Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration.

PURPOSE: Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. METHODS: An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. RESULTS: Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal-corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in pterygium (several small leucine-rich proteoglycan family members) or were expressed at considerably lower levels (ALDH3A1 and decorin). CONCLUSIONS: The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth.

The P23T cataract mutation causes loss of solubility of folded gammaD-crystallin.

Mutations in the human gammaD-crystallin gene have been linked to several types of congenital cataracts. In particular, the Pro23 to Thr (P23T) mutation of human gammaD crystallin has been linked to cerulean, lamellar, coralliform, and fasciculiform congenital cataracts. We have expressed and purified wild-type human gammaD, P23T, and the Pro23 to Ser23 (P23S) mutant. Our measurements show that P23T is significantly less soluble than wild-type human gammaD, with P23S having an intermediate solubility. Using synchrotron radiation circular dichroism spectroscopy, we have determined that the P23T mutant has a slightly increased content of beta-sheet, which may be attributed to the extension of an edge beta-strand due to the substitution of Pro23 with a residue able to form hydrogen bonds. Neither of the point mutations appears to have reduced the thermal stability of the protein significantly, nor its resistance to guanidine hydrochloride-induced unfolding. These results suggest that insolubility, rather than loss of stability, is the primary basis for P23T congenital cataracts.

Crystal structure of eta-crystallin: adaptation of a class 1 aldehyde dehydrogenase for a new role in the eye lens.

Eta-crystallin is a retinal dehydrogenase that has acquired a role as a structural protein in the eye lens of elephant shrews, members of an ancient order of mammals. While it retains some activity toward retinal, which is oxidized to retinoic acid, the protein has acquired a number of specific sequence changes that have presumably been selected to enhance the lens role. The crystal structure of eta-crystallin, in common with class 1 and 2 ALDHs, is a dimer of dimers. It has a better-defined NAD binding site than those of related mammalian ALDH1 enzymes with the cofactor bound in the "hydride transfer" position in all four monomers with small differences about the dimer dyads. Although the active site is well conserved, the substrate-binding site is larger in eta-crystallin, and there are some mutations to the substrate access tunnel that might affect binding or release of substrate and product. It is possible that eta-crystallin has lost flexibility to improve its role in the lens. Enhanced binding of cofactor could enable it to act as a UV/blue light filter in the lens, improving visual acuity. The structure not only gives a view of a "natural mutant" of ALDH1 illustrating the adaptive conflict that can arise in multifunctional proteins, but also provides a well-ordered NAD binding site structure for this class of enzymes with important roles in development and health.

A temperature-sensitive mutation of Crygs in the murine Opj cataract.

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for gammaS-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/+. Lens histology shows that whereas fiber cell morphology in Opj/+ mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of gammaS-crystallin which would be consistent with ideas that members of the betagamma-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.

Modernisation, the NHS Plan and healthy communities.

This paper reviews the NHS Plan from the perspective of the Government's wider programme of "modernising" public services. Although broadly focussed, particularly highlights older people. Two dimensions of modernisation are identified. The NHS Plan is seen to be patient-cited--rather than citizen-centred. Argues further, that, if the economic, social and environmental causes of ill health are to be addressed more generally and if citizens are to be enabled to live in healthy, sustainable communities, planning for health services should logically be subordinate to planning for health. Health improvement plans should, therefore, be integrated within the wider community strategies for which local authorities are to have lead responsibility. Similarly, as ill health is recognised to be an important aspect of poverty, inequality and social exclusion, there is a strong case for the integration of the regional offices of the NHSE within the wider structure of regional governance. Finally, the personal social services should ensure that the values of social work and social care are not displaced by medical and nursing models which, historically, have shown little understanding of community development processes.

Movement and change: independent sector domiciliary care providers between 1995 and 1999.

Promoting the development of a flourishing independent sector alongside good quality public services was a key objective of the community care reforms of the last decade. This paper charts some of the ways the independent domiciliary care sector is changing, as local authorities shift the balance of their provision toward independent sector providers and away from a reliance on in-house services. Two surveys of independent domiciliary care providers were carried out in 1995 and 1999. The aims of the studies were to describe the main features of provider organisations, such as size of business, client group and funding sources; to examine the nature of provider motivations and their past and future plans; to consider how local authorities manage the supply side of social care markets; and to examine the effects on providers of the development of the mixed economy. The first survey in 1995 was conducted in eight local authority areas, which by 1999 had increased to 11 because of the creation of three new unitary authorities. The findings are based on 261 postal surveys together with 111 interviews between the two studies. The research illustrates a domiciliary care market that is still relatively young with many small but growing businesses. There are considerable differences in the split between in-house and independent sector services in individual authorities and a common perception among independent providers that in-house services receive favourable treatment and conditions. Spot or call-off contracts continue to be the most common form of contract although there are moves toward greater levels of guaranteed service and more sophisticated patterns of contracting arrangements. There remains an ongoing need to share information between local authorities and independent providers so that good working relationships can develop with proven and competent providers.

Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23.

Genes causing nonsyndromic autosomal recessive deafness (DFNB12) and deafness associated with retinitis pigmentosa and vestibular dysfunction (USH1D) were previously mapped to overlapping regions of chromosome 10q21-q22. Seven highly consanguineous families segregating nonsyndromic autosomal recessive deafness were analyzed to refine the DFNB12 locus. In a single family, a critical region was defined between D10S1694 and D10S1737, approximately 0.55 cM apart. Eighteen candidate genes in the region were sequenced. Mutations in a novel cadherin-like gene, CDH23, were found both in families with DFNB12 and in families with USH1D. Six missense mutations were found in five families with DFNB12, and two nonsense and two frameshift mutations were found in four families with USH1D. A northern blot analysis of CDH23 showed a 9.5-kb transcript expressed primarily in the retina. CDH23 is also expressed in the cochlea, as is demonstrated by polymerase chain reaction amplification from cochlear cDNA.

Functional impairment of lens aquaporin in two families with dominantly inherited cataracts.

Opacities in the crystalline lens of eye appear with high frequency in the general population. Dominantly inherited cataracts with differing clinical features were found in two families carrying different point mutations in the gene encoding lens water channel protein AQP0 (major intrinsic protein, MIP). Families with E134G have a uni-lamellar cataract which is stable after birth, whereas families with T138R have multi-focal opacities which increase throughout life. To establish pathophysiological relevance of cataract formation, the Xenopus laevis oocyte expression system was employed to evaluate functional defects in the mutant proteins, E134G and T138R. Both substitutions cause loss of membrane water channel activity due to impaired trafficking of the mutant proteins to the oocyte plasma membrane. Although missense mutations in AQP1 and AQP2 proteins are known to result in recessive traits in vivo and in vitro, when E134G or T138R are co-expressed with wild-type AQP0 protein, the mutant proteins exhibit dominant negative behaviour. To our knowledge, these studies represent the first in vitro demonstration of functionally defective AQP0 protein from humans with congenital cataracts. Moreover, these observations predict that less severe defects in the AQP0 protein may contribute to lens opacity in patients with common, less fulminant forms of cataracts.

Cloning, genomic organization, and characterization of a human cholinephosphotransferase.

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.

Cloning, modeling, and chromosomal localization for a small leucine-rich repeat proteoglycan (SLRP) family member expressed in human eye.

PURPOSE: To examine a highly abundant novel transcript from human iris. METHODS: Expressed sequence tag (EST) analysis of an adult human iris cDNA library revealed an abundant (>0.7%) transcript for a novel member of the small leucine-rich proteoglycan (SLRP) family. Other 3' ESTs from retina were also detected in dbEST. The structure of the leucine-rich repeat (LRR) domain was investigated by molecular modeling. Antisera were raised against a specific peptide and used in western blots of human and rat eye tissues. RESULTS: From its prevalence in the eye and its superfamily relationships, this SLRP protein has been given the names oculoglycan or opticin (Optc). Sequence analysis suggests that Optc has a signal peptide and two structural domains, the larger of which is the LRR domain. Modeling of the LRR domain reveals structural variability in the repeat motifs, forming potential interaction sites for binding partners. Antiserum to a specific peptide detected a protein of approximately 48 kDa, in human iris, ciliary body and retina while the major protein detected in rat ocular tissues was 37 kDa in size. This may reflect a species difference in post-translational modification. Radiation hybrid mapping shows that the gene for OPTC is located on chromosome 1q31, close to the inherited eye diseases ARMD1 and AXPC1. CONCLUSIONS: Optc is a newly identified SLRP family member, which appears to have eye-preferred expression. Molecular modeling reveals local deviations from the familiar LRR structure, which are candidates for specific interaction sites. Western blotting with a specific peptide antibody detects Optc in iris, ciliary body and retina in the human eye and suggests that the protein is post-translationally modified. In rat, the antibody detects Optc in several eye tissues and in brain but the protein appears to have undergone much less modification, suggesting that this is not essential for all aspects of function. Considering its eye-preferred expression, the OPTC gene has the potential for involvement in inherited eye disease. Indeed, it maps close to at least two disease loci for which no gene has so far been identified.

The human gene for gammaS-crystallin: alternative transcripts and expressed sequences from the first intron.

PURPOSE: gammaS-crystallins are major components of adult vertebrate lenses. Here we examine the population of gammaS transcripts in adult human lens and the structure of the human CRYGS genes. METHODS: Adult lens human transcripts were obtained from NEIBANK, an Expressed Sequence Tag (EST) analysis of human eye tissues. The human CRYGS gene was isolated as a PAC clone and sequenced by direct and PCR-based methods. RESULTS: As judged by EST frequency, gammaS is one of the most abundant transcripts in the adult human lens, ranking just behind betaB2-, alphaB- and alphaA-crystallins. EST analysis reveals two transcript sizes resulting from alternative AATAAA and ATTAAA polyadenylation signals. In addition, one cDNA clone was found to contain a novel insert sequence that disrupted the open reading frame. Gene sequencing confirmed that this insert comes from intron 1 and is part of a sequence corresponding to a cluster of unidentified human transcripts in dbEST. Human and mouse gammaS gene proximal promoter sequences were compared and showed a high degree of evolutionary conservation, including consensus binding sites for transcription factors of the maf and SOX families. CONCLUSIONS: The human CRYGS gene can give rise to at least two transcripts through alternative polyadenylation. A minor transcript results from alternative splicing into sequences in intron 1. These sequences form part of a transcription unit (Mys) expressed in several non-lens tissues. The identity and function Mys of is not yet known, however, the cryptic splicing of CRYGS could produce a defective protein product, with potentially deleterious results for the adult human lens.

NHS plan. Exclusive offer?

Little consideration has been given to the proposals for social care in the NHS plan. The emphasis on social services' role in unblocking hospital beds ignores their responsibilities to the whole community. The plan is ambiguous on health improvement. There is a risk of health promotion being sidelined.

Home care and the reshaping of acute hospitals in England. An overview of problems and possibilities.

A changing boundary between hospital and home-care services over two decades has taken place enabling people to live in their own homes wherever possible, enabling "choice of independence". Against this background, five principal issues are raised regarding how hospital services have been reshaped over that time and how the pattern of service developments outside the hospital has altered over the same period.

Inter-agency services in England and The Netherlands. A comparative study of integrated care development and delivery.

In England and the Netherlands there is much comparable experience in developing and delivering integrated services, provided by different health care agencies to people with multiple care demands. The achievement of integrated care provision in such cases appears to be very difficult and laborious in both countries. This article may be considered a first step in exploring the reasons for this and in developing a framework that is not context specific, as a contribution to a more generally applicable analysis of obstacles to integration and the means for overcoming them. After analysing the English and Dutch health and social care systems and their development in recent decades, we conclude that basically there are clear system similarities which are hindering the integration of services, for instance the predominant complexity of the system with a lot of stakeholders having different roles, tasks, interests and power positions. We have identified common mechanisms that play a dominant role in both systems; not only the social, economic and political context, but also the local context, the legal context and funding streams. Other relevant factors are the procedural and structural arrangements at different system levels and the collaborative culture and tradition. The way these mechanisms work in practice, however, is different for England and the Netherlands, due to system differences. In the Netherlands for instance there is a clear emphasis on bargaining in the context of non-hierarchical structured networks, whilst in England hierarchies and the interplay between hierarchies, markets and networks play a more dominant role. In spite of the differences and problems in both countries we have found a similar recognition of interdependence and willingness to pursue integration of services for multi-problem patients.

Expression pattern of macrophage migration inhibitory factor during embryogenesis.

Although macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine that inhibits the migration of macrophages, its ubiquitous expression suggests it may have a role beyond the immune system. Here we report a detailed characterization of MIF expression during mouse embryogenesis. The MIF expression pattern was found to parallel tissues specification and organogenesis.

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications.

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.

Nursing acute psychiatric patients: a quantitative and qualitative study.

Political and managerial attention has focused on the consequences of the failure of community services to provide effective care to a small number of people with severe mental illness. However, the nature and value of care in hospitals have received less scrutiny. This paper addresses deficiencies in our knowledge about nursing care in acute psychiatric wards. It reports the findings from a recently completed study for the United Kingdom Department of Health. Four key developments are identified which, together, pose significant problems for nursing in acute wards: the increasingly diverse patient mix in wards; the volume of administrative duties performed by nurses; the weakness of multidisciplinary team working; and inappropriate education. In conclusion, the challenges for managers and clinicians responsible for local policy and practice and, by extension, those at the centre responsible for such services, are examined.

Progressive juvenile-onset punctate cataracts caused by mutation of the gammaD-crystallin gene.

Cataracts are a significant public health problem. Here, we describe the genetic alteration responsible for a progressive form of cataract, segregating as an autosomal dominant trait in a three-generation pedigree. Unlike most autosomal dominant cataracts, these are not clinically apparent at birth but are initially observed in the first year or two of life. The opacification evolves relatively slowly, generally necessitating removal of the lens in childhood or early adolescence. A genome-wide search in our kindred revealed linkage at 2q33-35 where the gamma-crystallin gene cluster resides. A single base alteration resulting in an Arg- 14 --> Cys (R14C) substitution in gammaD-crystallin was subsequently identified. Protein modeling suggests that the effect of this mutation is a subtle one, affecting the surface properties of the crystallin molecule rather than its tertiary structure, consistent with the fact that the patients' lenses are normal at birth. This is the first gene defect shown to be responsible for a noncongenital progressive cataract, and studying the defective protein should teach us more about the mechanisms underlying cataract formation.