulated translocation and an early lipidation step of the nascent particle with cholesterol or cholesterol ester have been implicated.

Studies on the translocation of the amino terminus of apolipoprotein B into the endoplasmic reticulum.

Apolipoprotein (apo) B is either co-translationally assembled into lipoproteins, or becomes associated with the membrane of the endoplasmic reticulum (ER) and is subsequently degraded. It has been proposed that apoB undergoes a novel process of translocation which generates cytoplasmically exposed apoB in the ER of hepatic and non-hepatic cells. Transmembrane forms of apoB can also be generated by in vitro translation (Chuck, S. L., and Lingappa, V. R. (1992) Cell 68, 9-21), which might explain the origin of untranslocated apoB in vivo. Here we have investigated a protocol which generates transmembrane forms of apoB during in vitro translation of truncated RNA transcripts. We observe that apoB can become transmembrane at sites of ribosome pausing and be held in this configuration by persistence of tRNA on the peptide chains. Ribosome pausing also occurs at these same sites in the absence of acceptor microsomes. Transmembrane topology can be generated at sites of ribosome pausing in a cytosolic protein, sea urchin cyclin when fused to a signal sequence. Mapping of the ribosome pause sites in apoB and in cyclin revealed no amino acid sequence homology. Chimeric constructs with engineered downstream glycosylation sites showed no evidence that ribosome pause sequences affect translocation of transcripts with termination codons. Transmembrane forms of apoB and cyclin were not generated during translocation into the ER in transfected COS cells.

Microsomal triglyceride transfer protein, the abetalipoproteinemia gene product, mediates the secretion of apolipoprotein B-containing lipoproteins from heterologous cells.

Apolipoprotein (apo) B is an obligatory component of triglyceride-rich lipoproteins. In the rare autosomal recessive disorder abetalipoproteinemia (ABL), no triglyceride-rich lipoproteins are secreted. Mutations in the gene encoding the 97-kDa subunit of a microsomal triglyceride transfer protein (MTP) cause ABL (Sharp, D., Blinderman, L., Combs, K. A., Klenzle, B., Ricci, B., Wager-Smith, K., Gil, C. M., Turck, C. W., Bouma, M. E., Rader, D. J., Aggerbeck, L. P., Gregg, R. E., Gordon, D. A., and Wetterau, J. R. (1993) Nature 365, 65-69; Shoulders, C. C., Brett, D. J., Bayliss, J. D., Narcisi, T. M., Jarmuz, A., Grantham, T. T., Leoni, P. R. D., Bhattacharya, S., Pease, R. J., Cullen, P. M., Levi, S., Byfield, P. G. H., Purkiss, P., and Scott, J. (1993) Hum. Mol. Genet. 2, 2109-2116). Here we have examined whether MTP is both necessary and sufficient to mediate the secretion of apoB-containing lipoproteins from cells that do not normally express either of these proteins. Carboxyl-terminal truncated forms of apoB, apoB17, and apoB41 on the centile system were expressed in COS-1 cells. ApoB17 was secreted whereas apoB41 was unable to traverse the secretory pathway. Cotransfection of apoB41 and MTP promoted the secretion of apoB41 as a buoyant lipoprotein particle with a modal density of 1.15 g/ml. When cotransfected COS-1 cells were cultured under conditions that increase the secretion of apoB100 from HepG2 cells, secretion of apoB41 was similarly increased. N-Acetyl-leucyl-leucyl-norleucinal (ALLN), a calpain I inhibitor, abolished intracellular degradation of apoB41 and increased secretion 2.5-fold. Oleate, a substrate for triglyceride synthesis, reduced degradation from 50 to 19% and increased secretion by 2.5-fold. The effects of ALLN and oleate were additive. We conclude that the secretion of apoB from COS-1 cells cotransfected with apoB and MTP is determined by the competitive processes of lipoprotein assembly and intracellular degradation in the endoplasmic reticulum and that MTP is the only tissue-specific component, other than apoB, required for the secretion of apoB-containing lipoproteins.

Fetal glycaemic control and neonatal complications in diabetic pregnancy.

To examine the relationship between fetal glycaemic control and macrosomia or neonatal hypoglycaemia, we measured umbilical cord glycosylated haemoglobin (GHb) by affinity chromatography in 44 diabetic and 40 normal pregnancies. Levels of GHb in cord blood were not significantly different between these two groups, suggesting good maternal glycaemic control was achieved in the diabetic patients. Moreover in the diabetic pregnancies, cord GHb levels did not differ in infants who were macrosomic or developed hypoglycaemia by comparison with those infants who showed neither phenomenon. We conclude that overall fetal glycaemic control in the 4-6 week period prior to delivery does not appear to influence these common neonatal complications of diabetic pregnancy.

Glycaemic control in diabetic patients transferred from therapy with animal insulins to human crystalline zinc insulin of recombinant DNA origin: a multicentre study.

87 stable insulin-dependent diabetic patients from 6 diabetic clinics within the UK were transferred from their existing once or twice daily regimen of subcutaneous animal insulin to a similar regimen of human insulin of recombinant DNA origin in neutral soluble and crystalline zinc suspension formulations. Seven point blood glucose profiles, glycosylated haemoglobin concentrations and insulin dose were examined, in each patient, before and after 6 weeks therapy with human insulins. The 67 patients on twice daily insulin showed no significant change in mean blood glucose values or glycosylated haemoglobin but required a significantly higher dose of human crystalline zinc suspension than their previous long-acting animal insulin. In contrast the 17 patients on a once daily regimen experienced a significant deterioration in glycaemic control without a significant change in insulin dose or glycosylated haemoglobin. Three patients on twice daily insulin withdrew shortly after transfer to human insulin. The combination of human soluble and crystalline zinc suspension appears to be a more satisfactory substitute for animal insulin when used in a multi-dose regime rather than on a once daily basis.

Outcome of pregnancy in insulin-dependent (type 1) diabetic women between 1971 and 1984.

We have looked at the outcome of pregnancy in women with type 1 (insulin-dependent) diabetes mellitus who were confined at Glasgow Royal Maternity Hospital between 1971 and 1984. Of 134 pregnancies, 123 proceeded beyond 28 weeks' gestation and yielded 125 live infants. The perinatal mortality rate was 16/1000, due solely to congenital foetal malformation. Malformations were not related either to the severity of maternal diabetes (as graded by the White classification) or the glycaemic control in the first trimester (as judged by maternal glycosylated haemoglobin concentration). Over the period of study, there has been a marked reduction in the frequency of ketoacidosis during pregnancy and in the delivery of small-for-dates babies; more mature lecithin:sphingomyelin ratios have been obtained by amniocentesis; and better Apgar scores have been demonstrated in the infants at delivery. The Caesarean section rate has fallen from 83 to 30 per cent, and babies now spend less time separated from their mothers in paediatric units. These improvements largely reflect better diabetic treatment (improved insulin regimens and glycaemic control) and closer obstetric assessment. Foetal malformation occurred overall in 11.4 per cent of pregnancies and did not become less frequent over the period of study. Further major improvement in the outcome of diabetic pregnancy will only come from a reduction in the congenital malformation rate, which implies better diabetic control at and around the time of conception.

A comparison of biosynthetic human insulin with porcine insulin in the blood glucose control of diabetic pregnancy.

Fifty insulin-dependent pregnant diabetic women were treated from presentation at the antenatal clinic with a regimen of either porcine or biosynthetic human soluble and isophane insulins. Blood glucose values, glycosylated haemoglobin, insulin requirements and outcome of pregnancy were compared in the two groups of patients. No difference was noted in blood glucose control, birth weight or neonatal complications. In the group treated with human insulin, fasting blood glucose could only be controlled by significantly greater doses of human isophane insulin injected before the evening meal. Furthermore, in two cases adequate glycaemic control required the injection of the evening human isophane insulin to be delayed until bedtime.

Glycosylated albumin and glycosylated proteins: rapidly changing indices of glycaemia in diabetic pregnancy.

Serial measurements of glycosylated albumin (GAlb), glycosylated plasma proteins (GPP) and glycosylated haemoglobin (GHb) were performed by affinity chromatography throughout the course of pregnancy in 14 insulin-dependent diabetic women. In patients whose glycaemic control improved markedly during pregnancy, the concentrations of GAlb and GPP decreased by more than 50 per cent after four weeks of good diabetic control. By contrast, the rate of decrease in GHb levels in the same patients was significantly less and did not indicate a comparable improvement in glycaemia until 12 weeks after good diabetic control was established. Elevated concentrations of GAlb or GPP decrease much more rapidly than GHb concentration when diabetic control is improved; thus measurement of glycosylated proteins is a valuable adjunct to serial blood glucose monitoring in clinical circumstances such as diabetic pregnancy, where early confirmation of good diabetic control is important.