Association of DRB1 shared epitope genotypes with early mortality in rheumatoid arthritis: results of eighteen years of followup from the early rheumatoid arthritis study.

OBJECTIVE: To determine whether the HLA-DRB1 shared epitope (SE) is associated with early mortality and specific causes of death in rheumatoid arthritis (RA). METHODS: HLA-DRB1 genotyping was carried out on blood samples from 767 patients recruited for the Early RA Study (ERAS), a multicenter, inception cohort study with followup over 18 years. Dates and causes of death (n = 186) were obtained from the Office of National Statistics. The association of HLA-DRB1 alleles with risk of mortality was assessed using Cox proportional hazards regression analyses. Multivariate stepwise models were used to assess the predictive value of HLA-DRB1 genotypes compared with other potential baseline risk factors. RESULTS: The SE was not significantly associated with overall mortality. However, the presence of 2 SE alleles was associated with risk of mortality from ischemic heart disease (hazard ratio [HR] 2.02 [95% confidence interval 1.04-3.94], P = 0.04), and malignancy (HR 2.18 [95% confidence interval 1.17-4.08], P = 0.01). Analysis of specific SE genotypes (corrected for age and sex) revealed that the HLA-DRB1*0101/*0401 and 0404/*0404 genotypes were the strongest predictors of mortality from ischemic heart disease (HR 5.11 and HR 7.55, respectively), and DRB1*0101/*0401 showed a possible interaction with smoking. Male sex, erythrocyte sedimentation rate, and Carstairs Deprivation Index were also predictive, but the Health Assessment Questionnaire score, rheumatoid factor, nodules, and swollen joint counts were not. Mortality due to malignancy was particularly associated with DRB1*0101 genotypes. CONCLUSION: The risk of mortality due to ischemic heart disease or cancer in RA is increased in patients carrying HLA-DRB1 genotypes with particular homozygous and compound heterozygous SE combinations.

Safety and tolerability of conversion from stable Sandimmun maintenance treatment to Sandimmun Neoral in patients with rheumatoid arthritis.

OBJECTIVE: To assess the safety and tolerability of converting patients with rheumatoid arthritis (RA) taking a stable dose of cyclosporin A (CyA) maintenance treatment (Sandimmun, SIM) to a new microemulsion capsule formulation, Sandimmun Neoral (Neoral), at an initial dose of 2.5 mg/kg/day. METHODS: In this single arm, open multicenter study, 28 patients were recruited to enter a 6 week pre-conversion period; of these, 22 patients completed 12 weeks' treatment with Neoral. RESULTS: During the 12 week post-conversion period, 11 patients experienced adverse events considered to be drug related; most were mild to moderate in severity and reflected the known safety profile for CyA. Only slight differences in efficacy variables were observed after conversion. The mean Neoral dose at Week 12 (2.84 mg/kg/day) was lower than the mean SIM pre-conversion dose (3.38 mg/kg/day). The study showed that, in patients with RA undergoing stable SIM maintenance treatment, conversion to an initial Neoral dose of 2.5 mg/kg/day did not give rise to any clinically relevant safety and tolerability concerns, and efficacy of the treatment was maintained compared with SIM. CONCLUSION: This conversion strategy constitutes a clinically acceptable alternative to a 1:1 dose conversion.

Dynamics of environmental supplementation of iodine: four years' experience of iodination of irrigation water in Hotien, Xinjiang, China.

Hotien prefecture, Xinjiang Province, China, in the Taklamakan Desert, is an area of severe iodine deficiency. Because usual methods of iodine supplementation failed here, we began supplementation in 1992 with potassium iodate, which was added to irrigation water (Lancet 1994; 334:107-110). We report 4 y experience with this method in 3 townships that contained a total treated population of 37,000. Potassium iodate was dripped into irrigation water (to a concentration 10-80 microg/l) during a 2- to 4-wk period. During the 3 y that followed, no further supplementation was made, and iodine concentrations increased several fold in crops and plants, sheep and chicken thyroid glands, and meat and in urine of children 2-6 y of age and of women who were of childbearing age. Infant mortality decreased 50%, and sheep production increased 43%. Iodine repletion of soil through irrigation water is an effective and cost-efficient way of providing iodine in appropriate situations.

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues.

A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by an increased level of 16:0 and a concomitant decrease of 18-carbon fatty acids as a consequence of a single recessive nuclear mutation at the fab1 locus. Quantitative analysis of the fatty acid composition of individual lipids established that lipids synthesized by both the prokaryotic and eukaryotic pathways were affected by the mutation. Direct enzyme assays demonstrated that the mutant plants were deficient in the activity of 3-ketoacyl-acyl carrier protein synthase II; therefore, it is inferred that fab1 may encode this enzyme. Labeling experiments with [14C]acetate and lipase positional analysis indicated that the mutation results in a small shift in the partitioning of lipid synthesis between the prokaryotic and eukaryotic pathways. Synthesis of chloroplast lipids by the prokaryotic pathway was increased with a corresponding reduction in the eukaryotic pathway.

A unique manual for self-assessment by dental practitioners.

The Self Assessment Manual and Standards is produced by the Faculty of General Dental Practitioners of the Royal College of Surgeons of England and the Department of Health as part of a clinical audit program. It is a self-help manual containing a set of clinical standards in 15 aspects of patient care. The standards are concerned with clinical outcomes, rather than techniques, and are intended to be educational, not regulatory. The book's contents and applications are described in detail, together with its political background and possible use in an international context.

Germinal and somatic activity of the maize element Activator (Ac) in Arabidopsis.

We have investigated the germinal and somatic activity of the maize Activator (Ac) element in Arabidopsis with the objective of developing an efficient transposon-based system for gene isolation in that plant. Transposition activity was assayed with a chimeric marker that consists of the cauliflower mosaic virus 35S promoter and a bacterial streptomycin phosphotransferase gene (SPT). Somatic activity was detected in seedlings germinated on plates containing streptomycin as green-resistant sectors against a background of white-sensitive cells. Germinal excisions resulted in fully green seedlings. The transposition frequency was extremely low when a single copy of the transposon was present, but appeared to increase with an increase in Ac copy number. Plants that were selected as variegated produced an increased number of green progeny. The methylation state of the Ac elements in lines with either low or high levels of excision was assessed by restriction analysis. No difference was found between these lines, indicating that the degree of methylation did not contribute to the level of Ac activity. Germinal excision events were analyzed molecularly and shown to carry reinserted transposons in about 50% of the cases. In several instances, streptomycin-resistant siblings carried the same transposed Ac element, indicating that excision had occurred prior to meiosis in the parent. We discuss parameters that need to be considered to optimize the use of Ac as a transposon tag in Arabidopsis.

General anaesthesia, sedation and resuscitation in dentistry.

In the eyes of the public there is a traditional link between general anaesthesia and dental practice. After 150 years' experience the link may be broken by Government's acceptance of the Poswillo Report and its major recommendations.

Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis inArabidopsis.

The seed fatty acid (FA) composition of various single mutant combinations ofArabidopsis thaliana that affect FA biosynthesis has been examined. Double mutant combinations offae, a mutation affecting CIS elongation, and a series of four other FA biosynthetic mutants were synthesized. The four other single mutants were:fad2 andfad3, which are deficient in 18∶1 and 18∶2 desaturation, respectively;fab1, which is elevated in 16∶0 and decreased in 18∶1; andfab2, which is elevated in 18∶0 and decreased in 18∶1. The superimposition of two blocks in the FA biosynthetic pathway leads to dramatic changes in the FA content of the double mutants. The tenArabidopsis stocks analyzed to date (wild-type, five single mutants, and four double mutants) make seed oils with a wide range of FA compositions, and illustrate the diversity of oils it is possible to obtain from a single plant species.

Isolation of EMS-induced mutants in Arabidopsis altered in seed fatty acid composition.

Mutants of Arabidopsis thaliana were identified by screening pedigreed M3 seed collections from EMS-treated plants for changes in fatty acid (FA) composition. The FA phenotypes of the most dramatic mutants are as follows: G30 and 1E5 (allelic) lack linolenic acid (18∶3) and are elevated in linoleic acid (18∶2); 4A5 is deficient in 18∶2 and 18∶3 and fourfold increased in oleic acid (18∶1); 9A1 lacks all FAs > C18 and is twofold increased in 18∶1; 1A9 is twofold increased in palmitic acid (16∶0) and decreased by one-half in 18∶1; 2A11 is two-to threefold increased in stearic acid (18∶0) and decreased by one-half in 18∶1. Based on segregation of F2 selfed plants derived from crosses to wild type, all of these phenotypes are the result of single gene mutations.

Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose.

Pseudomonas fluorescens HV37a inhibited growth of the fungus Pythium ultimum on potato dextrose agar (PDA). An antibiotic activity produced under these conditions was fractionated and partially characterized. Extracts prepared from the PDA on which HV37a was grown revealed a single peak of antibiotic activity on thin-layer chromatograms. Similar extracts were prepared from mutants of HV37a. Their analysis indicated that the antibiotic observed in thin-layer chromatograms was responsible for fungal inhibition observed on PDA. The production of the PDA antibiotic required the presence of glucose, whereas two other antibiotic activities were produced only on potato agar without added glucose. Two mutants (denoted AfuIa and AfuIb) previously characterized as deficient in fungal inhibition on PDA showed altered regulation of the production of all three antibiotics in response to glucose. These mutants were also deficient in glucose dehydrogenase. Mutants isolated as deficient in glucose dehydrogenase were also deficient in fungal inhibition and were grouped into two classes on the basis of complementation analysis with an AfuI cosmid. Glucose regulation of antibiotic biosynthesis therefore involves at least two components and requires glucose dehydrogenase.

Activity of mu- and delta-selective opioid agonists in the guinea pig ileum preparation: differentiation into peptide and nonpeptide classes with beta-funaltrexamine.

Previous studies have demonstrated that pretreatment of guinea pig longitudinal muscle-myenteric plexus ileal preparations with the highly selective noncompetitive mu antagonist beta-funaltrexamine (beta-FNA) causes an increase in the Ke value for the interaction of morphine with naloxone, suggesting that beta-FNA inactivates those receptors at which morphine interacts in the guinea pig ileum. The effect is selective for mu receptors since beta-FNA has no effect upon the interaction of naloxone with the kappa agonist nalorphine. In the present study, it was found that although beta-FNA attenuated the effects of morphine and other morphine-like agonists at mu receptors in the guinea pig longitudinal muscle-myenteric plexus ileal preparation, the mu-mediated actions of delta-selective peptide agonists and mu-selective peptide agonists were not completely attenuated by beta-FNA pretreatment. These data suggest that morphine-like mu agonists and other mu-selective and delta-selective peptide agonists in the guinea-pig ileum preparation either interact with similar opioid receptors but in a distinguishable manner or interact with different populations of opioid receptors or the peptides studied had greater intrinsic activity than the nonpeptides.

Relationship between Rapid, Firm Adhesion and Long-Term Colonization of Roots by Bacteria.

For rhizobacteria to exert physiological effects on plant growth, the bacteria must first effectively colonize the root surface. To examine the relationship between long-term colonization of root systems and adherence to roots in the short term, a binding assay was developed. Adherence was determined by incubating roots of intact radish seedlings with bacteria, washing and homogenizing the roots, and dilution plating the resulting homogenate. Irreversible binding of bacteria was rapid, reaching half-maximum by 5 min. All of the rhizosphere bacteria tested showed similar, concentration-dependent binding (ranging from 10 to 10 CFU/ml), as well as long-term colonization of radish roots under sterile conditions. Escherichia coli, which is not a root colonizer, showed about 10-fold less binding, but still demonstrated concentration-dependent binding and rapid kinetics of adherence at high concentrations (10 to 10 CFU/ml). The bacteria tested were very different with respect to source or habitat and plant response, yet they showed similar concentration-dependent binding. There was no correlation between the relative hydrophobicities of the cell surfaces of strains and the adherence of the strains to roots. Binding of Pseudomonas fluorescens E6-22 was promoted by divalent cations (Ca and Mg) at concentrations of 5 to 10 mM, whereas monovalent cations (Na and K) had little effect; electrostatic phenomena may partially explain adherence in the short term, an important prelude to long-term colonization of root surfaces.

Production of a Lectin in Tissue Cultures of Dolichos biflorus.

Callus cultures have been produced from the epicotyl and leaves, hypocotyl, and roots of germinating Dolichos biflorus seeds. These cultures were initiated on media containing 2,4-dichlorophenoxyacetic acid and kinetin, transferred to media with increased amounts of these hormones, and then maintained on hormone-free media. Extracts of these cultures were examined by radioimmunoassays specific for the lectin from the seeds of this plant and for a lectin that is present only in the stems and leaves of the intact plant. Although the seed lectin was not detected in any cultures, the stem and leaf lectin was produced in those cultures grown on the hormone free media. Lectin isolated from these cultures had subunits identical in electrophoretic mobilities to the subunits from the lectin isolated from intact stems and leaves. Levels of this lectin decreased when the cells were transferred back to media containing hormones and increased again upon transfer to the hormone-free media. The absence of exogenous hormones and the production of lectin were also correlated with the rapid growth and greening of the cells. Immunofluorescence and immunocytochemical studies on sections of cultured cells indicated that the stem and leaf lectin is associated with the cytoplasm as well as the cell wall as has been found in previous studies on the subcellular localization of this lectin in the intact plant.

Subcellular Localizations of Two Dolichos biflorus Lectins.

The subcellular localizations of the Dolichos biflorus seed lectin and the structurally related lectin (cross-reactive material [CRM]) from the stems and leaves of this plant were determined by immunofluorescence, immunocytochemistry, and cell fractionation procedures. Subcellular fractionation of the cotyledons using a nonaqueous procedure to minimize disruption of the protein bodies showed that the majority of the seed lectin was associated with the protein body fraction and some lectin was also present in the starch granules. Immunofluorescence and immunocytochemistry at the light microscopic level showed that the seed lectin was mainly localized at the peripheries of these organelles. Lectin was also found in the cytoplasm of the cells, although the amount appeared to be dependent upon the degree of protein body disruption.Immunofluorescence and immunocytochemistry studies of the stem and leaf lectin (CRM) indicated that a significant portion of this lectin may be associated with the cell walls, although lectin was also seen in the cytoplasm of plasmolyzed cells. Extraction and cell fractionation studies showed that a large portion of the CRM is readily solubilized and most of the remainder is pelleted at 1000g. The CRM can be extracted from these pellets by treatment with cellulase and pectinase; other reagents such as NaCl, detergents, and EDTA could also release significant amounts of CRM. These studies suggest that the CRM is noncovalently bound to the cell walls. A comparison of the distribution of exogenously supplied [(125)I]CRM with the endogenous CRM during extraction and cell fractionation indicates that soluble CRM is not adsorbed to the 1000g pellet during fractionation.The different subcellular distributions of these two structurally related lectins suggest that different tissues of the same plant may utilize lectins for different functions.

Centrifugation of normal and rheumatoid arthritis blood on Ficoll-Hypaque and Ficoll-Nycodenz solutions.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.