Understanding the biogenesis of polytopic integral membrane proteins.
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Cell Membrane↗
Biomedical subjects
R J Turner
Publications and source records attributed to R J Turner.
Cutaneous lichen planus and squamous cell carcinoma.
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Adult↗
The effects of sevoflurane on isolated gravid human myometrium.
The volatile anaesthetic agents are known to influence uterine muscle tone. All of the agents studied to date have been found to produce uterine relaxation. This property has been used to produce therapeutic uterine relaxation for difficult obstetric deliveries and the Ex Utero Intrapartum Treatment (EXIT) procedure. This study describes the effects of sevoflurane on isolated human myometrium at concentrations of 0.1, 0.25, 0.5, 0.75, 1.0, 1.5, 2.5 and 3.5 MAC. Sevoflurane produces dose-dependent depression of uterine muscle contractility with an ED50 of 0.94 MAC. Frequency of contraction was increased at concentrations of 2.5 MAC and greater. At concentrations of 3.5 MAC and above, uterine activity was virtually abolished.
Analysis of Variance↗
Airway management on placental support (AMPS)--the anaesthetic perspective.
Neonatal airway obstruction has been reported to have a high mortality. Antenatal diagnosis of this condition is now possible. Anaesthetic and surgical techniques have been developed that allow neonatal airway obstruction to be managed at delivery, while the fetus remains oxygenated via the placental circulation. Three case studies are presented, and the anaesthetic issues for mother and fetus/neonate are discussed with reference to previously published cases of airway management on placental support. In particular, techniques for uterine relaxation and maintenance of placental circulation are explored. The history of these procedures and issues of planning and logistics are also discussed.
Adult↗
Imaging tip formation in single-mode optical fibres.
The formation of probe tips is a crucial step in all forms of scanning probe microscopy (SPM). In this work single-mode optical fibres are chemically etched in a variable temperature bath of etchant solution (HF acid buffered with ammonium fluoride) to produce tips for optical SPM. Tip evolution is monitored by prematurely truncating the etching process and imaging the tip end-structure using atomic force microscopy (AFM). In the case of a visible regime single-mode fibre the AFM images show a remarkable ring structure in the central cladding region and a tip structure in the core with a central depression; this serves to demonstrate the efficacy of chemical etching for converting compositional variation to three-dimensional topography. In the case of a standard, single-mode optical communications fibre the (projected) tip cone angle is assessed from AFM images in the early stages of tip formation. Values of the cone angle thus determined, for different etch conditions, are compared to those predicted by a model in which the independently determined core and cladding etch rates, and core diameter are the sole determinants of the final tip geometry. The model was devised in the context of etching multi-mode fibres and is shown to be valid here for single-mode fibres within the range of experimental accuracy and etch conditions examined.
Journal Article↗
Identification of a twin-arginine leader-binding protein.
The transport and targeting of a number of periplasmic proteins is carried out by the Sec-independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin-arginine-containing leader. We hypothesized that specific leader-binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin-arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N-terminus of glutathione-S-transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell-free extract was passed. Proteins that bound to the leader were then separated and identified by N-terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI. This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N-oxide reductase). A strain carrying a dmsD knock-out mutation showed a loss of anaerobic growth on glycerol-DMSO medium and reduced growth on glycerol-fumarate medium. This work suggests that DmsD is a twin-arginine leader-binding protein.
Amino Acid Sequence↗
Low metabolic rate in scorpions: implications for population biomass and cannibalism.
Scorpions are abundant in arid areas, where their population biomass may exceed that of vertebrates. Since scorpions are predators of small arthropods and feed infrequently across multi-year lifespans, a parsimonious explanation for their observed, anomalously high biomass may be a depressed metabolic rate (MR). We tested the hypothesis that scorpion MR is significantly depressed compared with that of other arthropods, and we also measured the temperature-dependence of the MR of scorpions to quantify the interaction between large seasonal variations in desert temperatures and MR and, thus, long-term metabolic expenditure. Scorpion MR increased markedly with temperature (mean Q(10)=2.97) with considerable inter-individual variation. At 25 degrees C, the MRs of scorpions from two genera were less than 24 % of those of typical terrestrial arthropods (spiders, mites, solpugids and insects) of the same mass. It is likely, therefore, that the low MR of scorpions contributes to their high biomass in arid areas. The combination of high biomass and high production efficiency associated with low MR may also favor a density-dependent "transgenerational energy storage" strategy, whereby juveniles are harvested by cannibalistic adults that may be closely related to their juvenile prey.
Animals↗
Mohs' micrographic surgery for cutaneous squamous cell carcinoma: practical considerations.
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Carcinoma, Squamous Cell↗
A longitudinal study of the role and significance of mattering to others for depressive symptoms.
This paper addresses the hypothesis that the experience of mattering to others is inversely related to depressive symptomatology. We also assess the extent to which mattering overlaps or is redundant with other measures of personal and social resources. Based on multivariate analyses of two waves of data from a large urban community sample (n = 1,300), we identify three important findings. First, women experience substantially higher levels of mattering than do men. Therefore, if men and women had equal levels of mattering, women would report even higher depression scores relative to men. Second, it is clear that mattering and sub-dimensions of social support are substantially more confounded among men than among women. Finally, analyses confirm that mattering is conditionally predictive of depression by gender. Specifically, variations in mattering are predictive of depression for women, but not for men. This relationship is confirmed both cross-sectionally and over time.
Cross-Sectional Studies↗
Transmembrane topology of the secretory Na+-K+-2Cl- cotransporter NKCC1 studied by in vitro translation.
The secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is a member of a small gene family of electroneutral salt transporters. Hydropathy analyses indicate that all of these transporters have a similar general structure consisting of large hydrophilic N and C termini on either side of a central, relatively well conserved, hydrophobic domain. Programs that predict the transmembrane topology of polytopic membrane proteins identify 10-12 putative membrane-spanning segments (MSSs) in this hydrophobic domain; but to date, there is little experimental data on the structure of this region for any of these transporters. In this report, we have studied the transmembrane topology of NKCC1 using an in vitro translation system designed to test the membrane insertion properties of putative MSSs (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Fusion proteins consisting of putative NKCC1 MSSs inserted either (i) between an N-terminal cytosolic anchor sequence and a C-terminal reporter sequence containing multiple N-linked glycosidation sites or (ii) between an N-terminal signal anchor sequence and the same glycosidation flag were expressed in the presence of canine pancreatic microsomes. The glycosidation status of the reporter sequence, which indicated its luminal or extraluminal location in the microsomes, was then used to characterize the signal anchor or stop transfer activity of the inserted MSSs. The results of this experimental analysis yielded a topology scheme consisting of 12 membrane-spanning segments, two pairs of which apparently form rather tight hairpin-like structures within the membrane.
Amino Acid Sequence↗
The role of cysteine residues in tellurite resistance mediated by the TehAB determinant.
TehATehB is a tellurite (TeO(2-)(3)) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 microg K(2)TeO(3)/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 microg/ml. Both TehA and TehB have three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninies. The results showed that cysteine residues in both TehA and TehB play a role in tellurite resistance: A single cysteine change had no effect, however increasing combinations of two or three cysteine substitutions demonstrated strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 microg K(2)TeO(3)/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 microg/ml. Further investigations on the role of cysteines in resistance were studied using thiol reactive reagents on the soluble subunit TehB. These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite and S-adenosyl-l-methionine binding. Studies using native and SDS denaturing PAGE under reducing and oxidizing conditions suggested that a cysteine in TehB is involved in binding tellurite.
Alanine↗
One of two genes encoding glycyl-tRNA synthetase in Saccharomyces cerevisiae provides mitochondrial and cytoplasmic functions.
In the yeast Saccharomyces cerevisiae, two genes (GRS1 and GRS2) encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively). 59% of the sequence of GlyRS2 is identical to that of GlyRS1. Others have proposed that GRS1 and GRS2 encode the cytoplasmic and mitochondrial enzymes, respectively. In this work, we show that GRS1 encodes both functions, whereas GRS2 is dispensable. In addition, both cytoplasmic and mitochondrial phenotypes of the knockout allele of GRS1 in S. cerevisiae are complemented by the expression of the only known gene for glycyl-tRNA synthetase in Schizosaccharomyces pombe. Thus, a single gene for glycyl-tRNA synthetase likely encodes both cytoplasmic and mitochondrial activities in most or all yeast. Phylogenetic analysis shows that GlyRS2 is a predecessor of all yeast GlyRS homologues. Thus, GRS1 appears to be the result of a duplication of GRS2, which itself is pseudogene-like.
Amino Acid Sequence↗
Severe impairment of salivation in Na+/K+/2Cl- cotransporter (NKCC1)-deficient mice.
The salivary fluid secretory mechanism is thought to require Na(+)/K(+)/2Cl(-) cotransporter-mediated Cl(-) uptake. To directly test this possibility we studied the in vivo and in vitro functioning of acinar cells from the parotid glands of mice with targeted disruption of Na(+)/K(+)/2Cl(-) cotransporter isoform 1 (Nkcc1), the gene encoding the salivary Na(+)/K(+)/2Cl(-) cotransporter. In wild-type mice NKCC1 was localized to the basolateral membranes of parotid acinar cells, whereas expression was not detected in duct cells. The lack of functional NKCC1 resulted in a dramatic reduction (>60%) in the volume of saliva secreted in response to a muscarinic agonist, the primary in situ salivation signal. Consistent with defective Cl(-) uptake, a loss of bumetanide-sensitive Cl(-) influx was observed in parotid acinar cells from mice lacking NKCC1. Cl(-)/ HCO(3)(-) exchanger activity was increased in parotid acinar cells isolated from knockout mice suggesting that the residual saliva secreted by mice lacking NKCC1 is associated with anion exchanger-dependent Cl(-) uptake. Indeed, expression of the Cl(-)/ HCO(3)(-) exchanger AE2 was enhanced suggesting that this transporter compensates for the loss of functional Na(+)/K(+)/2Cl(-) cotransporter. Furthermore, the ability of the parotid gland to conserve NaCl was abolished in NKCC1-deficient mice. This deficit was not associated with changes in the morphology of the ducts, but transcript levels for the alpha-, beta-, and gamma-subunits of the epithelial Na(+) channel were reduced. These data directly demonstrate that NKCC1 is the major Cl(-) uptake mechanism across the basolateral membrane of acinar cells and is critical for driving saliva secretion in vivo.
Animals↗
Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins.
A number of previous reports have suggested that inositol 1,4, 5-trisphosphate receptors (IP(3)Rs) are present in the plasma membranes of cells. We confirm this directly in the present study by demonstrating that a significant proportion of the IP(3)Rs found in A431 cells, Jurkat cells, and rat parotid acini can be biotinylated by the extracellular application of sulfo-N-hydroxysuccinimide-biotin to intact cells. This labeling cannot be accounted for by the reaction of sulfo-N-hydroxysuccinimide-biotin with intracellular IP(3)Rs since calnexin and the SERCA2 ATPase, both integral membrane proteins of the endoplasmic reticulum, are not labeled under the same experimental conditions. Individual IP(3)R subtypes were detected using subtype-specific antibodies. A431 cells expressed only the type-3 IP(3)R, and 23% of this protein was in the biotinylated (plasma membrane) fraction. Jurkat cells and rat parotid cells expressed all three IP(3)R subtypes. Contrary to earlier results suggesting that only the type-3 IP(3)R might localize to the plasma membrane, we found that significant amounts (5-14%) of all three subtypes could be identified in the biotinylated fractions of Jurkat and rat parotid cells. Our results suggest a role for IP(3)Rs in plasma membrane as well as intracellular membrane function.
Amino Acid Sequence↗
Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli.
Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.
Amino Acid Sequence↗
The structural unit of the secretory Na+-K+-2Cl- cotransporter (NKCC1) is a homodimer.
The oligomeric state of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid plasma membranes was studied using the reversible chemical cross-linker DTSSP [3, 3'-dithiobis(sulfosuccinimidyl propionate)]. The monomeric apparent molecular mass of NKCC1 is approximately 170 kDa. However, we show here that this protein migrates as a approximately 355 kDa complex on SDS-PAGE gels after membrane treatment with DTSSP, indicating that NKCC1 exists as an oligomer in the plasma membrane. The stability of this oligomer is such that it is not disrupted by solubilization of the membrane by low concentrations of the nonionic detergent Triton X-100 (0.3%) or the mild ionic detergent deoxycholate (20 mM); however, higher concentrations of Triton X-100 or treatment with the denaturing detergent SDS do result in destabilization of the NKCC1 complex. In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis. In these studies, we were unable to detect the presence of any proteins other than NKCC1 itself in the 355 kDa oligomer, suggesting that this complex is an NKCC1 dimer. Strong evidence for this conclusion was provided by a quantitative analysis of the molecular sizes of oligomers formed by full-length NKCC1 and an N-terminally truncated version of NKCC1 expressed in HEK293 cells. Taken together, our data provide convincing evidence that the dominant structural unit of NKCC1 in the plasma membrane is a homodimer.
Animals↗
PUVA-related punctate keratoses of the hands and feet.
BACKGROUND: We have observed, in patients undergoing high-dose PUVA treatment, a type of keratosis not previously described. The lesions usually occur on the sides of the palms or soles and are clinically distinct. They are generally painless and often go unnoticed by patients. OBJECTIVE: We sought to further characterize these lesions both clinically and histologically. METHODS: Patients attending a PUVA clinic were screened for these keratoses. Other PUVA-related complications were recorded. Representative lesions were photographed, and biopsy specimens were taken. RESULTS: Biopsy specimens were taken from lesions in 10 patients. All had plaque psoriasis and had received high UVA doses (>1000 J/cm(2)) through PUVA therapy. All patients had PUVA-induced keratoses elsewhere, but the number varied greatly between patients. The hand and foot keratoses were well defined and circular and had a characteristic histologic appearance, with a sharp demarcation between normal and abnormal markedly pale-staining epidermis. CONCLUSION: These lesions are a further cutaneous manifestation of prolonged PUVA therapy.
Adult↗
A retrospective study of outcome of Mohs' micrographic surgery for cutaneous squamous cell carcinoma using formalin fixed sections.
The surgical management of recurrent or large squamous cell carcinoma (SCC) can be challenging as tumours often extend beyond visible margins. Micrographic surgery is a potentially effective method of ensuring complete clearance of tumour. A retrospective study of all cases of SCC treated by micrographic surgery in this department between 1986 and 1996 has been done. Sixty-one patients were treated using a formalin-fixed paraffin-embedded tissue technique with a median follow-up of 4 years. In two cases there was local recurrence and in three others metastasis to local lymph nodes. The overall cure rate was 92% (56 of 61), which compares favourably with published series using chemosurgery and frozen tissue techniques. The results show that this technique of micrographic surgery is a satisfactory and cost-effective alternative to conventional frozen section techniques in the treatment of SCC. The formalin-fixed tissue method has the advantage of providing high-quality permanent histological sections using existing conventional pathology services.
Adult↗