Search PubMed⌕ Search

Biomedical subjects

C Robinson

Publications and source records attributed to C Robinson.

At least 307 records · Page 17Linked to original sources
PubMed1990

The effect of fluoride on the developing mineralized tissues.

The work described considers the effects on calcified tissues of those concentrations of fluoride which are not overtly cyto-toxic, i.e., in the general region of up to 1-2 mumol/L. Plasma fluoride concentrations or those of the cellular environment are considered rather than dietary levels. The effect of fluoride ion on specific stages of tooth and bone development is discussed. Little effect has been observed on the modulation of gene expression as far as odontogenesis is concerned, although there is evidence that fluoride could be osteogenic in both embryonic and adult tissues. Expression of extracellular matrix protein genes seems not to be impaired, but subtle changes detected in the enamel matrix could be due to selective alterations in amino-acid uptake or interference with subsequent protein processing. This could also be due to an extension of the secretory period without concomitant changes in post-secretory matrix processing. Removal of matrix is apparently impaired, with concomitant incomplete maturation. While existing mineral phases can be affected, it is more likely that matrix and or mineral-matrix interaction is the site of action. Explant studies suggest that the effect may be reversible. Inhibition of proteolysis during enamel maturation may account for the reported inhibition of enamel crystal growth. This is supported by the finding that the normally incomplete maturation of porcine enamel is associated with a somewhat greater residual protein content. The use of animal models in the investigation of enamel dysplasia (fluoride-induced or otherwise) should therefore be viewed with caution.

Animals↗
PubMed1990

Extracellular processing of enamel matrix proteins and the control of crystal growth.

The origin, expression and role of matrix modifying enzymes in dental enamel was investigated by zymography and growth of enamel crystals in vitro. Gelatinase activity of the neutral metalloprotease type was detected at similar molecular weights in enamel organ, enamel and dentine. The activity was present throughout all developmental stages in enamel organ but was dramatically reduced in the maturation stage of the enamel. Activity of the serine protease type directed against enamel matrix was also detected in enamel, particularly in the maturation stage. No such activity was detected in the enamel organ. Phosphatase activity at alkaline pH was demonstrated at similar molecular weights in both enamel and enamel organ. This activity was maximal in the maturation stage. Further experiments showed that both serine proteases and alkaline phosphatase were able to facilitate enamel crystal growth in vitro. Matrix modification via temporally and spatially restricted enzymes may be directly involved in the control of enamel crystal growth and hence in the determination of final tissue architecture.

Animals↗
PubMed1990

The influence of cyclooxygenase inhibition on the loss of bronchoconstrictor response to repeated bradykinin challenge in asthma.

Bradykinin is a naturally occurring nonapeptide which may contribute to the pathogenesis of bronchial asthma. When inhaled by asthmatic subjects it is a potent bronchoconstrictor, but with repeated challenge airways responsiveness to the peptide decreases markedly. In vitro studies suggest that loss of bradykinin responsiveness may be due to the secondary generation of relaxant prostanoids. We have used the potent cyclooxygenase inhibitor flurbiprofen to investigate the potential role of prostanoid generation in bradykinin tachyphylaxis in eight asthmatic patients. The effects of oral flurbiprofen (150 mg) and matched placebo were observed on two consecutive dose response studies with inhaled bradykinin and histamine in a double-blind, randomized study. Venous blood was taken to measure the serum concentration of thromboxane B2 (TxB2) as a check on the extent of cyclooxygenase blockade achieved by flurbiprofen. Following recovery from the first challenge with bradykinin, the asthmatic airways showed a reduced response to a second challenge with this nonapeptide, the provocative concentration producing a 20% fall from baseline (PC20) increasing from 0.07 to 0.42 mg.ml-1 (p less than 0.01). The airway response to inhaled histamine after the second bradykinin challenge was not significantly changed. In the presence of demonstrable cyclooxygenase inhibition, flurbiprofen failed to prevent the development of reduced responsiveness to bradykinin observed on the second challenge, the PC20 increasing from 0.10 to 0.48 mg.ml-1 (p less than 0.01). This study demonstrates that repeated exposure to inhaled bradykinin results in loss of the bronchoconstrictor response which appears specific for this agonist and not secondary to the increased generation of protective prostanoids.

Adolescent↗
PubMed1989

The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.

Amino Acid Sequence↗
PubMed1989

Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex.

The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases.

Chlorophyll↗
PubMed1989

Site-specific variations in the concentrations of substances in the mouth.

Estimates of the concentration of soluble substances in the oral fluids have generally been obtained by the analysis of whole saliva, either mixed in the mouth or obtained directly from the salivary duct. Such values may give little indication of concentrations at any particular site in the mouth. This is partly because substances do not always move easily about the mouth and also because there are large regional differences between the rates of oral clearance or retention of substances dissolved in saliva. Differential patterns therefore develop and are related, via the patterns of salivary flow, to the anatomy and physiology of the mouth. There are general features in these patterns common to all mouths and variations, which relate to characteristics of the individual, which may influence the rates of reactions occurring at different sites. The patterns may be associated with the site-specific patterns of dental disease, and they may have implications with regard to the best use of pharmaceutical agents. This paper describes some of the more recent data, problems and future possibilities in this hitherto unexplored area of oral physiology.

Fluorides↗
PubMed1989

The metabolism of prostaglandin D2. Evidence for the sequential conversion by NADPH and NAD+ dependent pathways.

Cell-free 100,000 g supernatants from human lung converted PGD2 into a product which on HPLC was indistinguishable from 9 alpha, 11 beta-PGF2. The rate of reaction was relatively slow (0.30 +/- 0.03 pmol/min/mg protein). In the presence of NAD+, 9 alpha, 11 beta-PGF2 was itself metabolized at a rate of 1.46 +/- 0.3 pmol/min/mg protein. The product of this reaction was less polar than the substrate and eluted with an HPLC retention time similar to that seen in our previous study where it was identified by GC/MS as being 15-keto-9 alpha, 11 beta-PGF2. There was no evidence for the formation of 13,14-dihydro-15-keto-9 alpha, 11 beta-PGF2. The sequence of metabolism of PGD2 was further established in cell-free supernatants prepared from guinea-pig liver and kidney which have previously been shown to be rich in both 11-ketoreductase and 15-hydroxy-prostaglandin dehydrogenase (PGDH) activities. Reactions contained both NAD+ and a NADPH-regenerating system and demonstrated the sequential metabolism of PGD2 to 9 alpha, 11 beta-PGF2 and ultimately 13,14-dihydro-15-keto-9 alpha, 11 beta-PGF2. Under these conditions the rate of the C-15 oxidation reaction was slower than that of 11-ketoreduction. These observations provide further support for our previous suggestions that the 11-ketoreduction of PGD2 is followed by a PGDH-type reaction, and that these reactions are likely to occur sequentially in vivo provided that the appropriate cofactors are present.

Aged↗
PubMed1989

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles.

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.

Journal Article↗
PubMed1989

Tuft protein: its relationship with the keratins and the developing enamel matrix.

The relationship among tuft protein derived from mature human molars, human skin keratins, and the developing enamel matrix of the rat incisor was investigated using polyclonal antibodies in immunocytochemical and Western blotting techniques. Antibodies to tuft protein and keratin cross-reacted with proteins in the Mr range 50-70 K from demineralized developing enamel, enamel organ, human skin, and tuft extract. An immunocytochemical technique was used to locate the site of cross-reactivity in situ within secretory ameloblasts, enamel, and keratinized epithelium at the ultrastructural level. Antibodies to keratin cross-reacted with cytoplasmic tonofilaments and those inserted into desmosomes. Antibodies to tuft protein, however, did not cross-react with cytoskeletal components but produced labeling of the golgi and secretory vesicles. Labeling with this antibody was also observed within the stratum granulosum of the rat foot pad. It is concluded that tuft protein contains secretory products of the ameloblast that may represent a less specialized product of other epithelial tissues, perhaps related to the keratins.

Ameloblasts↗
PubMed1989

Site-specific variations in the biochemical composition of healthy sheep periodontium.

A topographical biochemical analysis of the periodontal soft tissues was carried out. The protein distribution in the tooth-supporting structures was determined from site by amino acid analysis and was compared with the collagen distribution in tissue protein, based upon hydroxyproline content. The biochemical composition of the periodontal ligament was heterogeneous but some specific patterns of protein and collagen distribution emerged. Protein concentration was highest at the gingival epithelium and adjacent to the cementum. Collagen concentrations were highest at the alveolar bone and below the junctional epithelium adjacent to the tooth. Such patterns may influence the way in which periodontal disease is propagated through the tissue.

Amino Acids↗
PubMed1989

Endopeptidases in the stroma and thylakoids of pea chloroplasts.

Three endopeptidases (EP2, EP3, and EP4) were identified after fractionation of pea (Pisum sativum, var Feltham First) chloroplast stromal extracts. All three were identified by their ability to cleave in vitro-synthesized preplastocyanin to lower molecular weight forms. EP2 is inhibited by phenylmethylsulfonylfluoride, and both EP2 and EP3 are inhibited by the heavy metal chelators 1,10-phenanthroline and EDTA. A further endopeptidase, EP5, was identified in Triton X-100 extracts of thylakoid membranes. Experiments involving contrifugation through a sucrose pad indicate that EP5 either has a high molecular weight or is associated with a thylakoid protein complex. EP5 is effectively inhibited by phenylmethylsulfonylfluoride, iodoacetate, and 1,10-phenanthroline, but not by EGTA. The implications of these results for the analysis of chloroplast protein maturation are discussed, and an improved protocol for the purification of the stromal processing peptidase is described which ensures the removal of EP2, the most active of the stromal peptidases analysed in this study.

Journal Article↗
PubMed1989

Differential release of histamine and eicosanoids from human skin mast cells activated by IgE-dependent and non-immunological stimuli.

1. Cells were dispersed from human foreskin using a mixture of collagenase and hyaluronidase and separated into mast cell-depleted (less than 1%) or enriched (greater than 75%) preparations by density-gradient centrifugation. 2. Challenge of gradient fractions with epsilon-chain-specific anti-human IgE stimulated the release of histamine, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The release of eicosanoids was significantly correlated with that of histamine, suggesting that they are derived from the mast cell population of the dispersate. In highly purified (76.2 +/- 4.2%) mast cell preparations, maximum net release of histamine, PGD2 and LTC4 was 3432 +/- 725, 84.9 +/- 10.8 and 6.6 +/- 1.2 pmol/10(6) nucleated cells. 3. The non-immunological stimuli substance P, vasoactive intestinal peptide (VIP), somatostatin, compound 48/80, morphine and poly-L-lysine released similar amounts of histamine to anti-IgE, but 12 to 21 fold less PGD2 and LTC4. 4. These studies suggest that IgE-dependent and non-immunological stimuli activate human skin mast cells by different secretory mechanisms, a hypothesis supported by our previous findings of differences in Ca2+ requirements and time-course of histamine release. Activation by the non-immunological mechanism may be of importance in vivo due to the close anatomical association between skin mast cells and dermal nerve-terminals containing neuropeptides.

Centrifugation, Density Gradient↗
PubMed1989

Enhancement of leukotriene C4 release from primate airway macrophages by cellular interactions.

1. Cells were obtained from the lungs of Macaque monkeys by bronchoalveolar lavage in order to study the role of cellular interactions in the release of leukotriene C4 (LTC4). 2. In normal monkeys, macrophages were the most abundant cell type recovered, whereas in monkeys sensitized with Ascaris suum there was an increase in the numbers of eosinophils and mast cells recovered. 3. Challenge of cells from both groups of animals with an optimal concentration of opsonized zymosan (OPZ) resulted in the time-dependent release of LTC4 from macrophages. However, release was significantly greater in cells obtained from sensitized donors compared to normal donors. 4. Density-gradient centrifugation of cells lavaged from sensitized donors was used to prepare fractions containing both eosinophils and mast cells. Addition of these cells to macrophage populations obtained from non-sensitized donors caused a significant enhancement of OPZ-induced LTC4 release. In the absence of macrophages no significant release of LTC4 was detected from eosinophil/mast cell-containing fractions stimulated with OPZ, despite the fact that the zymosan had been phagocytosed by the eosinophils. 5. There was a significant correlation between the percentage enhancement of LTC4 release and the number of eosinophils added. However, there was not a significant correlation with the number of mast cells added. 6. These results suggest that a cellular interaction between macrophages and eosinophils may be important in the regulation of mediator synthesis and release. The precise mechanism of this effect remains to be elucidated.

Animals↗
PubMed1989

Effect of a thromboxane receptor antagonist on PGD2- and allergen-induced bronchoconstriction.

In this study we investigated the effect of the selective and potent thromboxane A2 (TxA2) receptor antagonist GR32191 on smooth muscle contraction induced by the TxA2 analogue U46619, prostaglandin (PG) D2, PGF2 alpha, and methacholine (MCh) in guinea pig airways in vitro and the airways response provoked by inhaled PGD2 and MCh in asthmatic subjects in vivo. GR32191 antagonized competitively the contractile responses of all three prostanoids to a similar degree but had no effect on MCh-induced contractions. In asthmatic subjects GR32191, in a single oral dose of 80 mg, did not affect base-line airway caliber or MCh-induced broncho-constriction but caused significant inhibition of PGD2-induced bronchoconstriction, displacing the concentration-response curves to the right by greater than 10-fold. The effect of the same oral dose of GR32191 on allergen-induced immediate bronchoconstriction was subsequently investigated in allergic asthmatic subjects. In individual subjects, GR32191 inhibited to varying degrees the overall bronchoconstrictor response, with the maximum effect occurring between 10 and 30 min after allergen challenge. These studies suggest that prostanoids contribute to the immediate bronchoconstriction induced by inhaled allergen in allergic asthmatics, and that this effect is mediated by stimulation of a thromboxane receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗