Prevalence and predictors of advance directives in Australia.

BACKGROUND: Advance care planning is regarded as integral to better patient outcomes, yet little is known about the prevalence of advance directives (AD) in Australia. AIM: To determine the prevalence of AD in the Australian population. METHODS: A national telephone survey about estate and advance planning. Sample was stratified by age (18-45 and >45 years) and quota sampling occurred based on population size in each state and territory. RESULTS: Fourteen per cent of the Australian population has an AD. There is state variation with people from South Australia and Queensland more likely to have an AD than people from other states. Will making and particularly completion of a financial enduring power of attorney are associated with higher rates of AD completion. Standard demographic variables were of limited use in predicting whether a person would have an AD. CONCLUSIONS: Despite efforts to improve uptake of advance care planning (including AD), barriers remain. One likely trigger for completing an AD and advance care planning is undertaking a wider future planning process (e.g. making a will or financial enduring power of attorney). This presents opportunities to increase advance care planning, but steps are needed to ensure that planning, which occurs outside the health system, is sufficiently informed and supported by health information so that it is useful in the clinical setting. Variations by state could also suggest that redesign of regulatory frameworks (such as a user-friendly and well-publicised form backed by statute) may help improve uptake of AD.

Nuclear exchange of the U1 and U2 snRNP-specific proteins.

The snRNP particles include a set of common core snRNP proteins and snRNP specific proteins. In rodent cells the common core proteins are the B, D, D', E, F and G proteins in a suggested stoichiometry of B2D'2D2EFG. The additional U1- and U2-specific proteins are the 70-kD, A and C proteins and the A' and B" proteins, respectively. Previous cell fractionation and kinetic analysis demonstrated the snRNP core proteins are stored in the cytoplasm in large partially assembled snRNA-free intermediates that assemble with newly synthesized snRNAs during their transient appearance in the cytoplasm (Sauterer, R. A., R. J. Feeney, and G. W. Zieve. 1988. Exp. Cell Res. 176:344-359). This report investigates the assembly and intracellular distribution of the U1 and U2 snRNP-specific proteins. Cell enucleation and aqueous cell fractionation are used to prepare nuclear and cytoplasmic fractions and the U1- and U2-specific proteins are identified by isotopic labeling and immunoprecipitation or by immunoblotting with specific autoimmune antisera. The A, C, and A' proteins are found both assembled into mature nuclear snRNP particles and in unassembled pools in the nucleus that exchange with the assembled snRNP particles. The unassembled proteins leak from isolated nuclei prepared by detergent extraction. The unassembled A' protein sediments at 4S-6S in structures that may be multimers. The 70-kD and B" proteins are fully assembled with snRNP particles which do not leak from isolated nuclei. The kinetic studies suggest that the B" protein assembles with the U2 particle in the cytoplasm before it enters the nucleus.

A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework.

Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.

Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein.

The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera.

Synthesis of immobilized flavin derivatives and their use in purification of chicken egg-white ovoflavoprotein.

Affinity adsorbents for flavoproteins were prepared by the covalent attachment of polyacrylamide and agarose to flavin derivatives linked through position N(3) of the flavin nucleus. 3-Carboxymethyl-FMN covalently linked to aminoalkyl substituted agarose was successfully used for the separation and purification of the apo form of the ovoflavoprotein from chicken egg white. High yields and high purities were achieved by two different isolation procedures employing the affinity adsorbent.

Interaction of oxidized chicken ovotransferrin with chicken embryo red blood cells.

Iron-saturated chicken ovotransferrin was chemically oxidized with NaIO4, converting 50% of its methionine residues to their sulfoxide derivatives while maintaining 95% of its iron-binding activity. The oxidized chicken ovotransferrin was able to deliver iron to the chicken embryo red blood cell for heme synthesis. From competition experiments, oxidized diferric chicken ovotransferrin was estimated to be approx. 65% as efficient as unmodified diferric chicken ovotransferrin at competing with diferric (55Fe2) chicken ovotransferrin for the iron-donating sites of the chicken embryo red blood cells. The presence of apo chicken ovotransferrin preparations (native or oxidized) in the incubation medium had little effect on the rate of iron incorporation into heme from diferric chicken ovotransferrin. The effect of modifying the periodate-susceptible methionine residues in chicken ovotransferrin was small but significant. These methionine residues do not appear critical for the interaction of chicken ovotransferrin with the chicken embryo red blood cell receptors, the incorporation of chicken ovotransferrin into the cell, or the release of iron from chicken ovotransferrin for heme synthesis.

Antifreeze proteins differentially affect model membranes during freezing.

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.

Newly synthesized small nuclear RNAs appear transiently in the cytoplasm.

Newly synthesized small nuclear RNA (snRNA) species U1 and U2 are easily identified in cytoplasmic fractions prepared by standard aqueous cell fractionation. However, because the mature stable snRNA species leak from isolated nuclei during cell fractionation, the possibility exists that these newly synthesized species also leak from the nucleus. To overcome the problems of nuclear leakage, mouse L929 cells were fractionated by cell enucleation. Enucleation extrudes the nuclei from cytochalasin-treated cells and produces cytoplasts that, by several criteria, are a bona fide cytoplasmic fraction uncontaminated by nuclear material. All six of the major snRNAs are present in the cytoplasts (c-snRNAs) shortly after synthesis. The species are identified by immunoprecipitation with specific antisera against the ribonucleoproteins and by Northern blotting and hybrid selection using cloned probes. This confirms and extends similar studies that used non-aqueous cell fractionation and manual dissection to overcome nuclear leakage. Kinetic studies demonstrate that the c-snRNAs return to the interphase nucleus after approximately 20 minutes in the cytoplasm. The U2 precursor U2' is processed to mature-sized U2 in the cytoplast fractions before returning to the nucleus. The c-snRNAs occur in ribonucleoprotein particles with similar antigenicity to the mature nuclear particles within six minutes of transcription. This suggests that in mammalian cells, important steps in the assembly of these ribonucleoproteins occur in the cytoplasm.

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

Lipocortin-I inhibits the synthesis and release of prolactin from human decidual cells: evidence for autocrine/paracrine regulation by lipocortin-I.

The lipocortins are a family of calcium-dependent phospholipid-binding proteins that are induced by glucocorticoids and inhibit phospholipase-A2 activity. To determine whether the lipocortins affect the release of PRL from human decidua, decidual cells from term pregnancies were exposed to recombinant lipocortin-I for 96 h, with medium changes at 24-h intervals. Lipocortin-I (0.01-100 nM) caused a time- and dose-dependent inhibition of PRL release, with a half-maximal effective dose of 50 nM. PRL release was inhibited by 27%, 62%, 93%, and 98% at 24, 48, 72, and 96 h, respectively. The cells exposed to lipocortin-I did not release the enzymes alkaline phosphatase and lactic dehydrogenase, indicating that the inhibitory effect on PRL release was not due to cell death. In addition to inhibiting basal PRL release, lipocortin also completely inhibited the stimulation of PRL release by decidual PRL-releasing factor, a 23.5-kDa protein recently purified from human placenta that stimulates the synthesis and release of decidual, but not pituitary, PRL. Hydrocortisone and dexamethasone (0.1-10 microM) had no effect on PRL release, and arachidonic acid (2-100 microM) inhibited rather than stimulated PRL release. Western blot analysis demonstrated the presence of lipocortin-I in decidual cells and conditioned media. On Northern blot, decidual mRNA hybridized to an oligonucleotide for lipocortin-I. These results strongly suggest that lipocortin-I has an autocrine/paracrine role in regulation of the synthesis and release of PRL from human decidual cells.

On-site analysis of arsenic in groundwater using a microfabricated gold ultramicroelectrode array

Rapid on-site analysis of arsenic in groundwater was achieved with a small battery-powered unit in conjunction with a microfabricated gold ultramicroelectrode array (Au-UMEA). The sensor, consisting of 564 UME disks with a unique gold surface created by electron beam evaporation, was demonstrated to be highly sensitive to low-ppb As3+ using square wave anodic stripping voltammetry. The influence of the square wave frequency, pulse amplitude, and deposition potential on the arsenic peak stripping current was investigated. Varying those theoretical parameters yielded results surprisingly similar to those for the thin Hg film case. The performance of the Au-UMEA was evaluated for reproducibility and reliability. Three stability tests showed an average relative standard deviation of 2.5% for 15 consecutive runs. Limits of detection were investigated, and 0.05 ppb As3+ could be measured while maintaining a S/N of 3:1. Interference studies were performed in the presence of 50-500 ppb of Cu2+, Hg2+, and Pb2+. On-site analysis of groundwater containing arsenic was performed with a small battery-powered potentiostat. Quantification was done through standard additions, and these results were compared to the standard EPA methodology.

Reflections on chemical modification of proteinases and their protein inhibitors.

Proteolytic enzymes and their protein inhibitors have been studied by chemical modification for over four decades. Modifications have helped to identify the active (and reactive) sites and to understand their mechanisms of interaction. Inactive derivatives of the enzymes form stable complexes with some inhibitors. These inactive enzymes have also been used for affinity chromatographic adsorptions.

Cloning of a non-catalytic form of human trkB and distribution of messenger RNA for trkB in human brain.

A truncated form of the human trkB gene has been cloned and sequenced. This gene is related to the trk family of tyrosine kinases, the products of which act as receptors for the neurotrophins. Of these, brain-derived neurotrophic factor and mammalian neurotrophin-4 are the known ligands for the TrkB receptor. Catalytic and non-catalytic (or truncated) forms of the trkB gene have been cloned for rat and mouse. In this study, using in situ hybridization, we describe the distribution of trkB messenger RNA in fetal and adult human brain.

The development and evolution of U.S. Army field rations.

Armed conflicts create large masses of personnel that need to be fed nutritious foods in less than favorable conditions. The United States military, private industry, and academia continue to work together to develop rations that meet the needs of military personnel who find themselves in varying climates, conditions, and geography.

Normal beta-NGF content in Alzheimer's disease cerebral cortex and hippocampus.

Nerve growth factor (beta-NGF) is known to have beneficial effects on cholinergic cell survival and to function both in vivo and in vitro. It has been speculated that this protein, or the lack of it, may be involved in the aetiology of Alzheimer's disease (AD). We describe the measurement of beta-NGF content in 4 regions of the cerebral cortex and the hippocampus in AD brain compared with brain tissue from age-matched normal subjects using a sensitive sandwich immunoassay (ELISA). There was no difference in beta-NGF content in any region examined in AD compared with normal values despite the marked loss of cortical cholinergic function.

A method for improving the nutritional value of food proteins: covalent attachment of amino acids.

Casein was modified by use of a series of active N-hydroxy-succinimide esters of amino acids in order to study the effects of new covalently linked hydrophobic or hydrophilic groups on its physical and nutritional properties. Tryptophan was used to determine the best conditions for the chemical reaction and to study the stability of the newly formed amide linkage (isopeptide bond). Casein was also modified with glycine, alanine, methionine, N-acetyl-methionine and aspartic acid. In vitro hydrolysis studies using bovine chymotrypsin, pancreatine and rat bile-pancreatic juice indicated that digestibility of the modified casein derivatives was lower than that of the untreated protein. Since solubility was not significantly changed (except for tryptophyl-casein), the decreased in vitro digestibility is probably due to other factors such as steric hindrance as well as decrease in lysine residues available to trypsin in pancreatin and rat pancreatic juice. Plasma amino acid patterns for rats fed a 10% protein diet of highly modified glycyl-casein or methionyl-casein suggest that the epsilon-aminolysyl derivatives are readily hydrolyzed in vivo. This was confirmed by the growth response of rats fed the following isonitrogenous diets (protein source listed only): casein, casein + free methionine, methionyl-casein, casein + free N-acetyl-methionine, N-acety-methionyl-casein. Covalently attached methionine appeared to be as readily available as the free amino acid; bound N-acetyl-methionine was also available but to a slightly lower extent. Although this study is preliminary, the covalent attachment of amino acids to proteins appears to be a promising method for improving the biological value of food proteins.

An ergonomic assessment of BS 5810: 1979 access for the disabled to buildings, through a survey of architects.

This article describes a project which was undertaken to assess the British Standard 5810: 1979 Access for the Disabled to Buildings, with a view to it being improved at the next review. This code of practice 'concentrates on the essential provisions that need to be incorporated in buildings to ensure that they are conveniently usable by disabled people'. The aim of the project was to make suggestions for improvements to the Standard, both qualitatively and in its range of provision, by discovering what additional data, if any, architects require, what level of detail they need and the preferred method of presentation. Research was also undertaken to ascertain whether the information contained in the Standard is based on empirical data. The project involved a literature search, sending postal questionnaires to architects and visiting architectural practices to complete structured interviews. The method used, the results and the conclusions on how the British Standard could be improved are described.

Powered domestic lawnmowers: design for safety.

The paper describes a detailed accident investigation carried out by the Institute for Consumer Ergonomics for the Consumer Safety Unit at the Department of Trade. As such it serves to illustrate the application of two specific research techniques (i) analysis of product related accident data, and (ii) ergonomics evaluation of current models - and shows how these may be used to help in defining standards and criteria for the design of safer products. The study identified lawnmower features and activities associated with accidents recorded by the Home Accident Surveillance System. Ergonomics appraisal by expert assessment and user trials highlighted hazards associated with currently available powered lawnmowers. Performance criteria for safer design of selected features were developed with the aim of overcoming these hazards. At the end of the study liaison was sought with manufacturers to discuss how the results from the work could be used to effect.