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Solidarity between more and less vulnerable groups is fundamental to an effective public health response to a global pandemic. Yet in the case of COVID-19, a focus on deciding who can and who cannot be protected from harm has shaped the pandemic experience and continues to determine the post-pandemic trajectory of life with SARS-CoV-2. In this paper I discuss how this has affected our understanding and acceptance of solidarity.

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Bioethics traditionally focuses on establishing moral limits between different types of acts. However, boundaries are established by communities and individuals who differ in the constraints shaping their moral world. Phase boundaries, the sites of transition between two physical phases such as a liquid and a gas, provide a metaphor for 'drawing a line' in bioethics discourse. Phase boundaries occur where the physical constraints allow both phases to coexist in stable equilibrium. This relationship can also be considered in reverse, using the known position of the phase boundary to disclose the physical constraints. By analogy, instead of trying to locate the 'correct' moral boundary, the alternative perspective of 'reverse ethics' works from a commonly accepted boundary to examine the constraints of the moral world that are being used to establish it. Genetic interventions into the human body provide interesting examples of boundary establishment. In gene therapy, focusing on boundaries has resulted in a model of moral permissibility that ignores some alternative standpoints and increases the potential for conflict between them. Reverse ethics examines such conflicts in terms of the nature of moral worlds that have come into contact with each other, taking seriously the diversity of factors governing the location of a boundary, in ways that might help shift some entrenched lines of conflict.

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As the possibility of genetic intervention becomes more concrete, defining and regulating ethically permissible interventions must include a consideration of the implicit as well as explicit consequences. These include the moral implications of defining "enhancement" by reference to a standard of normality. Some authors have called into question the standard ethical concerns about genetic enhancement, but the distinction between enhancing and therapeutic interventions is still structured as relatively unproblematic. However, determining the boundary between therapy and enhancement will have feedback effects on the socially implemented definitions of what counts as normal in human embodiment. Positioning the interface between permissible and nonpermissible interventions at the same place as the boundaries between therapy and enhancement, and between normal and abnormal embodiment, (1) uses biology to justify a moral evaluation, (2) privileges the single standpoint of the genetically canonical person, and (3) enhances the dichotomy between "normal" and "not normal". Assuming that the limit of permissibility along the interventional continuum is coterminous with the definitions of enhancement and of normality, distracts from the work of uncovering the real grounds to setting limits to genetic manipulation.

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Enamel proteins are cleaved by proteinases soon after their secretion by ameloblasts. Intact proteins concentrate in the outer enamel at or near the growing tips of the enamel crystallites while cleavage products accumulate in the deeper enamel. In the transition and early maturation stages there is a dramatic increase in proteolytic activity. This activity, coupled with the diminished secretory and increased reabsorptive functions of ameloblasts, leads to a precipitous fall in the amount of enamel protein in the matrix. Recently we have cloned and characterized an mRNA encoding a tooth-specific serine proteinase designated enamel matrix serine proteinase 1 (EMSP1) [Simmer et al., JDR (1998) 77: 377]. EMSP1 can be detected in the inner enamel during the secretory stage and its activity increases sharply during the transition stage. Stage-specific Northern blot analysis demonstrates this increase is accompanied by a parallel increase in the amount EMSP1 mRNA. A 3-dimensional computer model of EMSP1, based upon the crystal structure of bovine trypsin, has been generated and analyzed. All six disulfide bridges as well as the active site are conserved. Changes in the peptide binding region and the specificity pocket suggest that interaction of the proteinase with protein substrates is altered, potentially causing a shift in substrate specificity. The calcium binding region of trypsin is thoroughly modified suggesting that the calcium independence of EMSP1 activity is due to an inability to bind calcium. The three potential N-linked glycosylation sites, N104, N139 and N184, are in surface accessible positions away from the active site.

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We have used reverse transcription followed by polymerase chain reaction amplification to investigate changes in expression of nerve growth factor (NGF) mRNA in immortalized hippocampal neurons after treatment with the glucocorticoids dexamethasone and corticosterone, the glucocorticoid antagonist RU38486, and the gonadal steroids progesterone and 17-beta oestradiol. We found that NGF mRNA levels rise after application of either dexamethasone or corticosterone, and that this rise is prevented by the antagonist. Thus, neurotrophin expression is modulated by the physiological glucocorticoid and is mediated by type II glucocorticoid receptors. Progesterone has no effect, while 17-beta oestradiol suppresses NGF mRNA in a postnatally-derived cell line but does not change levels in an embryonic line. An increase in neurotrophin expression is therefore not a general response to steroid hormone application, and may be a specific defence against the presence of metabolically endangering glucocorticoids.

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Nerve growth factor (NFF) is the prototypic member of a family of related neurotrophins (Nts). Although originally defined by its actions in the peripheral and central nervous systems, recent data indicate the presence of extensive interactions between NGF and the endocrine and immune systems steroid hormones are able to modulate the neurosomal expression NGF, while functional NGF receptors have been detected on cells of the immune system, and increased levels of NGF protein are found during the acute phase of diseases with a significant inflammatory component. These wider functions are likely to be of concern in any attempted therapeutic use of NGF.

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To see whether glucocorticoid hormones can influence the regulation of neurotrophin expression in hippocampal neurons, we have used reverse-transcription followed by polymerase chain reaction to investigate changes in the mRNA levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) in immortalized hippocampal neurons after dexamethasone application. Our results show that NGF mRNA levels rise in both embryonic and postnatal neurons, but with different time courses, while NT-3 levels rise in the embryonic but not in the postnatally derived cell line. Modulation of NT expression by glucocorticoids may therefore be developmentally regulated.

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Using a specific enzyme-linked immunosorbent assay (ELISA) for human nerve growth factor (NGF), serum levels in patients with systemic lupus erythematosus (SLE) were measured. We found a consistent increase in NGF levels in SLE patients compared with controls. A good correlation exists between serum NGF level and severity of clinical manifestation. We hypothesize that NGF might play a role in the pathogenesis of autoimmune disorders such as SLE.

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Defective retroviruses were used to investigate the effects of the oncogenes v-raf and v-mos on the behaviour of mammary epithelial cells. Cultures of the mammary epithelial cell lines #43 and NMuMG infected with either virus showed obvious areas of overgrowing cells, which were not seen when cells were infected with control viruses not containing an oncogene. Several clonal cell lines were derived from the areas of overgrowth on #43 cells. Most of these were tumorigenic on subcutaneous inoculation. In culture, several of the cloned lines showed marked morphological abnormalities, had altered rates of growth and saturation densities, and were able to clone in soft agar. Unexpectedly, one raf-containing and one mos-containing line grew more slowly in culture than did control or uninfected cells. Other lines showed only some of these changes. In the mos-containing lines the level of viral transcript corresponded approximately to the extent of transformation, whereas the raf-containing lines showed no such correlation.

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Mammalian aspartate transcarbamylase (ATCase) is part of a 243 kDa multidomain polypeptide, called CAD, that catalyzes the first three steps in de novo pyrimidine biosynthesis. The structural organization of the mammalian enzyme is very different from E. coli ATCase, a dodecameric, monofunctional molecule comprised of six copies of separate catalytic and regulatory chains. Nevertheless, sequence similarities and other properties suggested that the mammalian ATCase domain and the E. coli ATCase catalytic chain have the same tertiary fold. A model of mammalian ATCase was built using the X-ray coordinates of the E. coli catalytic chain as a tertiary template. Five small insertions and deletions could be readily accommodated in the model structure. Following energy minimization the RMS difference in the alpha carbon positions of the mammalian and bacterial proteins was 0.93 A. A comparison of the hydrophobic energies, surface accessibility index, and the distribution of hydrophilic and hydrophobic residues of the CAD ATCase structure with correctly and incorrectly folded proteins and with several X-ray structures supported the validity of the model. The mammalian ATCase domain associates to form a compact globular trimer, a prerequisite for catalysis since the active site is comprised of residues from adjacent subunits. Interactions between the clearly defined aspartate and carbamyl phosphate subdomains of the monomer were largely preserved while there was appreciable remodeling of the trimeric interfaces. Several clusters of basic residues are located on the upper surface of the domain which account in part for the elevated isoelectric point (pI = 9.4) and may represent contact regions with other more acidic domains within the chimeric polypeptide. A long interdomain linker connects the monomer at its upper surface to the remainder of the polypeptide. The configuration of active site residues is virtually identical in the mammalian and bacterial enzymes. While the CAD ATCase domain can undergo the local conformational changes that accompany catalysis in the E. coli enzyme, the high activity, closed conformation is probably more stable in the mammalian enzyme.

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Glutamine-dependent carbamoyl-phosphate synthetase (EC 6.3.5.5) catalyzes the first step in de novo pyrimidine biosynthesis. The mammalian enzyme is part of a 240-kDa multifunctional protein which also has the second (aspartate carbamoyltransferase, EC 2.1.3.2), and third (dihydroorotase, EC 3.5.2.3) activities of the pathway. Shigesada et al. (Shigesada, K., Stark, G.R., Maley, J.A., and Davidson, J.N. (1985) Mol. Cell Biol. 175, 1-7) produced a truncated cDNA clone from a Syrian hamster cell line that contained most of the coding region for this protein. We have completed sequencing this clone, known as pCAD142. The cDNA insert contained all of the coding region for the glutaminase (GLN) and carbamyl phosphate synthetase (CPS) domains but lacked a short amino-terminal segment. By comparing the primary structure of the mammalian chimera to monofunctional proteins we have identified the borders of the functional domains. The GLN domain is 21 kDa, close to the size of the functionally similar polypeptide products of the Escherichia coli pabA and hisH genes. The domain has the three regions of homology common to trpG-type glutamine amidotransferases, as well as a fourth region specific to the carbamyl phosphate synthetases. The CPSase domain is similar to other reported CPSases in size (120 kDa), primary structure (37-67% amino acid identity), and homology between its amino and carboxyl halves. Analysis of the nucleotide and amino acid sequence identities among the various carbamyl phosphate synthetases suggests that the gene fusion which joined the GLN and CPS domains was an early event in the evolution of eukaryotic organisms and that the Saccharomyces cerevisiae enzyme consisting of separate subunits arose by defusion from an ancestral multifunctional protein.

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Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.

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Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities. We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases. These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8. While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character. The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis. Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension. Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A. The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold.

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