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Biomedical subjects

C Robinson

Publications and source records attributed to C Robinson.

At least 55 records · Page 3Linked to original sources
PubMed2001

Multiple pathways used for the targeting of thylakoid proteins in chloroplasts.

The assembly of the chloroplast thylakoid membrane requires the import of numerous proteins from the cytosol and their targeting into or across the thylakoid membrane. It is now clear that multiple pathways are involved in the thylakoid-targeting stages, depending on the type of protein substrate. Two very different pathways are used by thylakoid lumen proteins; one is the Sec pathway which has been well-characterised in bacteria, and which involves the threading of the substrate through a narrow channel. In contrast, the more recently characterised twin-arginine translocation (Tat) system is able to translocate fully folded proteins across this membrane. Recent advances on bacterial Tat systems shed further light on the structure and function of this system. Membrane proteins, on the other hand, use two further pathways. One is the signal recognition particle-dependent pathway, involving a complex interplay between many different factors, whereas other proteins insert without the assistance of any known apparatus. This article reviews advances in the study of these pathways and considers the rationale behind the surprising complexity.

Amino Acid Sequence↗
PubMed2001

Protein targeting by the twin-arginine translocation pathway.

The twin-arginine translocation pathway operates in the thylakoid membrane of chloroplasts and in the plasma membrane of most free-living bacteria. Its main function is to transport fully folded proteins across the membrane. Three important tat genes have been identified and the sequences of the encoded proteins, together with the unusual properties of the pathway, indicate that the Tat system is completely different from other protein translocases.

Arginine↗
PubMed2001

Auxiliary functions in photosynthesis: the role of the FtsH protease.

Oxygenic photosynthesis can be described effectively by using two long-standing models: the Z-scheme and the chemiosmotic hypothesis. However, these models do not reveal the dynamic nature of the thylakoid membrane and the four major complexes that it binds. The composition of the photosynthetic apparatus is continually changing in response to a range of environmental stimuli. In addition, many photosynthetic components have some of the highest turnover rates in Nature. Changes in composition and turnover of photosynthetic components require the degradation of existing and damaged polypeptides and the resynthesis and co-ordinated assembly of new polypeptides and their associated cofactors. This is achieved by several auxiliary functions, including proteolysis, protein targeting and the action of molecular chaperones. Some of the components involved in these functions, such as translocons, chaperones and proteases, have been identified but many of the auxiliary functions of photosynthesis remain uncharacterized. Among the proteases known to be associated with the thylakoids is the zinc metalloprotease FtsH, which might also act as a chaperone. Here we provide an overview of the thylakoid FtsH protease and discuss its role in the maintenance and assembly of the photosynthetic apparatus.

ATP-Dependent Proteases↗
PubMed2001

The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.

Amino Acid Sequence↗
PubMed2001

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli.

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.

Amino Acid Sequence↗
PubMed2001

Cocaine use, hypertension, and end-stage renal disease.

Cocaine use has been associated with both acute renal failure and hypertension (HTN), but only recently have data suggested it may lead to a chronic insidious form of renal failure. We designed a cross-sectional study to compare the association of cocaine use in hemodialysis patients with and without a diagnosis of HTN-related end-stage renal disease (HTN-ESRD). Two hundred one black patients from two outpatient hemodialysis units in an urban community were evaluated. There were 193 eligible patients, 106 men and 87 women, aged 49.28 +/- 14.4 years. A history of significant cocaine use before dialysis was reported by 55 of 193 subjects (28.5%). A diagnosis of HTN-ESRD was reported in 49 of 55 cocaine users (89.1%) compared with 64 of 138 nonusers (46.38%; odds ratio, 9.44; 95% confidence interval, 3.79 to 23.49; P < 0.0005). Of the 113 subjects with HTN-ESRD, 49 subjects (43.4%) had a history of cocaine abuse, either alone or in combination with other drugs. Subjects with HTN-ESRD with cocaine use were younger than those without cocaine use (40.7 +/- 9.0 versus 53.8 +/-15.3 years; P < 0.0005) and had a shorter reported duration of HTN (5.3 +/- 5.4 versus 12.7 +/- 9.8 years; P < 0.0005, adjusted for age and sex). In our urban dialysis population, a clinical diagnosis of HTN-ESRD was strongly associated with a history of cocaine use and earlier onset of ESRD. Cocaine should be considered as a cause of ESRD in patients without a clear cause of renal failure.

Adult↗
PubMed2001

N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro.

N-t-butyl hydroxylamine (NtBHA) delays senescence-dependent changes in human lung fibroblasts (IMR90) (Atamna et al., J. Biol. Chem. 275, 6741-6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary fibroblasts (IMR90). In NtBHA-treated rats, the age-dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence-associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of growth factors. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10- to 15-fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical-induced toxicity to mitochondria.

Aging↗
PubMed2001

The effects of early motor intervention on children with Down syndrome or cerebral palsy: a field-based study.

This study reports the results of a field-based investigation of the effects of two motor intervention approaches--neurodevelopmental treatment and developmental skills--on children with two different diagnoses. The sample included 50 children, of whom 27 were diagnosed as having Down syndrome and 23 as having cerebral palsy. Children had a mean chronological age of 14 months at the beginning of the study. The severity of their motor impairment was assessed with the Gross Motor Function Classification System for Cerebral Palsy. Children's motor functioning was examined at entry into the study and after they received 1 year of motor intervention services. Dependent measures included children's general development, rate of motor development, and quality of movement. Pre-post comparisons indicated that children made significant changes in their motor development age and quality of movement over the course of intervention. However, there was no evidence that motor intervention accelerated development or improved quality of movement beyond what could be expected on the basis of maturation. Furthermore, no differential intervention effects were associated either with children's diagnosis or treatment model. Regression analyses indicated that the rate of motor development children attained after 1 year of intervention was highly related to their rate of development at the onset of intervention and, to a lesser degree, to the number of sessions of intervention children received. These results are discussed in terms of the need for the field of motor intervention to develop new treatment paradigms.

Cerebral Palsy↗
PubMed2001

Academic, motivational, and emotional correlates of adolescent dating.

This study is an examination of the relationship between dating status and academic achievement, academic motivation, depression, and self-esteem; it is an investigation of the differential effects wielded by gender and age (grade level) of the dating adolescent in each of these domains. Participants were a relatively large gender-balanced adolescent group (N = 380) from Grades 8, 10, and 12. Dating status was studied first as a binary variable (frequent versus infrequent dating) and second as a dating spectrum, including steady, frequent, and infrequent dating. Results showed that adolescents who dated frequently (more than once or twice a month), whether they were boys or girls, relatively young (8th grade) or more mature (l0th and 12th grades), exhibited consistently and significantly lower levels of academic achievement and academic motivation and higher levels of depressive symptoms. There were no significant effects of dating status on global self-esteem, but, as hypothesized, subscale analyses revealed important subscale-differentiated effects.

Adolescent↗
PubMed2000

A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen.

A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.

Amino Acid Sequence↗
PubMed2000

Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803.

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.

ATP-Dependent Proteases↗
PubMed2000

RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development.

The CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF(122), is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF(122) prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF(122) and its variant CBTF(98) are associated with translationally quiescent mRNP complexes. We show that CBTF(122) RNA binding activity is both necessary and sufficient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF(122) is insufficient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF(122). Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF(122). Thus, the nuclear translocation of CBTF(122) at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

5' Untranslated Regions↗
PubMed2000

TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export.

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.

Biological Transport↗
PubMed2000

Determination of the ionization state of 11-thioundecyl-1-phosphonic acid in self-assembled monolayers by chemical force microscopy

The present article describes the preparation and preliminary characterization of a novel phosphate-functionalized self-assembled monolayer (SAM) and the determination of the surface ionization states of the phosphate headgroup in aqueous solutions by chemical force microscopy (CFM). The phosphate headgroup used was PO(OH)2, a diprotic acid. The adhesion force between an AFM probe and a flat substrate, both of which were chemically modified with the same phosphate SAM, was also measured as a function of pH and ionic strength. At low ionic strength (10(-4) M), two peaks were observed in the force titration curve (adhesion force versus pH) at pH 4.5 and 8.4. The two peaks are positioned 2.4 and 1.2 pH units higher, respectively, than the acid dissociation constants obtained for the phosphate group free in aqueous solution. At high ionic strength (10(-1) M), the adhesion forces were reduced by 1 order of magnitude and the peaks were replaced by shoulders similar to those previously reported for acid force titrations. On the basis of JKR theory, the surface pKa values of the phosphate group in high ionic strength solutions were found to be 4.5 and 7.7, respectively. However, in light of the effects of ionic strength on the force titration curves, we discuss the applicability of JKR theory to nanoscopic measurements of adhesion force and surface pKa.

Journal Article↗
PubMed2000

Subunit interactions in the twin-arginine translocase complex of Escherichia coli.

A subset of Escherichia coli proteins, in particular cofactor-binding proteins with so-called twin-arginine signal peptides, is transported to the periplasm via the twin-arginine translocation (Tat) pathway. The tatA and tatB genes encode important components of the export system and we have analysed whether the proteins encoded by these genes physically interact. Using co-immunoprecipitation experiments, we show that TatA and TatB do indeed associate with each other. Gel filtration chromatography demonstrates that both proteins are present in a large complex with an apparent molecular mass of approximately 600 kDa, indicating the presence of other components and/or several TatA and TatB subunits. Finally, we show that TatA is stable in the absence of TatB and may participate in a separate complex lacking TatB in wild-type cells.

Bacterial Proteins↗