Biomedical subjects
S Thomson
Publications and source records attributed to S Thomson.
Subpopulations of proteasomes in rat liver nuclei, microsomes and cytosol.
Mammalian proteasomes are composed of 14-17 different types of subunits, some of which, including major-histocompatibility-complex-encoded subunits LMP2 and LMP7, are non-essential and present in variable amounts. We have investigated the distribution of total proteasomes and some individual subunits in rat liver by quantitative immunoblot analysis of purified subcellular fractions (nuclei, mitochondria, microsomes and cytosol). Proteasomes were mainly found in the cytosol but were also present in the purified nuclear and microsomal fractions. In the nuclei, proteasomes were soluble or loosely attached to the chromatin, since they could be easily extracted by treatment with nucleases or high concentrations of salt. In the microsomes, proteasomes were on the outside of the membranes. Further subfractionation of the microsomes showed that the proteasomes in this fraction were associated with the smooth endoplasmic reticulum and with the cis-Golgi but were practically absent from the rough endoplasmic reticulum. Using monospecific antibodies for some proteasomal subunits (C8, C9, LMP2 and Z), the composition of proteasomes in nuclei, microsomes and cytosol was investigated. Although there appear not to be differences in proteasome composition in the alpha subunits (C8 and C9) in the different locations, the relative amounts of some beta subunits varied. Subunit Z was enriched in nuclear proteasomes but low in microsome-associated proteasomes, whereas LMP2, which was relatively low in nuclei, showed a small enrichment in the microsomes. These differences in subunit composition of proteasomes probably reflect differences in the function of proteasomes in distinct cell compartments.
Processing of N3, a mammalian proteasome beta-type subunit.
Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G decreases TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.
Activities of nitric oxide in normal physiology and uremia.
Nitric oxide (NO) generated from arginine exerts a variety of renal and extrarenal physiological and pathophysiological effects. NO is generated by two types of nitric oxide synthases: acutely responsive, constitutive NOS and slower, more persistent inducible NOS (iNOS). The latter is transcriptionally dependent, often stimulated by cytokines. NO regulates glomerular ultrafiltration, tubular reabsorption, and intrarenal renin secretion; many of these renal effects are mediated by interactions with angiotensin II and adrenergic (alpha 2) activity. Decreased NO activity also enhances tubuloglomerular feedback activity, which could contribute to renal vasoconstriction, NaCl retention, and elevated blood pressure. Loss of renal function could influence NO activity via: (1) endothelial dysfunction; (2) decreased arginine synthesis by kidney; (3) responses to arginine analogs that act as NOS inhibitors; (4) increased cytokine activity; and (5) altered oxidation:reduction status of cells, etc. For example, platelet dysfunction in uremia may be caused by cytokine-induced iNOS activation. Moreover, acutely responsive, constitutive NOS activity may be depressed in progressive loss of renal function. Decreased NO activity might contribute to baroreceptor dysfunction observed in hypertension and progressive renal disease. Studies of the impact of uremia suggest that iNOS may be chronically stimulated by cytokines, whereas acutely responsive, constitutive NOS activity may be concurrently depressed.
LMP2+ proteasomes are required for the presentation of specific antigens to cytotoxic T lymphocytes.
BACKGROUND: Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. RESULTS: We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. CONCLUSIONS: Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression.
Catalytic components of proteasomes and the regulation of proteinase activity.
The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that beta-type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Pre1, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these beta-type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulated in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Homeostatic efficiency of tubuloglomerular feedback is reduced in established diabetes mellitus in rats.
We tested the hypothesis that the ability of the tubuloglomerular feedback (TGF) to stabilize renal function is impaired in rats with 7-8 wk of insulin-treated streptozotocin-diabetes. Proximal tubular flow was measured in free-flowing nephrons using a noninvasive optical technique. The homeostatic efficiency of TGF was determined from the fractional compensation for perturbations in ambient flow. Fractional compensation was substantially reduced in diabetic rats. To assess the roles of the proximal tubule and loop of Henle as determinants of TGF efficiency, we tested the effect of diabetes on proximal tubular reabsorption as determined by standard micropuncture and on the ionic content of early distal tubular fluid by employing a microelectrode for on-line measurement of electrical conductivity (TED). Diabetes caused glomerular hyperfiltration and increased fractional proximal tubular reabsorption (FPR), such that late proximal tubular flow (VLP) and early distal tubular flow were unaffected. The increase in FPR was a minor contributor to the overall effect on fractional compensation. Diabetes decreased the ambient TED without affecting the slope of the relationship between VLP and TED. These results demonstrate that the homeostatic, efficiency of the TGF system is reduced in diabetes and that this cannot be fully accounted for by changes in tubular reabsorption. Impaired TGF efficiency renders the diabetic glomerular microvasculature more susceptible to impact from fluctuations in systemic hemodynamics.
Inhibition of local nitric oxide synthase increases homeostatic efficiency of tubuloglomerular feedback.
Nephron filtration rate (SNGFR) and proximal tubular reabsorption are coordinated by the processes of tubuloglomerular feedback (TGF) and glomerulotubular balance (GTB). We examined the role of nitric oxide (NO) in TGF and GTB, by delivering the NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA) into late proximal nephrons by micropuncture in Inactin-anesthetized euvolemic rats. First, we determined the dependence of SNGFR on late proximal tubular flow rate (VLP) during orthograde microperfusion of Henle's loop downstream from an obstructing wax block (open-loop microperfusion). Second, we examined the homeostatic efficiency of the TGF-GTB system, (i.e., its ability to stabilize VLP) by perturbing VLP in free-flowing nephrons, while using a noninvasive optical technique to measure flow immediately upstream from the perturbation (closed-loop perturbation). Third, we tested whether L-NMMA could alter the afferent signal to TGF by changing the relationship between VLP and the flow rate (VED) or ionic content (CED) of early distal tubular fluid during open-loop microperfusion. During open-loop microperfusion, L-NMMA decreased SNGFR at VLP between 10 and 40 nl/min but did not alter the relationship between VLP and VED or CED. During closed-loop perturbation, L-NMMA did not affect ambient VLP but increased the homeostatic efficiency of the TGF-GTB system. The combined effects on SNGFR and ambient VLP suggest that L-NMMA reduces proximal reabsorption. However, this could account for only a small fraction of the increase in homeostatic efficiency, the greater share of which must be mediated within the juxtaglomerular apparatus. It appears that juxtaglomerular NO exerts an upward pressure on SNGFR and reduces the efficiency of the TGF-GTB system in stabilizing VLP at ambient flow rates under euvolemic conditions.
Erroneous automated eosinophil counts in HIV-infected individuals.
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The psychological impact of an abnormal smear report: a personal view from a general practitioner.
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Retinal topography with the Heidelberg Retina Tomograph.
The Heidelberg Retina Tomograph is a confocal laser scanning microscope designed for in vivo three-dimensional imaging of the posterior segment of the eye. Due to its ability to produce highly accurate and reproducible topographic images, this sophisticated technology can most effectively be applied to the evaluation of topography at and around the optic nerve head. The main application is seen to be the detection and monitoring of change which occurs in glaucoma.
Can pulse oximetry detect raised intracompartmental pressure?
Pulse oximetry has been advocated as a simple noninvasive investigation of vascular compromise. Its usefulness in aiding diagnosis of microvascular compromise in a developing compartment syndrome is questioned. This study investigates the reproducibility of pulse oximetry and the effect on arterial haemoglobin saturation of raising limb intracompartmental pressure by compression bandaging. In 32 out of 50 normal subjects there was a difference in percentage saturation between right and left arms, with a 2% difference in 6 people (12%). Percentage saturation fell significantly at average bandage pressures of 80 mmHg (P < 0.0001) and 60 mmHg (P < 0.001). At clinically relevant pressures, the test had a sensitivity of 40.4%. With a greater than 50% risk of a false-negative result, pulse oximetry is not an appropriate additional investigation in the detection of raised intracompartmental pressure.
cDNA cloning of a new type of subunit of mammalian proteasomes.
The primary structure of a new type of subunit (RN3) of rat proteasomes (multicatalytic proteinase complexes) has been determined from the nucleotide sequence of the cDNA. The cDNA encodes a protein of 232 amino acids but the directly determined N-terminal amino acid sequence suggests that the subunit is post-translationally processed to a M(r) = 24k form. Sequence alignments reveal a similarity of RN3 to other proteasome subunits. It can be designated a B-type proteasomal subunit but is not closely related to the beta subunit of the archaebacterial proteinase or to other members of the B group.
IV therapy: a financial feasibility study.
Results of a feasibility study comparing peripheral catheters to midline catheters are summarized. A step-by-step process involving audit studies, cost figures, data collection, calculation parameters, and cost analysis determined the effectiveness of the new technology.
High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.
We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.
Enhanced levels of multicatalytic proteinase mRNAs in Rous sarcoma virus transformed cells.
The multicatalytic proteinase (proteasome; MCP) is a high molecular mass proteinase which is found in all eukaryotic cells. Northern blot analysis of the levels of MCP mRNAs in a Rat-1 fibroblast cell line and in cells transformed with Rous sarcoma virus showed marked increases in the transformed cells. However, the results of immunoblot analysis with anti-MCP antibodies suggested that the MCP protein content of the two cell lines was similar.
Thermomechanical analysis of frozen aqueous systems.
Thermomechanical Analysis (TMA), using the parallel-plate-rheometer mode of the DuPont 943 TMA, was used to evaluate the softening properties of frozen aqueous carbohydrate model systems. A sample (of fixed dimension) of frozen solution was placed between the parallel plates and cooled to -80 degrees C. The sample was then heated at a linear heating rate of 2 degrees C/min to +15 degrees C. The dimension change was plotted as a function of temperature or time, and an estimate was made of the glass transition point and of the thermal softening behavior of the system. Good agreement was found with previously published glass transition data for similar frozen carbohydrate model systems measured by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA).
The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: a possible function in viral fusion?
Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.