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Biomedical subjects

C Robinson

Publications and source records attributed to C Robinson.

At least 109 records · Page 6Linked to original sources
PubMed1999

Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells: factors influencing its measurement.

Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 vs. 2.35 +/- 0.16 nmol/min/10(6) cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered gammaGCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells, at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that gammaGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether gammaGCS has an important role in the early phase of cell proliferation needs further investigation.

Cell Count↗
PubMed1999

Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane.

Over the past decade, some familiar themes have emerged on how proteins are inserted into or translocated across the plant chloroplast thylakoid membrane and bacterial inner membranes. In the SecA and signal recognition particle (SRP) pathways, nucleotides and soluble factors are used to translocate proteins across the membrane bilayer in the unfolded state. However, the delta pH-dependent pathway in thylakoids uses a radically different mechanism: transport of proteins across the membrane is driven by the transmembrane pH gradient, and neither stromal factors nor nucleotide triphosphates are needed. In addition, this pathway, which requires the membrane-bound protein Hcf106, appears to translocate proteins in a tightly folded form. Recently, a similar pathway has been shown to operate in eubacteria, and several of its components have been identified.

Bacterial Proteins↗
PubMed1999

An in vitro study of the use of photodynamic therapy for the treatment of natural oral plaque biofilms formed in vivo.

Seven-day oral plaque biofilms have been formed on natural enamel surfaces in vivo using a previously reported in situ device. The devices are then incubated with a cationic Zn(II) phthalocyanine photosensitizer and irradiated with white light. Confocal scanning laser microscopy (CSLM) of the biofilms shows that the photosensitizer is taken up into the biomass of the biofilm and that significant cell death is caused by photodynamic therapy (PDT). In addition, the treated biofilms are much thinner than the control samples and demonstrate a different structure from the control samples, with little evidence of channels and a less dense biomass. Transmission electron microscopy (TEM) of the in vivo-formed plaque biofilms reveals considerable damage to bacteria in the biofilm, vacuolation of the cytoplasm and membrane damage being clearly visible after PDT. These results clearly demonstrate the potential value of PDT in the management of oral biofilms.

Biofilms↗
PubMed1999

Characterisation of the PsbX protein from Photosystem II and light regulation of its gene expression in higher plants.

The location and expression of the previously uncharacterised photosystem II subunit PsbX have been analysed in higher plants. We show that this protein is a component of photosystem II (PSII) core particles but absent from light-harvesting complexes or PSII reaction centres. PsbX is, however, localised to the near vicinity of the reaction centre because it can be cross-linked to cytochrome b559, which is known to be associated with the D1/D2 dimer. We also show that the expression of this protein is tightly regulated by light, since neither protein nor mRNA is found in dark-grown plants.

Arabidopsis↗
PubMed1999

Inhibition of gelatinase activity in human airway epithelial cells and fibroblasts by dexamethasone and beclomethasone.

The effects of dexamethasone and beclomethasone on gelatinase activity released from lung epithelial cells (A549, NCI-H292 and Calu-3 cell lines and NHBE primary cultures) and human lung fibroblasts (HLF) were investigated. All cells spontaneously released gelatin-degrading activity but the amounts were unaffected by treatment with glucocorticoids. Phorbol myristate acetate (PMA) increased the amount of gelatinase activity in conditioned media prepared from all cell types examined. In epithelial cells, PMA induced the expression of gelatinase B, whereas in HLF the increased gelatinase activity resulted from increased activation of gelatinase A. Dexamethasone and beclomethasone produced concentration-dependent inhibition of PMA-induced gelatinase activity in HLF and epithelial cell lines. In the epithelial cell lines, the inhibition of activity was associated with an attenuation of enzyme induction by PMA. In contrast, primary cultures of human bronchial epithelial cells were unresponsive to dexamethasone at concentrations that were maximally effective at inhibiting gelatinase activity induced in other cells.

Beclomethasone↗
PubMed1999

A plastidial lysophosphatidic acid acyltransferase from oilseed rape.

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotic-type acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1999

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Allergens↗
PubMed1999

Use of a noncontact radiant heat bandage for the treatment of chronic venous stasis ulcers.

A noncontact radiant heat bandage was used for the treatment of chronic venous stasis ulcers (mean duration 4.44 years) in inpatients who had failed aggressive inpatient and outpatient conventional therapy. The noncontact radiant heat bandage was placed over the ulcer for 5 hours daily: three 1-hour heating periods separated by two 1-hour nonheating periods during this 2-week trial. Wound size, status, and pain severity were recorded for each patient. A total of 17 patients with 31 total wounds were enrolled. No adverse effects were noted in any patient. There was improvement in 14/17 total patients during the 2-week inpatient trial and 8/17 patients healed completely after discharge. There was 1 recurrence during an 18-month follow-up. Pain scores were improved in most patients after the bandage was applied. The use of a noncontact radiant heat bandage is a safe and efficacious inpatient therapy for the management of chronic venous stasis ulcers in patients who have failed conventional therapy.

Aged↗
PubMed1998

The sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane.

A subset of lumen proteins is transported across the thylakoid membrane by a Sec-independent translocase that recognizes a twin-arginine motif in the targeting signal. A related system operates in bacteria, apparently for the export of redox cofactor-containing proteins. In this report we describe a key feature of this system, the ability to transport folded proteins. The thylakoidal system is able to transport dihydrofolate reductase (DHFR) when an appropriate signal is attached, and the transport efficiency is almost undiminished by the binding of folate analogs such as methotrexate that cause the protein to fold very tightly. The system is moreover able to transport DHFR into the lumen with methotrexate bound in the active site, demonstrating that the DeltapH-driven transport of large, native structures is possible by this pathway. However, correct folding is not a prerequisite for transport. Truncated, malfolded DHFR can be translocated by this system, as can physiological substrates that are severely malfolded by the incorporation of amino acid analogs.

Aminopterin↗
PubMed1998

Beta2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils.

Beta2-microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11 years. The primary structure of the N-terminal portion of the protein and mass determination by electrospray mass spectrometry demonstrate that beta2-microglobulin, extracted as fibrils by the water extraction procedure, was not glycated and that Asn17 was not deamidated. Limited proteolysis was observed in more than 20% of beta2-microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. Beta2-microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pathway of the native protein. Beta2-microglobulin is stable at neutral pH where it displays a lower tendency to self-aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields beta2-microglobulin with a tertiary structure identical to that of the native form. The CD spectrum in the near-ultraviolet region and the spectrum of the intrinsic fluorescence of Trp overlap those of the native protein, but the CD spectrum in the far-ultraviolet region is affected by the contribution of oligomers created by beta2-microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native beta2-microglobulin.

Amino Acid Sequence↗
PubMed1998

Targeting of thylakoid proteins by the delta pH-driven twin-arginine translocation pathway requires a specific signal in the hydrophobic domain in conjunction with the twin-arginine motif.

Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec- or delta pH-dependent pathway. A twin-arginine motif is essential but not sufficient to direct delta pH-dependent targeting; here we show that a second determinant is located in the hydrophobic region. A highly hydrophobic amino acid is found either two or three residues C-terminal to the twin-arginine in all known transfer peptides for the delta pH-dependent system, and substitution of this residue in the 23-kDa (23K) peptide markedly inhibits translocation. Further, whereas the insertion of twin-arginine in a Sec-dependent precursor does not permit efficient delta pH-dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient delta pH-dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective. We conclude that the core signal for this pathway is a twin-arginine together with an adjacent hydrophobic determinant.

Amino Acid Sequence↗
PubMed1998

Targeting signals for a bacterial Sec-independent export system direct plant thylakoid import by the delta pH pathway.

Preproteins targeted to the Sec-independent protein transport systems of plant thylakoids and of bacteria both have unusual transfer peptides bearing a consensus twin-arginine motif. Possible mechanistic similarity between the two Sec-independent transport pathways was investigated by assessing the ability of bacterial twin-arginine transfer peptides to direct thylakoid import. High efficiency import was observed. This process was demonstrated to occur specifically via the Sec-independent deltapH pathway and to depend on an intact twin-arginine motif on the transfer peptide. These results provide strong evidence for the operation of mechanistically related Sec-independent protein transport pathways in chloroplasts and bacteria.

Amino Acid Sequence↗
PubMed1998

Sec-independent insertion of thylakoid membrane proteins. Analysis of insertion forces and identification of a loop intermediate involving the signal peptide.

A group of membrane proteins are synthesized with cleavable signal sequences but inserted into the thylakoid membrane by an unusual Sec/SRP-independent mechanism. In this report we describe a key intermediate in the insertion of one such protein, photosystem II subunit W (PSII-W). A single mutation in the terminal cleavage site partially blocks processing and leads to the formation of an intermediate-size protein in the thylakoid membrane during chloroplast import assays. This protein is in the form of a loop structure: the N and C termini are exposed on the stromal face, whereas the cleavage site has been translocated into the lumen. In this respect the insertion of this protein resembles that of M13 procoat, which also adopts a loop structure during insertion, and we present preliminary evidence that a similar mechanism is used by another thylakoid protein, PSII-X. However, whereas the negatively charged region of procoat is translocated by an apparently electrophoretic mechanism using the DeltamuH+, the corresponding region of PSII-W is equally acidic but insertion is DeltamuH+ independent. We furthermore show that neutralization of this region has no apparent effect on the insertion process. We propose that a central element in this insertion mechanism is a loop structure whose formation is driven by hydrophobic interactions.

Amino Acid Sequence↗
PubMed1998

An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria.

Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded in Escherichia coli by an unassigned reading frame, yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-arginine translocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.

Bacterial Proteins↗
PubMed1998

Overlapping functions of components of a bacterial Sec-independent protein export pathway.

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.

Bacterial Proteins↗