Role and Function of the Clinical Tutor in Mental Health Nursing in Ireland.

The clinical tutor (CT) in mental health nursing is a role aimed at supporting the learning needs of mental health nursing students undertaking a 12-month post-registration programme. This paper aims to examine the role of the clinical tutor in mental health nursing in Ireland by describing the experience of nursing students and key service stakeholders. A qualitative descriptive design was employed using focus group discussions and semi-structured interviews. Two focus groups were conducted with 14 nursing students in the final week of their one-year programme. Semi-structured interviews were undertaken with seven service stakeholders and service leaders. Participants reported positive experiences of working with the clinical tutor and valued the role in terms of educational and pastoral support. Participants suggested the role strengthened the link between theory and practice and enhanced the relationship between the higher education institute and clinical sites. However, a lack of clarity existed in terms of role description. Participants suggested the CT role enhanced the link between the university and clinical areas providing benefits to both student and service stakeholders. Implementing similar roles may benefit post-registration mental health nursing students in other jurisdictions. Further investigation on how the role operates from the perspective of those in the post may provide more clarity and enhance the development of such roles in the future.

A national house-staff audit of medical prophylaxis in medical patients for the PREVENTion of Venous ThromboEmbolism (PREVENT-VTE).

We established a national audit to assess the thromboprophylaxis rate for venous thromoembolism (VTE) in at risk medical patients in acute hospitals in the Republic of Ireland and to determine whether the use of stickers to alert physicians regarding thromboprophylaxis would double the rate prophylaxis in a follow-up audit. 651 acute medical admission patients in the first audit and 524 in the second re-audit were recruited. The mean age was 66.5 yrs with similar numbers of male and female patients and 265 (22.6%) patients were active smokers. The first and second audits identified 549 (84%) and 487 (93%) of patients at-risk for VTE respectively. Of the at-risk patients, 163 (29.7%) and 132 (27.1%) received LMWH in the first and second audit respectively. Mechanical thromboprophylaxis was instigated in 75 (13.6%) patients in the first and 86 (17.7%) patients in the second audit. The placement of stickers in patient charts didn't produce a significant increase in the number of at risk patients treated in the second audit. There is unacceptably low adherence to the ACCP guidelines in Ireland and more complex intervention than chart reminders are required to improve compliance.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptors, and the soluble receptor osteoprotegerin in human gestational membranes and amniotic fluid during pregnancy and labor at term and preterm.

We have studied TNF-related apoptosis-inducing ligand (TRAIL) and its membrane-bound (R1-R4) and soluble receptors [osteoprotegerin (OPG)] in gestational membranes to assess their significance in preterm parturition and premature rupture of membranes (PROM). TRAIL was detected by ELISA in extracts of term choriodecidual (but not amnion) tissues and explant-conditioned media. Concentrations of OPG (determined using ELISA) in gestational membranes were 20- to 50-fold greater than those of TRAIL. Median OPG concentrations in amniotic fluid (AF) at 15-17 wk gestation were similar to those at term before and during labor, whereas levels in pregnancies sampled preterm were significantly elevated. OPG levels in AF from women with preterm PROM were similar to those from women in preterm labor. In contrast, in pooled AF samples (n = 23-33), TRAIL concentrations at term with and without labor were elevated compared with samples from preterm deliveries. TRAIL-R3 and -R4 decoy receptors were detected in term amnion and choriodecidual extracts by immunoblotting and were localized by immunohistochemistry to amnion epithelial cells and chorionic trophoblasts. TRAIL (100 ng/ml) had little or no effect on amnion or choriodecidual cell viability or apoptosis, although these tissues responded to TNF-alpha with increased prostaglandin E(2) production. Our findings suggest that OPG is abundant in gestational membranes and, in concert with TRAIL decoy receptors, may protect resident cells of the fetal membranes against the proapoptotic effects of TRAIL and other related ligands during pregnancy.

Three families with autosomal dominant nephrogenic diabetes insipidus caused by aquaporin-2 mutations in the C-terminus.

The vasopressin-regulated water channel aquaporin-2 (AQP2) is known to tetramerize in the apical membrane of the renal tubular cells and contributes to urine concentration. We identified three novel mutations, each in a single allele of exon 4 of the AQP2 gene, in three families showing autosomal dominant nephrogenic diabetes insipidus (NDI). These mutations were found in the C-terminus of AQP2: a deletion of G at nucleotide 721 (721 delG), a deletion of 10 nucleotides starting at nucleotide 763 (763-772del), and a deletion of 7 nucleotides starting at nucleotide 812 (812-818del). The wild-type AQP2 is predicted to be a 271-amino acid protein, whereas these mutant genes are predicted to encode proteins that are 330-333 amino acids in length, because of the frameshift mutations. Interestingly, these three mutant AQP2s shared the same C-terminal tail of 61 amino acids. In Xenopus oocytes injected with mutant AQP2 cRNAs, the osmotic water permeability (Pf) was much smaller than that of oocytes with the AQP2 wild-type (14%-17%). Immunoblot analysis of the lysates of the oocytes expressing the mutant AQP2s detected a band at 34 kD, whereas the immunoblot of the plasma-membrane fractions of the oocytes and immunocytochemistry failed to show a significant surface expression, suggesting a defect in trafficking of these mutant proteins. Furthermore, coinjection of wild-type cRNAs with mutant cRNAs markedly decreased the oocyte Pf in parallel with the surface expression of the wild-type AQP2. Immunoprecipitation with antibodies against wild-type and mutant AQP2 indicated the formation of mixed oligomers composed of wild-type and mutant AQP2 monomers. Our results suggest that the trafficking of mutant AQP2 is impaired because of elongation of the C-terminal tail, and the dominant-negative effect is attributed to oligomerization of the wild-type and mutant AQP2s. Segregation of the mutations in the C-terminus of AQP2 with dominant-type NDI underlies the importance of this domain in the intracellular trafficking of AQP2.

Association of calnexin with wild type and mutant AVPR2 that causes nephrogenic diabetes insipidus.

Over 155 mutations within the V2 vasopressin receptor (AVPR2) gene are responsible for nephrogenic diabetes insipidus (NDI). The expression and subcellular distribution of four of these was investigated in transfected cells. These include a point mutation in the seventh transmembrane domain (S315R), a frameshift mutation in the third intracellular loop (804delG), and two nonsense mutations that code for AVPR2 truncated within the first cytoplasmic loop (W71X) and in the proximal portion of the carboxyl tail (R337X). RT-PCR revealed that mRNA was produced for all mutant receptor constructs. However, no receptor protein, as assessed by Western blot analysis, was detected for 804delG. The S315R was properly processed through the Golgi and targeted to the plasma membrane but lacked any detectable AVP binding or signaling. Thus, this mutation induces a conformational change that is compatible with endoplasmic reticulum (ER) export but dramatically affects hormone recognition. In contrast, the W71X and R337X AVPR2 were retained inside the cell as determined by immunofluorescence. Confocal microscopy revealed that they were both retained in the ER. To determine if calnexin could be involved, its interaction with the AVPR2 was assessed. Sequential coimmunoprecipitation demonstrated that calnexin associated with the precursor forms of both wild-type (WT) and mutant receptors in agreement with its general role in protein folding. Moreover, its association with the ER-retained R337X mutant was found to be longer than with the WT receptor suggesting that this molecular chaperone also plays a role in quality control and ER retention of misfolded G protein-coupled receptors.

Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants.

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.

Coupling semiconductor nanocrystals to a fused-silica microsphere: a quantum-dot microcavity with extremely high Q factors.

We demonstrate a quantum-dot microcavity by coupling core-shell semiconductor nanocrystals to a fused-silica microsphere. We show that the composite microcavity can feature Q factors of the order of 10(8), providing a model system for investigating cavity QED and microlasers at the level of single quantum dots.

Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.

Mutations in the aquaporin-2 (AQP2) water channel gene cause autosomal recessive nephrogenic diabetes insipidus (NDI). Here we report the first patient with an autosomal dominant form of NDI, which is caused by a G866A transition in the AQP2 gene of one allele, resulting in a E258K substitution in the C-tail of AQP2. To define the molecular cause of NDI in this patient, AQP2-E258K was studied in Xenopus oocytes. In contrast to wild-type AQP2, AQP2-E258K conferred a small increase in water permeability, caused by a reduced expression at the plasma membrane. Coexpression of wild-type AQP2 with AQP2-E258K, but not with an AQP2 mutant in recessive NDI (AQP2-R187C), revealed a dominant-negative effect on the water permeability conferred by wild-type AQP2. The physiologically important phosphorylation of S256 by protein kinase A was not affected by the E258K mutation. Immunoblot and microscopic analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not retarded in the endoplasmic reticulum, but retained in the Golgi compartment. Since AQPs are thought to tetramerize, the retention of AQP2-E258K together with wild-type AQP2 in mixed tetramers in the Golgi compartment is a likely explanation for the dominant inheritance of NDI in this patient.

Quantitative study of the resolving power of arrays of carbon black-polymer composites in various vapor-sensing tasks.

A statistical metric, based on the magnitude and standard deviations along linear projections of clustered array response data, was utilized to facilitate an evaluation of the performance of detector arrays in various vapor classification tasks. This approach allowed quantification of the ability of a 14-element array of carbon black-insulating polymer composite chemiresistors to distinguish between members of a set of 19 solvent vapors, some of which vary widely in chemical properties (e.g., methanol and benzene) and others of which are very similar (e.g., n-pentane and n-heptane). The data also facilitated evaluation of questions such as the optimal number of detectors required for a specific task, whether improved performance is obtained by increasing the number of detectors in a detector array, and how to assess statistically the diversity of a collection of detectors in order to understand more fully which properties are underrepresented in a particular set of array elements. In addition, the resolving power of arrays of carbon black-polymer composites was compared to the resolving power of specific collections of bulk conducting organic polymer or tin oxide detector arrays in a common set of vapor classification tasks.

Nature and recurrence of AVPR2 mutations in X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine-vaso-pressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. We analyzed 31 independent NDI families to determine the nature and recurrence of AVPR2 mutations. Twenty-one new putative disease-causing mutations were identified: 113delCT, 253del35, 255de19, 274insG, V88M, R106C, 402delCT, C112R, Y124X, S126F, W164S, S167L, 684delTA, 804insG, W284X, A285P, W293X, R337X, and three large deletions or gene rearrangements. Five other mutations--R113W, Y128S, R137H, R181C, and R202C--that previously had been reported in other families were detected. There was evidence for recurrent mutation for four mutations (R113W, R137H, S167L, and R337X). Eight de novo mutation events were detected (274insG, R106C, Y128S, 167L [twice], R202C, 684delTA, and R337X). The origins were maternal (one), grandmaternal (one), and grandpaternal (six). In the 31 NDI families and 6 families previously reported by us, there is evidence both for mutation hot spots for nucleotide substitutions and for small deletions and insertions. More than half (58%) of the nucleotide substitutions in 26 families could be a consequence of 5-methyl-cytosine deamination at a CpG dinucleotide. Most of the small deletions and insertions could be attributed to slipped mispairing during DNA replication.

Mutations in the vasopressin V2 receptor gene in families with nephrogenic diabetes insipidus and functional expression of the Q-2 mutant.

Nephrogenic diabetes insipidus (NDI) is characterized by a resistance of the kidney towards arginine vasopressin (AVP). Following molecular cloning of the vasopressin V2 receptor, we identified different mutations in the V2 receptor gene in families with X-linked NDI, which segregated with the disease. The Hopewell mutation (W71X) causes the disease in the largest North American NDI pedigree, with most of its members residing on Nova Scotia. Different mutations were found in three families from the Quebec area (Q-2: R137H, Q-3: R113W, Q-5: 804delG) and in the large Cannon kindred residing in Utah (L312X). In an Iranian family (O-1), another mutation was detected (A132D). Three of the six mutations (Hopewell, Cannon, Q-5) are predicted to cause the expression of a truncated V2 receptor and are therefore unlikely to function. The functional consequences of missense mutations (Q-2, Q-3, O-1) are less obvious. We therefore introduced the Q-2 mutation into wild-type cDNA. When expressed in COS.M6 or Ltk cells, the Q-2 mutant bound AVP with normal affinity. However, cells expressing the Q-2 mutant failed to respond to AVP with an increase in adenylyl cyclase activity. Thus the Q-2 mutant is unable to interact with or to activate the stimulatory G-protein Gs. The present data indicate that X-linked NDI is frequently attributable to a mutation in the V2 receptor gene. In addition, the data prove biochemically that the Q-2 mutation is the cause of NDI in the Q-2 family.

Predicting operative mortality after coronary artery bypass surgery in males.

The general practitioner often requires a simple and reliable method of determining the potential risks of surgical intervention. We derived and tested a simple clinical scoring system for the preoperative prediction of 30-day mortality after coronary artery bypass surgery. From a national register of all open heart operations in the Republic of Ireland 1984-1989, we identified 4276 male patients who had primary isolated non-emergency coronary artery bypass surgery. Using logistic regression, we derived a clinical scoring system to predict operative (30-day) mortality in patients operated on between 1984 and 1987. We then prospectively evaluated the score on patients seen over the next two years. Variables identified for our scoring system were age, recent myocardial infarction, left ventricular failure, extensive distal coronary artery disease and body surface area. Five risk categories were defined; mortality in the high-risk group was 9.7-fold (95% CI: 4.6-20.7) greater than in the low-risk group. When tested on new patients, the relative mortality between the two risk groups was 15.2 (4.6-50.5). The observed and predicted mortalities in each risk group showed close agreement. This clinical scoring system, easily used by a general practitioner, can predict operative mortality in males for whom primary isolated coronary artery bypass surgery is contemplated.

A regulatory element in the beta 2-microglobulin promoter identified by in vivo footprinting.

Expression of the beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I genes is coordinately regulated. By ligation-mediated polymerase chain reaction, we have analyzed in vivo factor binding to the promoter region of the murine beta 2-m gene. In adult spleen, in which beta 2-m is expressed, strong protection was found in three elements. Two of these elements, the beta 2-m NF-kappa B binding site and the interferon consensus sequence, are homologous to the regulatory elements of the MHC class I genes and were also found to be protected in spleen. A third protected element, PAM, identified in this work, is unique to the beta 2-m gene. None of the elements showed protection in brain tissue, in which neither the beta 2-m nor the MHC class I gene is expressed. In vivo footprinting was also performed with F9 embryonal carcinoma cells, in which expression of the beta 2-m and MHC class I genes is induced at a low level only upon stimulation with retinoic acid (RA). No in vivo protection was detected before and after RA treatment of F9 cells, indicating that RA induction of beta 2-m (and MHC class I) expression occurs without detectable in vivo factor occupancy, whereas EL4 T lymphocytes expressing beta 2-m at a high level exhibited strong protection similar to that in spleen. Despite the lack of in vivo occupancy, the nuclear factors specific for each of the three elements were present in brain tissue and F9 cells as well as in spleen tissue and EL4 cells. We show that PAM, an element identified by its in vivo protection, binds nuclear factors ranging from 40 to 50 kDa in size and is capable of enhancing transcription of a reporter in F9 and other cells. Taken together, these results indicate that in vivo factor occupancy for the beta 2-m and MHC class I promoters is coordinated and occurs through a mechanism other than mere expression of relevant factors.

X-linked nephrogenic diabetes insipidus mutations in North America and the Hopewell hypothesis.

In X-linked nephrogenic diabetes insipidus (NDI) the urine of male patients is not concentrated after the administration of the antidiuretic hormone arginine-vasopressin. This disease is due to mutations in the V2 receptor gene that maps to chromosome region Xq28. In 1969, Bode and Crawford suggested that most NDI patients in North America shared common ancestors of Ulster Scot immigrants who arrived in Halifax in 1761 on the ship Hopewell. A link between this family and a large Utah kindred was also suggested. DNA was obtained from 17 affected male patients from the "Hopewell" kindred and from four additional families from Nova Scotia and New Brunswick who shared the same Xq28 NDI haplotype. The Utah kindred and two families (Q2, Q3) from Quebec were also studied. The "Hopewell" mutation, W71X, is a single base substitution (G-->A) that changes codon 71 from TGG (tryptophan) to TGA (stop). The W71X mutation was found in affected members of the Hopewell and of the four satellite families. The W71X mutation is the cause of X-linked NDI for the largest number of related male patients living in North America. Other families (Utah, Q2 and Q3) that are historically and ethnically unrelated bear other mutations in the V2 receptor gene.

Regulation of MHC class I and beta 2-microglobulin gene expression in human neuronal cells. Factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes.

MHC class I molecules are coexpressed with beta 2-microglobulin (beta 2-M) on many somatic cells. However, these proteins are normally not present on cells of the central nervous system (CNS). Cells derived from human neuroblastomas were used as a model for investigating the molecular basis for the paucity of MHC class I and beta 2-M gene expression in neural cells and for the induction of these genes by two cytokines, IFN-gamma, and TNF-alpha. These cytokines independently increased MHC class I and beta 2-M cell surface expression on the neuroblastoma cell lines. IFN-gamma or TNF-alpha also increased MHC class I and beta 2-M steady-state RNA levels and the expression of MHC class I and beta 2-M CAT reporter constructs transiently transfected into the neuroblastoma cell lines, indicating that the cytokines acted by increasing the transcription of these genes. MHC class I and beta 2-M genes share two conserved regulatory elements, an NF kappa B-like site and the IFN consensus sequence, that act as a constitutive enhancer and an IFN-responsive element, respectively. Low MHC class I and beta 2-M gene expression in these cells was accounted for by undetectable to low factor binding activity specific for the above regulatory elements of these genes. TNF-alpha increased factor binding activity specific for the NF kappa B-like elements and IFN-gamma increased factor binding activity specific for the IFN consensus sequence elements of the MHC class I and beta 2-M genes, but not vice versa. Taken together, our results indicated that IFN-gamma and TNF-alpha increased MHC class I and beta 2-M gene expression in the neuroblastoma cell lines by inducing factor binding to the regulatory elements present in both genes.