[Assisted suicide in psychiatry; current situation and notes on a recent case]. / Hulp bij zelfdoding in de psychiatrie; stand van zaken en bespreking van een recente casus.

BACKGROUND: The Dutch legal system grants psychiatric patients the right to assisted suicide but imposes strict conditions. The matter, however, remains controversial. Many psychiatrists are reluctant to become involved in assisted suicide because they are not familiar with the procedure that has to be followed, nor are they clear about the precise meaning of 'acting responsibly'. AIM: To provide an overview of the procedure to be followed and of the criteria to be met when a psychiatrist is wondering whether to grant a patient's request for assistance with suicide. METHOD: We analyse the law, jurisprudence and medical ethics and the special problems that arise in the case of psychiatric patients. RESULTS: We describe the developments in law and psychiatric practice which have led to greater recognition of the autonomy of the patient. The final decision depends on an assessment of the patient's competence and his ability to articulate his request clearly and on the hopelessness engendered by unbearable suffering. CONCLUSION: In the future there may be an increase in the number of cases of assisted suicide in psychiatry now that procedures and criteria have been established which are in accordance with the Dutch legal system.

Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein.

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides. The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon.

FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation.

The Paracoccus denitrificans fnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high-affinity cbb3-type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox-sensitive [4Fe-4S] cluster in FNR. NNR, another FNR-like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane-bound nitrate reductase and the cbb3-type oxidase was significantly reduced, whilst the activity of the bb3-type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3-type oxidase, cytochrome C550, cytochrome C552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP- and NNR-regulated genes are highly similar to or even identical to each other, the absence of cross-talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.

Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria.

The taxonomy of Paracoccus denitrificans and related bacteria is discussed. Evidence is given which shows that the physiological differences between P. denitrificans and Thiosphaera pantotropha are less fundamental than previously thought. A proposal to consider a species P. pantotropha is mentioned. The properties of the denitrifying enzymes and the genes involved in their formation in P. denitrificans is discussed. The synthesis of the membrane-bound-nitrate reductase is regulated by FNR, that of the nitrite- and nitric oxide reductase by NNR. Evidence is given that FNR acts as a redox sensor rather than an oxygen sensor. The occurrence of aerobic denitrification and coupled heterotrophic nitrification-denitrification in the original strain of Thiosphaera pantotropha are explained by a limiting respiratory activity which activates FNR. Aerobic denitrification leads to a lower growth yield and an increase in mumax in batch culture when a limiting respiratory activity is assumed and when excess substrate is present. Coupled heterotrophic nitrification-denitrification gives a smaller increase in mumax and a more drastic reduction in yield. Both processes are thus advantageous to the organism. In a chemostat with limiting substrate these processes are disadvantageous. T. pantotropha has lost the ability for aerobic denitrification during extended cultivation. Possibly the substrate concentration was limiting during extended cultivation giving a selective advantage to variants which have lost these properties. The calculations predict that P. denitrificans should be able to grow chemolithotrophically with hydroxylamine.

Mutational analysis of the nor gene cluster which encodes nitric-oxide reductase from Paracoccus denitrificans.

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

Regulation of oxidative phosphorylation: the flexible respiratory network of Paracoccus denitrificans.

Paracoccus denitrificans is a facultative anaerobic bacterium that has the capacity to adjust its metabolic infrastructure, quantitatively and/or qualitatively, to the prevailing growth condition. In this bacterium the relative activity of distinct catabolic pathways is subject to a hierarchical control. In the presence of oxygen the aerobic respiration, the most efficient way of electron transfer-linked phosphorylation, has priority. At high oxygen tensions P. denitrificans synthesizes an oxidase with a relatively low affinity for oxygen, whereas under oxygen limitation a high-affinity oxidase appears specifically induced. During anaerobiosis, the pathways with lower free energy-transducing efficiency are induced. In the presence of nitrate, the expression of a number of dehydrogenases ensures the continuation of oxidative phosphorylation via denitrification. After identification of the structural components that are involved in both the aerobic and the anaerobic respiratory networks of P. denitrificans, the intriguing next challenge is to get insight in its regulation. Two transcription regulators have recently been demonstrated to be involved in the expression of a number of aerobic and/or anaerobic respiratory complexes in P. denitrificans. Understanding of the regulation machinery is beginning to emerge and promises much excitement in discovery.

Isolation and characterization of a novel insertion sequence element, IS1248, in Paracoccus denitrificans.

A new suicide vector, pRVS3, was constructed to facilitate gene replacements in the genome of Paracoccus denitrificans. In control experiments, incorporation of this suicide vector into the genome did not depend on the presence of homologous DNA. Using appropriate restriction enzymes, the suicide vector and flanking DNA were recovered from the genomic DNA. Sequence analysis demonstrated that both up- and downstream of the ex-integrant vector there was an element that showed high homology with bacterial insertion sequences (IS). Southern blot analysis of wild-type and integrant strains revealed that at least four copies of this IS element reside in the P. denitrificans genome, one of which, designated IS1248, had been involved in the transpositional event described here. IS1248 is 830 bp long, has 13-bp imperfect inverted repeats at the borders, and contains five open reading frames. With respect to the organization and primary sequences of the open reading frames, IS1248 closely resembles IS869 and IS427 of Agrobacterium tumefaciens, IS402 of Pseudomonas cepacia, and ISmyco found in Mycobacterium tuberculosis.

Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.

The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N-terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb3-type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrification in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.

The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.

Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.

Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans.

By using the gene encoding the C-terminal part of the cd1-type nitrite reductase of Pseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene from Paracoccus denitrificans was isolated. This gene, nirS, codes for a mature protein of 63144 Da having high homology with cd1-type nitrite reductases from other bacteria. Directly downstream from nirS, three other nir genes were found in the order nirECF. The organization of the nir gene cluster in Pa. denitrificans is different from the organization of nir clusters in some Pseudomonads. nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the heme d1 biosynthesis in Pa. denitrificans. The third gene, nirC, codes for a small cytochrome c of 9.3 kDa having high homology with cytochrome c55X of Ps. stutzeri ZoBell. The 4th gene, nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity. nirS mutants lack the cd1-type nitrite reductase while nirE, nirC and nirF mutants produce a small amount of cd1-type nitrite reductase, inactive due to the absence of heme d1. Upstream from the nirS gene the start of a gene was identified which has limited homology with nosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene and nirS.