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Biomedical subjects

C Robinson

Publications and source records attributed to C Robinson.

At least 271 records · Page 15Linked to original sources
PubMed1991

Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.

Amino Acid Sequence↗
PubMed1991

Targeting of ricin A chain into pea chloroplasts.

A chimaeric gene was constructed encoding the pre-sequence of the 33 kDa oxygen-evolving complex protein from wheat (a thylakoid lumen protein) linked to ricin A chain. The fusion protein is efficiently imported by isolated pea chloroplasts and localised partly in the stroma, with the remainder bound to the stromal surface of the thylakoids. The imported protein is fully processed by both the stromal and thylakoidal processing peptidases, indicating that partial or complete translocation across the thylakoid membrane has taken place.

Amino Acid Sequence↗
PubMed1991

Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol and targeted into the thylakoid lumen by bipartite presequences. In this report, we describe conditions for the efficient import of each of these proteins by isolated pea thylakoids. Import of the 33-kDa protein requires both light and stromal extract. The probable function of the stromal extract is to provide stromal processing peptidase to remove the first "envelope transit" signal of the presequence. Import of the 23-kDa protein is also driven by light, but stromal extract is not required for import; furthermore, efficient import is still observed if the precursor is modified to completely block cleavage by residual stromal processing peptidase activity. The intermediate form of the 23-kDa protein, which is generated by incubation of the precursor protein with stromal processing peptidase, is also efficiently imported. The results indicate that the thylakoidal protein transport system can import both the precursor and intermediate forms of the 23-kDa protein, but probably only the intermediate form of the 33-kDa protein.

Adenosine Triphosphate↗
PubMed1991

A proton gradient is required for the transport of two lumenal oxygen-evolving proteins across the thylakoid membrane.

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol as larger precursors and transported into the thylakoid lumen via stromal intermediate forms. We have investigated the energetics of protein transport across the thylakoid membrane using import assays that utilize either intact chloroplasts or isolated thylakoids. We have found that the light-driven import of the 23-kDa protein into isolated thylakoids is almost completely inhibited by electron transport inhibitors or by the ionophore nigericin but not by valinomycin. These compounds have similar effects in chloroplast import assays: precursors of both the 33- and 23-kDa proteins are imported and processed to intermediate forms in the stroma, but transport into the thylakoid lumen is blocked when electron transport is inhibited or nigericin is present. These results indicate that the transport of these proteins across the thylakoid membrane requires a protonmotive force and that the dominant component in this respect is the proton gradient and not the electrical potential.

Biological Transport↗
PubMed1991

Transport of proteins into chloroplasts. The thylakoidal processing peptidase is a signal-type peptidase with stringent substrate requirements at the -3 and -1 positions.

The transport of proteins across the thylakoid membrane in higher plant chloroplasts is usually mediated by an amino-terminal peptide extension which is subsequently removed by a specific thylakoidal processing peptidase. We have previously shown that the reaction specificity of this enzyme is very similar to those of signal peptidases located in the endoplasmic reticulum and bacterial plasma membrane. In the present report, the reaction mechanism of the thylakoidal peptidase has been investigated by substituting a variety of amino acids for the alanine residues at the -3 and -1 positions of a thylakoid lumen protein precursor. Small neutral side chains are known to be essential at these positions for cleavage by signal peptidases, and we find that these residues likewise play a critical role in defining the thylakoidal processing peptidase cleavage site. However, the requirements of the thylakoidal enzyme at these sites are significantly more restrictive than those of the bacterial or endoplasmic reticulum peptidases. Whereas leucine at the -3 position in the substrate is tolerated by the latter two enzymes, cleavage by the thylakoidal peptidase is almost completely inhibited. At the -1 position the presence of alanine appears to be critical; substitution of this residue by glycine, serine, threonine, leucine, lysine, or glutamate leads to either substantial or complete inhibition of cleavage at this site. Substitutions at either -3 or -1 which blocked cleavage at the correct site led to cleavage taking place at an alternative site, probably after the -21 residue.

Amino Acid Sequence↗
PubMed1991

The full precursor of the 33 kDa oxygen-evolving complex protein of wheat is exported by Escherichia coli and processed to the mature size.

A full-length cDNA encoding the precursor of the lumenal 33 kDa oxygen-evolving complex protein from wheat was inserted into a prokaryotic expression vector. Cell-free transcription-translation of this construct generates a precursor protein of the correct size. However, when expressed in Escherichia coli, the protein is quantitatively exported into the periplasm and processed to the mature size. The results indicate that the thylakoid transfer sequence of this precursor can function as an internal E. coli export signal.

Biological Transport↗
PubMed1991

Inflammatory mediators and cellular infiltration of the lungs in a guinea pig model of the late asthmatic reaction.

Alterations in cell numbers, vascular permeability, and concentrations of various inflammatory mediators in the lung were measured in a guinea pig model of the late asthmatic reaction. Animals sensitized by inhalation of ovalbumin were challenged with an aerosol of ovalbumin or saline, and bronchoalveolar lavage fluid (BALF) and peripheral blood were collected after periods ranging from 5 min to 72 h. Increased vascular leakage within the lungs was indicated by elevated BALF/plasma albumin ratios at all time points, and was maximal 6 h after challenge. There were increased numbers of eosinophils in BALF by 6 h after challenge and they remained elevated at least until 72 h. A corresponding increase in the proportion of blood leukocytes represented by eosinophils was observed at 6 and 17 h, which suggests that these cells may be drawn to the lung following their release into the circulation, but by 72 h the proportion in blood had returned to normal. A transitory neutrophilia was evident in BALF and blood 6 h after allergen exposure, but there were no allergen-induced changes in BALF numbers of macrophages, lymphocytes, epithelial cells, or mast cells (as assessed by concentrations of cell-associated histamine). beta-Glucuronidase activity was significantly increased in BALF of guinea pigs at 2 h and 17 h following challenge. The degree to which eicosanoids can be recovered in BALF was investigated by instilling a range of tritiated compounds into the lungs of normal guinea pigs at the time of lavage. Ratio high-performance liquid chromatography revealed that there had been little metabolism of the eicosanoids recovered in BALF. However, there was evidence for a rapid removal of these mediators from the lung, a process which will militate against their accurate quantitation in BALF. Histamine, prostaglandin D2, and thromboxane B2 were detected in BALF but did not differ between treatment groups, and levels showed no simple relationship with the other inflammatory changes measured.

Albumins↗
PubMed1991

Distribution of fluoride in sound and periodontally diseased human cementum.

Fluoride distribution was investigated by an abrasive micro-sampling technique. The fluoride concentration increased with age in both sound and diseased cementum. In sound teeth, the fluoride profiles (distribution of fluoride from the surface to interior) of the middle and apical cementum were similar. In the diseased cementum the profile of the middle region tended to be more variable than that of the apical cementum, suggesting a possible effect of the oral environment on the fluoride profile at this site.

Adult↗
PubMed1991

Effect of periodontal disease ("broken mouth") on the distribution of matrix macromolecules in the sheep periodontium.

A topographical, biochemical analysis of the soft connective tissues of sheep periodontia diagnosed clinically as showing periodontitis was carried out and compared with healthy periodontal tissue. Protein content was mapped on a site-to-site basis and compared with the distribution of collagen for both healthy and diseased tissue using amino acid analysis. Diseased tissue was more biochemically homogeneous than corresponding healthy tissue. Protein content per cent was in general greater but collagen content was greatly reduced. Tissue adjacent to radicular cementum appeared to be most markedly affected by the disease process. The apparent selective loss of non-proteinaceous components along with loss of collagen attachment may explain the direction in which periodontal disease is propagated through the tissue.

Amino Acids↗
PubMed1991

Characterization of the enamel ultrastructure and mineral content in hypoplastic amelogenesis imperfecta.

Amelogenesis imperfecta (AI) comprises a diverse group of hereditary enamel disorders that are characterized by hypoplastic and in some cases hypomineralized defects. The specific biochemical abnormalities remain unknown for all AI types, making histologic and chemical analyses of affected dentitions essential for resolving the etiology of AI. The purpose of this investigation was to characterize the ultrastructure and mineral content of smooth hypoplastic AI enamel. The AI enamel showed no evidence of surface pitting and was uniformly reduced in thickness by approximately 60% compared with control enamel. Imbibition studies indicated that the AI enamel was generally porous. The first 30 microns of AI enamel adjacent to the dentinoenamel junction was translucent with poorly formed prisms. Abnormal prism structure was seen throughout the AI enamel. Amorphous, presumably organic material that may have been retained enamel protein was also seen. Although the crystallite widths were similar in both AI and normal enamel, the AI teeth showed areas where the crystallite order and continuity appeared disrupted. The mean mineral content was similar for all variables measured except sodium, which was significantly lower in the AI teeth. The calcium concentration was very low in the AI enamel directly adjacent to the dentinoenamel junction and showed a steeper concentration gradient moving from the dentin to the surface compared with control teeth. It may be concluded that the ameloblasts in smooth hypoplastic AI produce a tissue of reduced thickness, which is excessively porous and displays alterations in its ultrastructural organization.

Ameloblasts↗
PubMed1991

Visual control of locomotion: strategies for changing direction and for going over obstacles.

Dynamics of gait adjustments required to go over obstacles and to alter direction of locomotion when cued visually were assessed through the measurement of ground reaction forces, muscle activity, and kinematics. The time of appearance of obstacles of varying heights, their position within the step cycle, and cue lights for direction change were varied. Direction change must be planned in the previous step to reduce the acceleration of the body center of mass toward the landing foot to 0. The inability of steering within the step cycle is due to the incapacity of muscles to rotate the body and translate it along the mediolateral axes. For obstacle avoidance, Ss systematically manipulated the gait patterns as a function of obstacle height and position and the time available within the ongoing step. Greater supraspinal involvement in control of locomotion is found.

Adult↗
PubMed1991

Fe-Chelate Reductase Activity of Plasma Membranes Isolated from Tomato (Lycopersicon esculentum Mill.) Roots : Comparison of Enzymes from Fe-Deficient and Fe-Sufficient Roots.

Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (-Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3-))(2). NADH and Fe(3+)(citrate(3-))(2)K(m) values for reductase in PM from +Fe and -Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from -Fe tomatoes. The pH optimum for Fe-chelate reductase was 6.5. Fe-chelate reductases from -Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n-octyl beta-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and -Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from -Fe PM. We conclude that PM from roots of +Fe and -Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM.

Journal Article↗
PubMed1991

Structural and secretory characteristics of bovine lung and skin mast cells: evidence for the existence of heterogeneity.

We have examined cells dispersed enzymatically from three different sites in the bovine lung (tracheal mucosa, bronchial mucosa and parenchyma) and the skin, in order to ascertain whether the bovine model could be used to study mast cell heterogeneity. Histochemically there were two sub-populations of mast cells present in both lung and skin (on the basis of toluidine blue staining and the sensitivity to formalin fixation), but their proportions were similar in all sites studied. Skin mast cells contained approximately twice the amount of histamine than their counterparts in the lung (P less than 0.05). Functional heterogeneity was examined by in vitro release of histamine following secretagogue challenge. Calcium ionophore induced a substantial release of histamine; skin mast cells releasing significantly more histamine than any of the lung mast cells (at 10 microM ionophore, 37.1% and 20.7% net histamine release, respectively, P less than 0.05), although the time-course of release from the two tissues was similar. The neuropeptides vasoactive intestinal peptide and somatostatin induced a modest but statistically significant release of histamine from both skin and lung mast cells, whilst substance P only induced histamine secretion from skin mast cells. A range of other potential immunological and non-immunological secretagogues was unsuccessful in eliciting histamine release from mast cells in any of the tissues. We conclude that there were no convincing histochemical differences between mast cells from the sites examined in the lung or skin. Additionally, there was no discernable functional heterogeneity between mast cells within the lung, but functional differences were evident between mast cells of the bovine lung and skin. However, in the absence of a suitable immunological stimulus the bovine model cannot be regarded as a good model of mast cell heterogeneity.

Animals↗
PubMed1991

In vitro modulation of the eosinophil-dependent enhancement of the permeability of the bronchial mucosa.

1. Basolateral to apical albumin flux has been measured in sheets of bovine bronchial and tracheal mucosa mounted in vitro. 2. Addition of guinea-pig peritoneal eosinophils or neutrophils to the basolateral side of such tissues had no significant influence on the transmucosal flux of albumin in either the bronchial or tracheal mucosa. 3. Stimulation of eosinophils or neutrophils by the calcium ionophore A23187, or by their presentation to an opsonized airways mucosa, resulted in a significant increase in the transbronchial flux of albumin. This effect was seen after only 60 min incubation of the leucocytes with the bronchial mucosa, and was no greater when the contact time was extended to 180 min. Incubation of bronchial mucosal tissues with 1 mg ml-1 polyarginine for 3 h produced a significant increase in albumin flux, but was ineffective at 0.5 mg ml-1. 4. In contrast to the bronchial mucosa, the tracheal mucosa appeared resistant to the effects of stimulated eosinophils and neutrophils. 5. The lipoxygenase inhibitor AA-861 failed to influence the ability of eosinophils to augment the transmembrane flux of albumin. However, insertion of a Millipore filter mask between the eosinophils and the bronchial mucosa significantly inhibited the eosinophil-dependent enhancement of mucosal permeability. 6. The broad spectrum antiproteinase alpha 2-macroglobulin achieved almost total ablation of the action of stimulated eosinophils in the bronchial mucosa. These results suggest that proteinases may make a significant contribution to the genesis of epithelial injury, whereas leukotrienes do not.

Animals↗
PubMed1991

Distribution of fluoride in the cementum of human deciduous canines.

This study was undertaken to determine the fluoride distribution in human deciduous cementum. Ten pairs of sound deciduous canines, extracted from children aged 6-9 years, were examined. The teeth were sectioned, and specimens of cementum were removed from the buccal or lingual aspect of the sections. Samples were removed sequentially by abrasion, from the cementum surface to the cementum-dentine junction, and their fluoride content determined as described earlier. The concentrations of fluoride were highest at or near the cementum surface and decreased towards the interior of the tissue. Concentrations and patterns of fluoride distribution were characteristic of the individual subjects. The distribution patterns of fluoride in the contralateral teeth from the same subject were also fairly similar.

Child↗