Ethically problematic treatment decisions in different medical specialties.

BACKGROUND: Ethical dilemmas are an integral part of medicine. Whether physicians actually feel that they have made ethically problematic treatment decisions or choices in their work is largely unknown. Identifying physicians with ethical problems, and the types of problems and underlying factors, might benefit organisational and educational efforts to help physicians solve ethical dilemmas in a constructive way. We investigated how the frequency and types of ethically difficult treatment decisions vary by specialty. METHOD: A mail survey of all non-retired Finnish physicians (n = 17,172, response rate 75.6%) was conducted in 2004. Of those who had made any ethically problematic treatment decisions, the types of decisions and reasons given for these decisions were asked for. Factor analysis was used to investigate clustering of ethically problematic treatment decisions, and logistic regression to investigate the effect of specialty, adjusted for age and gender. RESULTS: Psychiatrists experienced ethically problematic treatment decisions most frequently, followed by pulmonologists, internists and neurologists. Problems were reported least often by pathologists, laboratory physicians and ophthalmologists. Overtreatment was more common than undertreatment in most specialties, with the exception of psychiatrists who emphasised undertreatment and patient rights issues. CONCLUSION: Physicians of different specialties differ significantly regarding frequency and types of ethically problematic treatment decisions they have made. Psychiatrists differ from all other specialists in reporting more undertreatment and patient rights issues. Experiencing ethically problematic decisions might affect the quality of care and physician well-being in many ways. The findings could be useful for both under- and postgraduate ethics education.

Molecular cloning, expression and chromosomal localization of a human gene encoding a 33 kDa putative metallopeptidase (PRSM1).

The zincins are a superfamily of structurally-related Zn(2+)-binding metallopeptidases which play a major role in a wide range of biological processes including pattern formation, growth factor activation and extracellular matrix synthesis and degradation. In this paper we report the identification and complete primary structure of a novel 33 kDa protein which contains the zinc-binding HEXXH motif found in the zincin superfamily. We have named this novel protein PRSM1 (PRoteaSe, Metallo, number 1). The gene was identified by the immunoscreening of a human placental cDNA library using polyclonal antibodies raised to the 70 kDa human matrix metalloendopeptidase, type III procollagen N-proteinase [Halila, R. and Peltonen, L. (1986) Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum. Biochem. J. 239, 47-52]. The protein is found in placenta and cultured osteosarcoma cells. PRSM1 could share sequence homology with the type III procollagen N-proteinase. The prsm1 gene is represented once in the human genome and is localized on chromosome 16 (q24.3).

A sensitive and rapid method for assaying the activity of type III procollagen amino-terminal proteinase.

A rapid assay procedure was developed for measuring the rate of cleavage of the amino-terminal propeptide of type III procollagen. The method was based on the sequential precipitation of type III collagen and uncleaved pN-collagen by 30% ammonium sulfate, while the free amino-terminal propeptide remained in solution and could be further precipitated by 60% ammonium sulfate. Consistently better results were obtained than with the earlier method in which absolute ethanol was used as the precipitant, and selective precipitation was confirmed by polyacrylamide gel electrophoresis of the pellets. The high sensitivity of this method facilitates relatively rapid assays even from small amounts of cultured cells.

Neutral protease cleaving the N-terminal propeptide of type III procollagen: partial purification and characterization of the enzyme from smooth muscle cells of bovine aorta.

Procollagen type III amino-terminal protease was detected in cultures of smooth muscle cells of fetal calf aorta, and this protease was purified about 400-fold. Only about half of the enzyme activity was consistently attached to concanavalin A-agarose (Con A-agarose). After affinity chromatography on type III pN-collagen-Sepharose, the Con A bound fraction showed only one major band with a molecular weight of about 72000, this value corresponding well with the elution position of enzyme activity in gel filtration. The enzyme did not cleave procollagens type I or type IV, and denatured type III pN-collagen also remained uncleaved. The Km of the enzyme activity for iodo[14C]acetamide-labeled type III pN-collagen was 0.76 microM. Neutral pH and Ca2+ wer required for maximal enzymic activity. The metal chelators ethylenediaminetetraacetic acid and ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid inhibited the activity as well as did dithiothreitol, but there was only little if any inhibition by several other proteinase inhibitors tested.

Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.

Serum procollagen type III is an early and sensitive marker for veno-occlusive disease of the liver in children undergoing bone marrow transplantation.

Veno-occlusive disease of the liver (VOD) is a life-threatening complication occurring in patients undergoing bone marrow transplantation (BMT). Although clinical signs and laboratory parameters such as elevation of serum bilirubin often suggest this condition, it would be useful to identify early biochemical markers for VOD. Fibrous alterations in the hepatic venules and small lobular veins occur during development of VOD; these changes are accompanied by the deposition of types I and III collagen in the liver tissue. Since the N-terminal propeptide of type III procollagen (PIIINP) is a sensitive marker of liver and lung fibrosis, we undertook a study to evaluate the usefulness of measurements of serum PIIINP in children with VOD. Seven of the 28 children who underwent BMT, both allogenic and autologous, developed VOD. All seven had an increase of more than 100 ng/mL in the serum PIIINP level, whereas only one of the remaining 21 children not affected by VOD had an increment of PIIINP more than 100 ng/mL (P = .0001). The levels of serum PIIINP were higher in the VOD group during the follow-up period of up to 91 days after BMT. The elevation of PIIINP also occurred at a stage of the disease usually preceding any other laboratory or clinical signs of VOD. Serum concentration of PIIINP thus seems to be of value as an early marker for VOD in children undergoing BMT.

Enzymes converting procollagens to collagens.

Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I-III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time. Two amino-terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy-terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy-terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl-terminal propeptides. The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis. In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.

Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII.

The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age-matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino-terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion.

N-terminal propeptide of type III collagen in tracheal fluid and serum in preterm infants at risk for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common pulmonary complication in preterm infants that leads to fibrosis of the bronchoalveolar walls and often to severe clinical consequences. Type III collagen is deposited early in progressive fibrosis. Because the N-terminal propeptide of type III collagen (PIIINP), a by-product of type III collagen synthesis, reflects the degree of pulmonary fibrosis in adults, we hypothesized that PIIINP in tracheal aspirates and/or serum may be a useful early marker of developing BPD in neonates. We serially measured PIIINP in tracheal fluid and serum samples during the first weeks of life in 41 consecutive respirator-treated preterm infants (mean birth weight 1067 g, mean gestational age 28.3 wk). Eight of the infants died and 22 infants fulfilled the criteria for BPD at age 28 d. The mean level of PIIINP decreased with advancing postnatal age in tracheal fluid but not in serum. The mean tracheal fluid PIIINP during d 1 and 2 of life, respectively, was 175 and 200 ng/mg protein in infants who were still in a respirator at age 28 d (n = 13), 122 and 97 ng/mg protein in those who were weaned earlier (n = 20), and 50 and 30 ng/mg protein in those who died before age 28 d (n = 8). These differences are not statistically significant, and the variability of the values was large. The PIIINP concentrations in tracheal aspirates of infants subsequently developing BPD did not differ from those without BPD. Neither did the levels correlate with the degree of BPD or radiologically defined fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Human leucocyte aspartylglucosaminidase. Evidence for two different subunits in a more complex native structure.

Human leucocyte aspartylglucosaminidase (AGA: 1-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) was purified to homogeneity by using affinity chromatography, gel filtration, chromatofocusing and reverse-phase h.p.l.c. As shown by SDS/PAGE, the homogeneous purified enzyme preparation consists of four polypeptide chains with molecular masses of 25, 24, 18 and 17 kDa. In the native polyacrylamide gel these polypeptides migrate as one active enzyme complex, and by gel filtration the peak of enzyme activity can be detected in a position of about 65 kDa. Digestion with endoproteinase Lys-C or endoproteinase Asp-N, followed by peptide analysis with reverse-phase h.p.l.c., reveals an identical peptide pattern for the 24 and 25 kDa bands as well as for the 17 and 18 kDa bands. This treatment further demonstrated a totally different peptide pattern for the 24/25 kDa versus the 17/18 kDa subunit. The N-terminal sequences of the 17 kDa and the 18 kDa peptides were identical, as determined by Edman degradation. The N-termini of the 24 kDa and the 25 kDa peptides were blocked. The enzyme was partly resistant to endoglycosidases H and F, but N-glycosidase F transformed the 24/25 kDa band into one 23 kDa band and the 17/18 kDa band into one 16 kDa band. Also, immunological data obtained with antisera produced against these subunits showed that AGA consists of two non-identical polypeptides.

Human aspartylglucosaminidase. A biochemical and immunocytochemical characterization of the enzyme in normal and aspartylglucosaminuria fibroblasts.

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is an essential enzyme in the degradation of asparagine-linked glycoproteins. In man, deficient activity of this enzyme leads to aspartylglucosaminuria (AGU), a recessively inherited lysosomal storage disease. Here we used affinity-purified polyclonal antibodies against the native AGA and its denatured subunits to establish the molecular structure and intracellular location of the enzyme in normal and AGU fibroblasts. Inactivation of the enzyme was found to coincide with the dissociation of the heterodimeric enzyme complex into subunits. Although the subunits were not linked by covalent forces, the intrapolypeptide disulphide bridges were found to be essential for the normal function of AGA. AGA was localized into lysosomes in control fibroblasts by both immunofluorescence microscopy and immuno-electron microscopy, whereas in AGU cells the location of antigen was different, suggesting that, owing to the mutation, a missing disulphide bridge, most of the enzyme molecules get retarded in the cis-Golgi region and most probably face intracellular degradation.

The human gene for xanthine dehydrogenase (XDH) is localized on chromosome band 2q22.

Mutations in the xanthine dehydrogenase gene (XDH), which codes for the last enzyme of the purine catabolic pathway in man, cause the autosomal recessive disease xanthinuria. We obtained cDNA clones from a human breast cDNA library and confirmed one of the two different sequences proposed for human XDH. Using a somatic cell hybrid mapping panel and specific primers for human XDH, we assigned the gene to chromosome 2. By fluorescence in situ hybridization, the gene was localized to bands 2p22.3-->p22.2. The FLpter probe location was 0.135 (SD = 0.016), as determined by digital image analysis.

Spectrum of mutations in aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is an inherited lysosomal storage disorder caused by the deficiency of aspartylglucosaminidase. We have earlier reported a single missense mutation (Cys163----Ser) to be responsible for 98% of the AGU alleles in the isolated Finnish population, which contains about 90% of the reported AGU patients. Here we describe the spectrum of 10 AGU mutations found in unrelated patients of non-Finnish origin. Since 11 out of 12 AGU patients were homozygotes, consanguinity has to be a common denominator in most AGU families. The mutations were distributed over the entire coding region of the aspartylglucosaminidase cDNA, except in the carboxyl-terminal 17-kDa subunit in which they were clustered within a 46-amino acid region. Based on the character of the mutations, most of them are prone to affect the folding and stability and not to directly affect the active site of the aspartylglucosaminidase enzyme.

Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease.

We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients.