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C Robinson

Publications and source records attributed to C Robinson.

At least 127 records · Page 7Linked to original sources
PubMed1998

Characterisation of an Arabidopsis cDNA encoding a thylakoid lumen protein related to a novel 'pentapeptide repeat' family of proteins.

We have cloned an Arabidopsis cDNA encoding a novel thylakoid lumen protein, P17.4, that has been previously isolated from lumen extracts of spinach chloroplasts. The protein is synthesised with a bipartite presequence containing a Sec-type lumen-targeting signal peptide and the precursor protein is imported into the lumen of pea chloroplasts. The encoded protein is homologous to an Anabaena protein that is essential for correct glycolipid localisation, and is also related to at least 16 unassigned open reading frames in Synechocystis. This family of proteins is characterised by the presence of numerous pentapeptide repeats with the consensus structure AXLXX, and its members are predicted to be located in the cytosol, plasma membrane and periplasm/lumen. P17.4 is therefore the first higher plant member of an extended family of putative cyanobacterial proteins that may serve important roles in lipid transport or assembly.

Amino Acid Sequence↗
PubMed1998

Sec/SRP-independent insertion of two thylakoid membrane proteins bearing cleavable signal peptides.

Two imported thylakoid membrane proteins, PSII-X and PSII-W, are synthesised with cleavable N-terminal signal peptides that closely resemble those of Sec-dependent lumenal proteins. In this report we have reconstituted the insertion of pre-PSII-X and pre-PSII-W into isolated thylakoids. We show that insertion does not require either nucleoside triphosphates or stromal extracts, both of which are required for Sec- and signal recognition particle (SRP)-dependent targeting mechanisms. Insertion is furthermore unaffected by protease treatments that destroy the known protein translocation apparatus in the thylakoid membrane. We conclude that these membrane proteins are inserted by an unusual Sec/SRP-independent mechanism that probably resembles that used by CFoII, and we discuss possible parallels with the biogenesis of phage M13 procoat.

Chloroplasts↗
PubMed1998

An Arabidopsis cDNA encodes an apparent polyprotein of two non-identical thylakoid membrane proteins that are associated with photosystem II and homologous to algal ycf32 open reading frames.

We have characterised an Arabidopsis thaliana cDNA homologous to the ycf32 open reading frames present in the Synechocystis genome and the plastid genomes of several eukaryotic algae. The predicted protein is also homologous to a novel protein reported to be associated with photosystem II. The protein is synthesised as a 23 kDa precursor with an N-terminal presequence that appears to be bipartite in structure, and the protein is targeted into the thylakoid membrane of pea chloroplasts. Although the Ycf32 presequence contains an apparent signal peptide, we find that this protein is not imported by either of the standard Sec- or deltapH-dependent pathways. The mature protein is also unusual in two respects. First, there are two distinct, non-identical copies of typical single-span Ycf32 sequences in the Arabidopsis sequence, separated by an additional hydrophobic region. Secondly, the imported protein runs as a doublet of 6 kDa and 7 kDa polypeptides whereas the mature protein is predicted to be 14 kDa. We speculate that the protein undergoes further maturation once inserted into the thylakoid membrane to yield two separate Ycf32-like polypeptides.

Amino Acid Sequence↗
PubMed1998

Inwardly rectifying whole cell potassium current in human blood eosinophils.

1. Membrane currents were studied in single human blood eosinophils using the whole cell voltage clamp technique. The whole cell current-voltage relationship exhibited rectification about the membrane potential which followed the potassium equilibrium potential when [K+]o was raised. Elevation of [K+]o considerably potentiated inward current amplitude, and in some cells channel activity was discernible in the whole cell membrane current recordings. The single channel conductance was 24 +/- 1 pS ([K+]o, 100 mM; [K+]i, 140 mM), and eosinophils were found to have as few as three, and on average twenty, inward rectifier channels each. 2. The inward current was inhibited in a voltage-dependent manner by extracellular cations in order of potency Ba2+ > Cs+ > Na+. Intracellular acidification inhibited while alkalization augmented the inward current. Mg2+ contributed to rectification as dialysis with nominally Mg(2+)-free pipette solution was associated with an increase in the outward current during membrane polarization. 3. By reverse transcription-polymerase chain reaction (RT-PCR) using suitable primers on human eosinophils mRNA, an inward rectifier channel, Kir2.1, was identified, which is known from expression studies to have very similar properties to those found in this study. 4. Superoxide anion production or its stimulation by phorbol 12-myristate 13-acetate (PMA) was not significantly affected by depolarization with 140 mM [K+]o, or by 1 mM BaCl2. 5. It is concluded that the single channel currents and the whole cell current rectification observed in human blood eosinophils resulted from the presence of an inwardly rectifying potassium channel, probably Kir2.1.

Action Potentials↗
PubMed1998

Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene.

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence).

Amino Acid Sequence↗
PubMed1998

Sera from patients with malignant mesothelioma can contain autoantibodies.

Malignant mesothelioma (MM) is resistant to all conventional forms of therapy though there is considerable evidence from clinical trials and animal models of the disease that an immune response can be elicited to the tumour. In order to define those target antigens expressed by MM cells which might provide a focus for an effective immune response we tested patients' sera for the presence of MM autoantibodies by Western blot analysis. Eight of 29 (28%) patients with MM had serum antibodies of the IgG class in high titre and each antiserum recognised different protein antigens. In those individuals where sequential samples were available, the antibody titre increased with the progression of the disease though the number of target antigens remained constant. Sera from the eight patients were studied further: six of the antigen complexes were expressed at least partially in the nucleus; two showed some specificity for the tumour in that they discriminated antigens that were highly expressed in all human MM cell lines, but were not expressed in a human SV40 transformed mesothelial line; four of the antisera recognised a homologue in mouse tissue and each of these had a different pattern of expression. Collectively, these antisera define a subset of nuclear autoantigens that are over-expressed in dividing cells.

Adult↗
PubMed1998

Prediction and assessment of the severity of post-operative pain and of satisfaction with management.

A prospective observational study of cohorts of patients undergoing hip replacement (30), knee replacement (31), and spinal nerve root decompressive surgery (30) were interviewed pre-operatively to identify factors which might correlate with and potentially predict severe post-operative pain and dissatisfaction with analgesic management. The hip patients comprised 33% females and averaged 64 years, while the knee patients were 45% female and older (mean 71 years) and the spinal patients were 43% female and averaged 50 years. The three groups were similar with respect to all other pre-operative variables. Pain intensity was assessed mainly by self-report using the Present Pain Intensity (PPI) and Visual Analogue Scales (VAS) of the McGill Pain Questionnaire. The PPI was preferred by patients and nurses and, as there were no analytical advantages for the VAS, the PPI data are presented. The average post-operative pain during routine management mainly with patient controlled intravenous opiate, was mild to moderate and declined over days 1-5, declined further at discharge but rose slightly 1 month after discharge. The hip replacement patients experienced significantly (P < 0.01) less pain overall than the patients in the other two groups. Nurses' assessments of pain severity from observed behaviour were low and agreed poorly with the patients' self reports. Assessed on Likert Scales (0-6), the patients generally indicated good or excellent pain control, better than expected pain experience, and high levels of satisfaction with analgesic management. Significant (P < or = 0.01) multivariate correlates of severe post-operative pain assessed by logistic regression analysis of 11 variables were female gender, high pre-operative pain severity, and younger age. Significant (P < or = 0.01) multivariate correlates of both worse than expected pain experience and low satisfaction were female gender, high pre-operative pain severity, high anxiety about risks and problems, low expected pain severity, age (younger) and high willingness to report pain. These variables may reasonably be tested in further studies as potential predictors of adverse post-operative pain experience.

Adult↗
PubMed1998

Warm-Up Active Wound Therapy: a novel approach to the management of chronic venous stasis ulcers.

This preliminary study investigates the use of a radiant-heat bandage, Warm-Up Active Wound Therapy, as a new approach to the treatment of patients with chronic venous ulcers. Thirteen patients were randomly assigned to either conventional therapy or Warm-Up Active Wound Therapy for inpatient treatment of chronic venous stasis ulcers. Our results indicated that Warm-Up Active Wound Therapy is more effective than conventional therapy in healing chronic venous ulcers, and patients reported a significant reduction in wound pain. Warm-Up Active Wound Therapy also was found to be a safe treatment modality with no adverse events occurring in any patient.

Adult↗
PubMed1998

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium.

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.

Animals↗
PubMed1998

Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.

BACKGROUND: Allergenic and non-allergenic proteinases from house dust mites (HDM) cause loss of adhesion between airway epithelial cells that may result in a loss of functional cohesion between the cells and thus assist in allergen presentation. Improved cellular assay systems are needed to ascertain the mechanisms involved. OBJECTIVES: To survey a series of epithelial cell lines (Calu-3, 16HBE14o-, NCI-H292 and A549 from human airways, and MDCK from dog kidney) and establish their utility for studies of the effects of HDM proteinases from D. pteronyssinus on epithelial permeability. To develop an improved method for measuring changes in epithelial permeability induced by HDM proteinases and other provocants. METHODS: The permeability of epithelial monolayer cultures to mannitol was calculated from measurements of clearance using a technique that permits mathematical estimation and reduction of non-cellular diffusional constraints. Permeability was studied under control conditions and after perturbation of monolayers with HDM proteinases (separated into serine- and cysteine-proteinase classes) or chelation of extracellular Ca2+. Fluorescent antibody staining was used to investigate whether the cells expressed tight junctions (staining of ZO-1), desmosomes (staining of desmoplakin) and zonulae adherentes (staining of E-cadherin). RESULTS: The Calu-3 line was identified as an airway cell line that expressed functional tight junctions, desmosomes and zonulae adherentes. Calu-3 monolayers exhibited a low clearance and permeability to mannitol, similar to that seen in the extensively characterized MDCK cell line. Clearance and permeability were significantly increased by treatment with either HDM proteinase fraction or by calcium chelation. 16HBE14o- cells also had a low permeability to mannitol under control conditions and expressed a similar repertoire of functional proteins from major intercellular junctions. In contrast, NCI-H292 and A549 cell lines were functionally deficient in tight junctions, although they did express desmosomes and zonulae adherentes to a greater extent. Epithelial permeability was found to be a more appropriate and sensitive index of epithelial perturbation than was tracer clearance. CONCLUSION: These results suggest that the Calu-3 and 16HBE14o- cell lines are useful tools in studying the mechanism of HDM proteinases on airway epithelial cell function. HDM proteinases of both cysteine and serine mechanistic classes were found to perturb epithelial adhesion and function.

Adult↗
PubMed1998

Identification of cDNAS encoding plastid-targeted glutathione peroxidase.

A cDNA was isolated from pea leaf RNA which encodes a phospholipid hydroperoxide glutathione peroxidase (PHGPX; E.C. 1.1.1.1.9). The N-terminal section of this PHGPX encodes a recognisable chloroplast transit peptide. Efficient import in vitro of the pre-PHGPX protein into the stroma of isolated pea chloroplasts confirmed that the PHGPX is a chloroplast-located enzyme. The pea PHGPX has highly conserved homologues in Arabidopsis, citrus and Nicotiana sylvestris and the authors suggest that these proteins are also localised in the chloroplast and not in the cytosol as previously supposed.

Amino Acid Sequence↗
PubMed1998

The developing enamel matrix: nature and function.

The hydroxyapatite crystals of mature enamel are unusually large, uniform and regularly disposed within the tissue, implying that their development is a highly controlled process. The organic matrix of developing enamel is presumed to play an important role in the modulation of mineral deposition and growth during tooth morphogenesis but the precise functions of individual matrix proteins remain unclear. The aim of this review was to survey the current knowledge of enamel matrix proteins with a view to suggesting possible functions. The organic matrix is highly heterogeneous, comprising proteins derived from a number of different genes, including amelogenin, enamelin, ameloblastin (amelin/sheathlin), tuftelin, dentine sialophosphoprotein, enzymes and serum proteins such as albumin. Each of these classes appears to undergo post-secretory sequential degradation which contributes further towards matrix heterogeneity. Possible functions of these proteins include de novo mineral nucleation/initiation (dentine sialophosphoprotein, tuftelin), mineral ion binding as crystal precursors (amelogenin, enamelin), control of crystal growth (amelogenin, enamelin, ameloblastin), support of growing crystals (amelogenin, enamelin), determination of prismatic structure (ameloblastin), cell signalling (tuftelin, ameloblastin), control of secretion (breakdown products) and protection of the mineral phase (amelogenin, enamelin). Failure of these mechanisms could lead to incomplete maturation of the enamel and the eruption of dysplastic tissue.

Ameloblasts↗
PubMed1998

The maternal CCAAT box transcription factor which controls GATA-2 expression is novel and developmentally regulated and contains a double-stranded-RNA-binding subunit.

The transcription factor GATA-2 is expressed at high levels in the nonneural ectoderm of the Xenopus embryo at neurula stages, with lower amounts of RNA present in the ventral mesoderm and endoderm. The promoter of the GATA-2 gene contains an inverted CCAAT box conserved among Xenopus laevis, humans, chickens, and mice. We have shown that this sequence is essential for GATA-2 transcription during early development and that the factor binding it is maternal. The DNA-binding activity of this factor is detectable in nuclei and chromatin bound only when zygotic GATA-2 transcription starts. Here we report the characterization of this factor, which we call CBTF (CCAAT box transcription factor). CBTF activity mainly appears late in oogenesis, when it is nuclear, and the complex has multiple subunits. We have identified one subunit of the factor as p122, a Xenopus double-stranded-RNA-binding protein. The p122 protein is perinuclear during early embryonic development but moves from the cytoplasm into the nuclei of embryonic cells at stage 9, prior to the detection of CBTF activity in the nucleus. Thus, the accumulation of CBTF activity in the nucleus is a multistep process. We show that the p122 protein is expressed mainly in the ectoderm. Expression of p122 mRNA is more restricted, mainly to the anterior ectoderm and mesoderm and to the neural tube. Two properties of CBTF, its dual role and its cytoplasm-to-nucleus translocation, are shared with other vertebrate maternal transcription factors and may be general properties of these proteins.

Animals↗
PubMed1998

Effect of insulin on glycerol production in obese adolescents.

Impaired stimulation of glucose metabolism and reduced suppression of lipolytic activity have both been suggested as important defects related to the insulin resistance of adolescent obesity. To further explore the relationship between these abnormalities, we studied seven obese [body mass index (BMI) 35 +/- 2 kg/m2] and seven lean (BMI 21 +/- 1 kg/m2) adolescents aged 13-15 yr and compared them with nine lean adults (aged 21-27 yr, BMI 23 +/- 1 kg/m2) during a two-step euglycemic-hyperinsulinemic clamp in combination with 1) a constant [2H5]glycerol (1.2 mg.m-2.min-1) infusion to quantify glycerol turnover and 2) indirect calorimetry to estimate glucose and net lipid oxidation rates. In absolute terms, basal glycerol turnover was increased and suppression by insulin was impaired in obese adolescents compared with both groups of lean subjects (P < 0.01). However, when the rates of glycerol turnover were adjusted for differences in body fat mass, the rates were similar in all three groups. Basal plasma free fatty acid (FFA) concentrations were significantly elevated, and the suppression by physiological increments in plasma insulin was impaired in obese adolescents compared with lean adults (P < 0.05). In parallel with the high circulating FFA levels, net lipid oxidation in the basal state and during the clamp was also elevated in the obese group compared with lean adults. Net lipid oxidation was inversely correlated with glucose oxidation (r = -0.50, P < 0.01). In conclusion, these data suggest that lipolysis is increased in obese adolescents (vs. lean adolescents and adults) as a consequence of an enlarged adipose mass rather than altered sensitivity of adipocytes to the suppressing action of insulin.

Adolescent↗
PubMed1998

Identification of human serum albumin in human caries lesions of enamel: the role of putative inhibitors of remineralisation.

Carious attack on enamel is not a unidirectional process but involves both demineralisation and remineralisation. The chemistry of carious attack on enamel has, to a large extent, now been clarified as far as mineral components are concerned but little attention, however, has been paid to the identity of organic material in carious lesions and its possible role in the caries process. The only clear information available is that organic material accumulates with time within enamel lesions. The present study was aimed at identifying a specific protein component known to bind to hydroxyapatite (albumin) in carious lesions with a view to investigating its role in the disease process. The distribution of albumin within both white spot and fissure lesions and adjacent sound enamel of extracted human teeth was investigated using SEM immunohistochemistry on undermineralised sections of human enamel and employing a polyclonal antibody to human serum albumin. The nature of the protein, i.e. whether it was in the form of intact molecules or degraded fragments, was investigated by Western blotting, employing the same antibody. The immunohistochemistry revealed the presence of albumin within both interproximal white spot and fissure lesions with little if any present in sound enamel. The Western blotting indicated that the albumin was in the intact form with no evidence of degradation products. The ability of albumin to bind and to inhibit growth of calcium phosphate crystals raises the question as to the possible role of such a molecule in the development of carious lesions.

Antibodies↗
PubMed1998

Atomic force microscopy studies of crystal surface topology during enamel development.

During the secretory stage of enamel development, the hydroxyapatite crystals appear as thin ribbons which grow substantially in width and thickness during the later maturation stage. In this study, the atomic force microscope (AFM) was used to investigate developmentally-related changes in deproteinized enamel crystal surface topography in normal animals and in those receiving daily doses of fluoride. The AFM revealed previously undescribed surfaces features, some of which may represent growth sites or different crystalline phases. Secretory stage crystals had greater surface rugosity and were more irregular, with spherical sub-structures of 20-30 nm diameter arranged along the "c"-axis. Maturation stage crystals were smoother and larger but revealed both subnanometer steps and lateral grooves running parallel to the "c"-axis. Crystals from fluorotic tissue showed similar features but were more irregular with a higher degree of surface roughness, suggesting abnormal growth. The AFM may prove an important adjunct in determination of the mechanisms controlling crystal size and morphology in skeletal tissues.

Animals↗