A prospective surveillance study of inhibitor development in haemophilia A patients following a population switch to a third-generation B-domain-deleted recombinant factor VIII.

INTRODUCTION: Following a provincial tender, most subjects with haemophilia A in Quebec switched their treatment to a third-generation recombinant B-domain-deleted factor VIII (FVIII). AIM: Our objective was to evaluate the incidence of inhibitor development and FVIII recovery in patients following the switch of factor replacement therapy. METHODS: One hundred and thirty-five subjects were enrolled and tested for FVIII activity and inhibitors every 6 months during 1 year. Subjects with mild haemophilia A or current inhibitors were excluded. Data on demographics, bleeds and FVIII usage were collected. RESULTS: A total of 125 switchers and 10 non-switchers were enrolled. Most subjects had severe haemophilia A (95.6%) and were on prophylaxis (89.6%). Mean FVIII recovery was similar at 0, 6 and 12 months postswitch. Two switchers developed de novo inhibitors in the 6 months postswitch, one of which was transient. No recurrent inhibitor was observed. A small but significant increase in FVIII usage was observed for adult switchers and the whole cohort of switchers and non-switchers. There was an increase in the annualized bleeding rate (ABR) for non-joint bleeds for the whole cohort of switchers. However, no significant differences were observed in ABR for joint bleeds. CONCLUSION: Our surveillance study shows comparable inhibitor development to similar published studies. A significant increase in FVIII utilization was noted for the whole cohort, switchers and non-switchers. Lastly, no clinically significant changes were observed in ABR for joint bleeds, but a difference for non-joint bleed ABRs was observed in switchers.

Alloantibody developed in a factor XIII A subunit deficient patient during substitution therapy; characterization of the antibody.

INTRODUCTION: In factor XIII A subunit (FXIIIA) deficiency, the development of alloantibodies is extremely rare. Only four reports have been published and the antibodies were not characterized. AIM: The aim of this study was to describe the clinical course and the laboratory diagnosis of a FXIII-A deficient patient who developed alloantibodies. METHODS: FXIII activity was assessed with an ammonia release assay. FXIII-A, FXIII B subunit (FXIII-B) and the complex plasma FXIII (FXIII-A2 B2 ) antigens were determined by ELISA. The causative mutation was detected by fluorescent DNA sequencing. The binding of alloantibody to FXIII-A2 and FXIII-A2 B2 was studied by surface plasmon resonance. The cleavage of FXIII-A by thrombin and Ca2+ -induced activation of thrombin-cleaved FXIII were followed by western blotting and activity measurement, respectively. RESULTS: FXIII activity, FXIII-A2 B2 and FXIII-A antigens were below the limit of detection in the patient's plasma. The severe FXIII-A deficiency was due to a novel homozygous mutation resulting in early stop codon (c.127C>T, p.Gln42STOP). The alloantibody bound to FXIII-A2 and FXIII-A2 B2 with equally high affinity (Kd ~10-8 ). It accelerated the elimination of administered FXIII concentrate from the circulation, interfered with thrombin and Ca2+ -induced activation and inhibited FXIII activity. Attempts to eliminate the alloantibody resulted only in transient improvement. Patient developed intracerebral haemorrhage after a minor trauma and died in spite of aggressive replacement therapy with FXIII concentrate. CONCLUSION: The anti-FXIII-A alloantibody caused an unmanageable bleeding complication. The antibody was of combined subtype which accelerated the elimination of FXIII and exerted a multiple inhibitory effect on FXIII activation/activity.

Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

Comparison of endocrine and cellular mechanisms regulating the corpus luteum of primates and ruminants.

The corpus luteum (CL) is a transient endocrine organ that is essential for maintenance of pregnancy in both ruminants and primates. The cellular and endocrine mechanisms that regulate the CL in these species have commonalities and some distinct and intriguing differences. Both species have similar cellular content with large luteal cells derived from the granulosa cells of the follicle, small luteal cells from follicular thecal cells, and large numbers of capillary endothelial cells that form the vasculature that has an essential role in optimal CL function. Intriguingly, the large luteal cells in ruminants grow larger than in primates and acquire a capacity for high constitutive progesterone (P4) production that is independent of stimulation from LH. In contrast, the primate CL and the granulosa lutein cells from primates continue to require stimulation by LH/CG throughout the luteal phase. Although the preovulatory follicle of women and cows had similar size and steroidogenic output (10 to 20 mg/h), the bovine CL had about ten-fold greater P4 output compared to the human CL (17.4 vs. 1.4 mg/h), possibly due to the development of high constitutive P4 output by the bovine large luteal cells. The continued dependence of the primate CL on LH/CG/cAMP also seems to underlie luteolysis, as there seems to be a requirement for greater luteotropic support in the older primate CL than is provided by the endogenous LH pulses. Conversely, regression of the ruminant CL is initiated by PGF from the nonpregnant uterus. Consequently, the short luteal phase in ruminants is primarily due to premature secretion of PGF by the nonpregnant uterus and early CL regression, whereas CL insufficiency in primates is related to inadequate luteotropic support and premature CL regression. Thus, the key functions of the CL, pregnancy maintenance and CL regression in the absence of pregnancy, are produced by common cellular and enzymatic pathways regulated by very distinct luteotropic and luteolytic mechanisms in the CL of primates and ruminants.

Growth, morphogenesis, and differentiation during mouse prostate development in situ, in renal grafts, and in vitro.

BACKGROUND: In vitro organ culture and renal grafting of the urogenital sinus (UGS) have both been used as models of prostate development. However, neither has been rigorously examined for its fidelity to replicate the canonical process of prostate differentiation in situ. METHODS: We assessed size, morphology, histology, and the mRNA expression of differentiation marker genes of the E14 male mouse UGS grown for 0-28 days as sub-renal capsule allografts in nude mice or in culture containing androgen and compared these to UGS development in situ. RESULTS: Development of grafted tissues was morphologically and histologically similar to development in situ but differentiation occurred more rapidly. UGS growth in organ culture resulted in bud formation, but did not trigger cellular differentiation. However, the potential for differentiation was maintained and could be rescued by grafting tissues into nude mice. CONCLUSIONS: In vitro organ culture and renal grafting of UGS tissues may be appropriate models for studying prostatic bud formation, but only grafting is an appropriate model for prostatic differentiation.

Externalization of host cell protein kinase C during enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in children in developing countries. Protein kinase C (PKC), a serine- and threonine-directed protein kinase, is rapidly activated following EPEC infection and this is accompanied by its translocation to a membrane-bound location where it is tightly bound to phosphatidylserine (PS). EPEC infection causes host cell death, one of whose features is externalization of PS. We hypothesized that externalization of PS would be accompanied by externalization of PKC as well. We report that EPEC infection triggers the externalization of PKC to the outer surface of the host cell. Ecto-PKC remains firmly tethered to the cell but can be released by incubation with peptide or protein substrates for the enzyme. Ecto-PKC is intact and biologically active and able to phosphorylate protein substrates on the surface of the host cell. Phosphorylation of whole EPEC bacteria or EPEC-secreted proteins could not be detected. Externalization of PKC could be reproduced by the combination of an apoptotic stimulus (ultraviolet (UV) irradiation) and phorbol myristate acetate (PMA), a procedure which resulted in externalization of >25% of the total cellular content of PKC-alpha. In the presence of ATP, ecto-PKC inhibited UV-induced cell shrinkage, membrane blebbing, and propidium iodide uptake but not the activation of caspases 3 and 7. This is the first report that expression of an ecto-protein kinase is altered by a microbial pathogen and the first to note that externalization of PKC can accompany apoptosis.

Apolipoprotein A-I deficiency. Biochemical and metabolic characteristics.

Familial HDL deficiencies are associated with variable susceptibility to premature coronary heart disease, but the mechanism underlying this association remains poorly understood. Three homozygotes with isolated complete apo A-I deficiency caused by an autosomal codominant apo A-I Q[-2]X mutation and one heterozygote developed coronary heart disease before age 40 years. We characterized the effects of this mutation on lipoprotein metabolism. LDL FC, phospholipid, and apo B were all significantly higher in homozygotes than in heterozygotes. The HDLs of the heterozygotes were apo A-I poor relative to apo A-II. Lecithin-cholesterol acyltransferase activity was 59% lower in homozygotes than in normal subjects or heterozygotes. Cholesteryl ester transfer activity was increased in a homozygote compared with a normolipidemic control subject. Postprandial lipid metabolism was studied in one homozygote and one heterozygote. Post-prandial TG response in the homozygote was significantly exaggerated, while residual plasma HDL level remained unaffected. The homozygote also had delayed clearance of retinyl ester, a marker of chylomicron remnant metabolism. Thus, homozygosity and heterozygosity for apo A-I Q[-2]X are associated with qualitative, as well as quantitative, disturbances in plasma HDLs, LDLs, lipid-modifying enzyme activities, and postprandial retinyl ester metabolism. The observed elevation of atherogenic lipoproteins and reduction in antiatherogenic lipoproteins in the affected members of the apo A-I Q[-2]X kindred are consistent with the primary deficiency in apo A-I having pleiotropic effects that markedly enhance susceptibility for coronary heart disease.

Constitutive expression of a microinjected glucose-regulated protein (grp78) fusion gene during early Xenopus laevis development.

In this study we have found that a rat glucose-regulated protein (grp) 78 chloramphenicol acetyltransferase (CAT) fusion gene deleted to -456 bp at the 5' end and injected into fertilized Xenopus eggs was first expressed in a constitutive manner in late blastula stage embryos and displayed increased expression as the embryos developed to the gastrula and neurula stages. Using a series of internal deletion mutants and linker-scanner mutants of the rat grp78 promoter, we have found that a CCAAT box and CCAAT-like element within the region -129 to -90 were essential for constitutive expression of the chimeric genes in neurula stage embryos. These results suggest conservation of the regulatory sequences within the grp78 promoter between rat and Xenopus. Interestingly, deletion or alteration of sequences between -130 and -149 had a dramatic stimulatory effect on basal promoter activity. This effect, which was not observed previously in rat cells, may be the result of upstream elements that are transcriptionally active in Xenopus and that can compensate for the mutated or deleted sequences. It is also possible that these results indicate the presence of a negative regulatory element that is recognized by the Xenopus transcriptional apparatus.

Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11.

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

Kinetics of lipolysis of very low density lipoproteins by lipoprotein lipase. Importance of particle number and noncompetitive inhibition by particles with low triglyceride content.

Our objective was to identify the major compositional factor(s) of very low density lipoprotein which determines its properties as a substrate for lipoprotein lipase. Human very low density lipoprotein was fractionated by preparative electrophoresis. The apparent Km was significantly lower for pre-beta very low density lipoprotein compared with beta very low density lipoprotein when calculated on the basis of triglyceride concentration. When the triglyceride concentration was adjusted for the triglyceride/apolipoprotein B ratio, the apparent Km was not different among very low density lipoprotein fractions. This implied that very low density lipoprotein particle number was of primary importance. To test this hypothesis further, rabbit cholesterol-rich very low density lipoprotein and human intermediate density lipoprotein and low density lipoprotein, from a patient with hepatic lipase deficiency, were added to the incubations. Each of these fractions functioned as noncompetitive inhibitors of lipolysis. We speculate that the saturation of lipoprotein lipase by an excess number of particles is a characteristic of human hyperlipoproteinemias that predispose to coronary heart disease and that are commonly classified as familial combined hyperlipoproteinemia or hyperapobetalipoproteinemia.

Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia.

We report a Canadian kindred with a novel mutation in the apolipoprotein (apo) A-I gene causing analphalipoproteinemia. The 34-yr-old proband, product of a consanguineous marriage, had bilateral retinopathy, bilateral cataracts, spinocerebellar ataxia, and tendon xanthomata. High density lipoprotein cholesterol (HDL-C) was < 0.1 mM and apoA-I was undetectable. Genomic DNA sequencing of the proband's apoA-I gene identified a nonsense mutation at codon [-2], which we designate as Q[-2]X. This mutation causes a loss of endonuclease digestion sites for both BbvI and Fnu4HI. Genotyping identified four additional homozygotes, four heterozygotes, and two unaffected subjects among the first-degree relatives. Q[-2]X homozygosity causes a selective failure to produce any portion of mature apoA-I, resulting in very low plasma level of HDL. Heterozygosity results in approximately half-normal apoA-I and HDL. Gradient gel electrophoresis and differential electroimmunodiffusion assay revealed that the HDL particles of the homozygotes had peak Stokes diameter of 7.9 nm and contained apoA-II without apoA-I (Lp-AII). Heterozygotes had an additional fraction of HDL3-like particles. Two of the proband's affected sisters had documented premature coronary heart disease. This kindred, the third reported apoA-I gene mutation causing isolated complete apoA-I deficiency, appears to be at significantly increased risk for atherosclerosis.

Identification of disulfide-linked apolipoprotein species in human lipoproteins.

We wished to determine whether apolipoprotein C-IIToronto, a mutant form of apolipoprotein C-II that contains a C-terminal cysteine residue, exists as a monomeric species or as multiple disulfide-linked species in plasma lipoproteins. The plasma lipoproteins from a heterozygous carrier and two homozygous carriers of apoC-IIToronto were investigated. The mutant apolipoprotein was found in homodimeric form and as heterodimers with apolipoprotein A-II, apolipoprotein B-100, and apolipoprotein E. Of particular interest was the demonstration of the existence of the disulfide-linked species apolipoprotein B-100:A-II and B-100:C-IIToronto in the very low density and low density lipoproteins in subjects who were carriers of apoC-IIToronto. We also observed that apoE3:C-IIToronto and apoE3:A-II dimers were present in the chylomicrons and very low density lipoproteins of these subjects. The observation of the existence of apolipoprotein B-100:A-II was extended to other hypercholesterolemic and hypertriglyceridemic subjects. The highest proportion of apolipoprotein B-100:A-II was observed in the very low density lipoproteins of hypertriglyceridemic subjects. The concentration of this species was significantly higher in hyperlipidemic subjects than in normolipidemic controls. These results demonstrate that the molecular species of cysteine-containing apolipoproteins are complex and should be considered in studies of human lipoprotein composition and function.

Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics.

Hepatic lipase (HL) is an important enzyme in the metabolism of triglyceride-rich lipoproteins and high density lipoproteins. The clinical syndrome of HL deficiency is rare and difficult to identify. We studied carriers of mutant HL to ascertain whether there are distinctive clinical and/or biochemical characteristics of the heterozygous state. In an Ontario kindred, compound heterozygosity for two HL mutations, S267F and T383M, underlies the clinical syndrome of complete HL deficiency. We report that simple heterozygotes for either HL mutant do not have a discrete lipoprotein abnormality, except for relative triglyceride enrichment of lipoprotein fractions with d > 1.006 g/mL. Postheparin HL activity is depressed to a greater degree in carriers of S267F compared with carriers of T383M. Retinyl palmitate loading studies in a compound heterozygote revealed impaired clearance of chylomicron remnants. The dyslipoproteinemia in a compound heterozygote was ameliorated by lovastatin. There was no difference in the quantity and distribution of HL mRNA in the liver of a compound heterozygote when compared with that of a normal subject. Thus, HL deficiency associated with structural variation of the HL gene is characterized by premature atherosclerosis, triglyceride enrichment of lipoprotein fractions with d > 1.006 g/mL, the presence of circulating beta-very low density lipoproteins, and abnormal catabolism of postprandial triglyceride-rich lipoproteins.

Specific types of colonic fermentation may raise low-density-lipoprotein-cholesterol concentrations.

To assess the effects of increased colonic fermentation on serum lipids, eight healthy volunteers were placed on two identical 2-wk metabolic diets, one of which was supplemented with lactulose (18-25 g/d). Lactulose raised day-long concentrations of breath hydrogen and serum glutamine as indicators of increased colonic fermentation by 78 +/- 13% (P less than 0.001) and 24.7 +/- 9.5% (P less than 0.05), respectively). Unexpectedly, however, fasting serum total and low-density-lipoprotein cholesterol and apolipoprotein B concentrations were higher at 2 wk by 8.9 +/- 1.5% (P less than 0.001), 10.9 +/- 2.2% (P less than 0.005), and 18.9 +/- 5.9% (P less than 0.02), respectively, compared with the control diet. With lactulose, mean free fatty acid concentrations were reduced over the day by 19.5 +/- 5.9% (P less than 0.02), with no change in mean day-long blood glucose, serum insulin, or C-peptide concentrations. We conclude that certain rapidly fermented substrates may raise rather than lower serum lipids, possibly through increasing the amount of acetate absorbed from the colon.

A hepatic lipase gene mutation associated with heritable lipolytic deficiency.

Absent hepatic lipase (HL) activity results in dyslipidemia and premature atherosclerosis. DNA sequencing of the HL gene from subjects with heritable HL deficiency identified a new C to T substitution within exon 8 that in the mature enzyme caused a threonine to methionine change at position 383 (T383M). With a rapid DNA detection method we observed that all 6 individuals with complete HL deficiency from 2 families had the T383M mutation. None of 50 random unrelated unaffected subjects had this mutation. We propose that T383M is specific to families with heritable HL deficiency. Furthermore, structural variation at the HL gene, possibly in combination with other factors, appears to be etiologic in HL deficiency.

Effect of mRNA secondary structure on the efficiency of translational initiation by eukaryotic ribosomes.

We have used site-directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected as an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129-nucleotides-long 5'-untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS-1 cells. Analysis of the proopiomelanocortin-related peptides present in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non-mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5'-untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation.

Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis.

The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA.

The SP6 polymerase/promoter system was used to synthesize porcine pro-opiomelanocortin mRNAs with nucleotide sequence deletions in the 5'- as well as 3'-untranslated and coding regions. The translational efficiency of the mutant mRNAs was evaluated by cell-free translation or by monitoring the rate and extent of ribosome binding in the presence of sparsomycin. The results of these experiments indicate that specific nucleotide sequences in the 5'-untranslated and coding regions of the pro-opiomelanocortin mRNA decrease its rate of translation. Structure mapping of the mRNA with double-strand and single-strand specific nucleases suggests that these sequences can form stable secondary structures.