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Biomedical subjects

A E Clark

Publications and source records attributed to A E Clark.

At least 19 recordsLinked to original sources
PubMed1992

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.

Amino Acid Sequence↗
PubMed1992

Development of an intracellular pool of glucose transporters in 3T3-L1 cells.

The membrane-impermeant bis-mannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine (ATB-BMPA) has been used to study the development of an intracellular pool of glucose transporters in 3T3-L1 cells. The subcellular distributions of the transporter isoforms GLUT1 and GLUT4 were determined by comparing the labeling obtained in cells in which the impermeant reagent only had access to the cell surface and the labeling obtained in digitonin-permeabilized cells. ATB-BMPA labeling showed that only GLUT1 was present in preconfluent fibroblasts and that most of the transporters were distributed to the cell surface. In preconfluent fibroblasts, the 2-deoxy-D-glucose transport activity was approximately 5 times higher than in confluent fibroblasts. ATB-BMPA labeling showed that the decrease in transport as cells reached confluence was associated with a decrease in the proportion of GLUT1 distributed to the cell surface. The sequestration of these transporters was associated with the development of an insulin-responsive transport activity which increased by approximately 2.5-fold compared with unstimulated confluent cells. ATB-BMPA labeling showed that insulin stimulation resulted in an approximately 2-fold increase in surface GLUT1 so that about one-half of the available transporters became recruited to the cell surface. Measurements of the changes in the distribution of both GLUT1 and GLUT4 throughout the differentiation of confluent fibroblasts into adipocytes showed that both transporters were sequestered in parallel. Basal levels of transport and photolabeling remained low throughout the differentiation period when the total pool of transporters (GLUT1 plus GLUT4) was increased by approximately 5-fold. These results suggest that the sequestration process was present before new transporters were synthesized. Thus, the sequestration mechanism develops in confluent growth-arrested fibroblasts although the capacity to sequester additional transporters may increase as differentiation proceeds.

3T3 Cells↗
PubMed1992

Kinetic resolution of the separate GLUT1 and GLUT4 glucose transport activities in 3T3-L1 cells.

A bis-mannose-photolabel-displacement method has been developed for resolving the separate kinetic properties of the glucose transporters GLUT1 and GLUT4, which are both present in 3T3-L1 cells. We have quantified the cell-surface transporter abundance (Bmax.) for the two isoforms by displacing radiolabelled 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D- mannos-4-yloxy)-2-propylamine (ATB-BMPA) by non-labelled ATB-BMPA. In cells acutely treated with insulin, the GLUT1 Bmax. was 0.19 microM and the GLUT4 Bmax. was 0.17 microM. In cells which were chronically treated with insulin, the GLUT1 Bmax. was increased by approximately 4-fold to 0.7 microM, whereas the GLUT4 was decreased by approximately 50% (Bmax. = 0.1 microM). However, this large increase in total concentrations of cell-surface transporters (the sum of GLUT1 and GLUT4 concentrations) was not reflected in a large increase in 3-O-methyl-D-glucose transport, suggesting that GLUT1 makes a smaller contribution to transport than does GLUT4. In acutely insulin-treated cells at 37 degrees C, the apparent kinetic parameters for 3-O-methyl-D-glucose transport were Vapp.max. = 0.52 mM.s-1 and Kapp.m = 12.3 mM. In chronically insulin-treated cells the Vapp.max. = 1.24 mM.s-1 and Kapp.m = 23.0 mM. We have measured the displacement of ATB-BMPA by different concentrations of 3-O-methyl-D-glucose to resolve the separate affinity constants of GLUT1 and GLUT4 for this transported ligand. In acute- and chronic-insulin-treated cells the GLUT1 Km for 3-O-methyl-D-glucose was approximately 20 mM, and the GLUT4 Km for 3-O-methyl-D-glucose was approximately 7 mM. An analysis of these data and the 3-O-methyl-D-glucose transport rates was carried out to calculate transport capacity (TK values) for the two isoforms at 37 degrees C. In acute- and chronic-insulin-treated cells the TK values were 0.36 x 10(4) mM-1.min-1 for GLUT1 and 1.13 x 10(4) mM-1.min-1 for GLUT4. Thus GLUT1 has an approximately 3-fold lower transport capacity than GLUT4 at low concentrations of transported sugar. The lower GLUT1 transport capacity was shown to be mainly due to the high Km of GLUT1. The calculated turnover numbers were 7.2 x 10(4) min-1 for GLUT1 and 7.9 x 10(4) min-1 for GLUT4.

3-O-Methylglucose↗
PubMed1992

Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins.

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.

3T3 Cells↗
PubMed1992

Compositional dependence of calcium phosphate layer formation in fluoride Bioglasses.

Bioglasses form a double layer composed of apatite and a silica-rich layer when placed in a simulated physiological solution as well as in living tissue [A.E. Clark, C.G. Pantano, and L. L. Hench, "Auger spectroscopic analysis of bioglass corrosion films," J. Am. Ceram. Soc., 59(1-2), 37-39 (1976).]. In the present work, the mechanisms of the calcium phosphate layer and the silica-rich layer formation of fluoride Bioglasses in Tris-buffer solution are studied as a function of the SiO2 content. Fourier Transform Infrared Reflection Spectroscopy (FTIRS) is used to investigate the mechanism of formation of calcium phosphate and silica-rich layers on the glass surface. Ion concentration in reacted solution and elemental depth profiles are obtained by Induced Coupled Plasma Atomic Emission Spectrometry (ICP) and Auger Electron Spectroscopy (AES), respectively. Si--O bonds with one nonbridging oxygen and Si--O--Si bonds form at the early stage of reaction. Strong phosphorus ion uptake occurs when an amorphous calcium phosphate layer crystallizes. Glasses with high silica content (conventional glass) form the silica-rich layer first followed by a calcium phosphate layer on top. However, glasses with low silica content (invert glass) form both layers simultaneously. The rate of apatite formation decreases with increasing SiO2 content, especially in the region of conventional glass compositions. Ion release rates decreases as SiO2 content increases, with a significant change occurring at the compositional boundary between invert and conventional glasses.

Biocompatible Materials↗
PubMed1992

Injectable bioglass as a potential substitute for injectable polytetrafluoroethylene.

Injectable polytetrafluoroethylene (Teflon) and collagen have inherent problems that may prevent their long-term success. In search of a different injectable biomaterial we confirmed in rabbits the safety of injected Bioglass particles suspended in sodium hyaluronate. A second study was performed testing the ability of the Bioglass suspension to increase urethral resistance in pigs. Bioglass particles in suspension have the potential to substitute for polytetrafluoroethylene or collagen in the treatment of urinary incontinence or vesicoureteral reflux.

Animals↗
PubMed1991

Determination of the rates of appearance and loss of glucose transporters at the cell surface of rat adipose cells.

We have used an impermeant bis-mannose compound (2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos+ ++- 4-yloxy)-2- propylamine; ATB-BMPA) to photolabel the glucose transporter isoforms GLUT4 and GLUT1 that are present in rat adipose cells. Plasma-membrane fractions and light-microsome membrane fractions were both labelled by ATB-BMPA. The labelling of GLUT4 in the plasma membrane fraction from insulin-treated cells was approximately 3-fold higher than that of basal cells and corresponded with a decrease in the labelling of the light-microsome fraction. In contrast with this, the cell-surface labelling of GLUT4 from insulin-treated intact adipose cells was increased approximately 15-fold above basal levels. In these adipose cell preparations, insulin stimulated glucose transport activity approximately 30-fold. Thus the cell-surface labelling, but not the labelling of membrane fractions, closely corresponded with the stimulation of transport. The remaining discrepancy may be due to an approx. 2-fold activation of GLUT4 intrinsic transport activity. We have studied the kinetics of trafficking of transporters and found the following. (1) Lowering the temperature to 18 degrees C increased basal glucose transport and levels of cell-surface glucose transporters by approximately 3-fold. This net increase in transporters probably occurs because the process of recruitment of transporters is less temperature-sensitive than the process involved in internalization of cell-surface transporters. (2) The time course for insulin stimulation of glucose transport activity occurred with a slight lag period of 47 s and a t 1/2 3.2 min. The time course of GLUT4 and GLUT1 appearance at the cell surface showed no lag and a t 1/2 of approximately 2.3 min for both isoforms. Thus at early times after insulin stimulation there was a discrepancy between transporter abundance and transport activity. The lag period in the stimulation of transport activity may represent the time required for the approximately 2-fold stimulation of transporter intrinsic activity. (3) The decrease in transport activity after insulin removal occurred with a very high activation energy of 159 kJ.mol-1. There was thus no significant decrease in transport or less of cell-surface transporters over 60 min at 18 degrees C. The decrease in transport activity occurred with a t1/2 of 9-11 min at 37 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗
PubMed1991

Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes.

A new method for photoaffinity labeling of glucose transporters has been used to compare the effects of glucose-starvation, acute-insulin, and chronic-insulin treatments on the cell-surface glucose transporters in 3T3-L1 adipocytes. Starvation alone increased the cell-surface levels of GLUT1 and GLUT4 by approximately 4- and approximately 2-fold, respectively. As shown by Calderhead, D, M., Kitagawa, K., Tanner, L.T., Holman, G.D., and Lienhard, G.E. (1990) J. Biol. Chem. 265, 13800-13808) acute-insulin treatment increased cell-surface GLUT1 and GLUT4 by approximately 5- and approximately 15-fold respectively. In contrast to this, chronic-insulin treatment gave a further 3-4-fold increase in both cell-surface and total cellular GLUT1, but availability of GLUT4 at the cell-surface was down-regulated to half the level found in the acute treatment but with no change in the total cellular level. This effect occurred in starved and non-starved cells and suggests that starvation, acute-insulin, and chronic-insulin treatments regulate glucose transporter availability through independent mechanisms. The down-regulation of GLUT4 reached a maximally reduced cell-surface level in 6 h while the rise in GLUT1 reached a maximum after 24-48 h. The rise in GLUT1 appeared to compensate for the decline in cell-surface GLUT4 as glucose transport activity was further increased during the long term treatment with insulin. The down-regulation of GLUT4 due to the chronic-insulin treatment is associated with a marked resistance of the cells to restimulate glucose transport and particularly to recruit further GLUT4 to the cell-surface following an additional insulin treatment. The defect appears to be in the signaling mechanism that is responsible for translocation.

Adipose Tissue↗
PubMed1990

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester.

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).

Adipose Tissue↗
PubMed1990

Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose.

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.

Azides↗
PubMed1989

An investigation of Bioglass powders: quality assurance test procedure and test criteria.

Bioglass, in bulk form, has been used to bond to the body's hard and soft tissues in many surgical and dental applications over the past 18 years. There is also a large potential use of Bioglass, in powder or paste from, to fill irregular-shaped bony defects caused by local or systemic disease, congenital malformations, or trauma in the fields of otolaryngology and dental sciences. A quality assurance test procedure to analyze powdered Bioglass to guarantee that the bioactivity of the glass has been maintained during manufacturing was developed. Herein states the quality assurance test procedure, test variables, and test criteria to assure the bioactivity of Bioglass powders made from known bioactive bulk Bioglass samples. Also described herein is the path taken to develop the test procedure and test criteria and experiments performed to study the test variables.

Bone Cements↗
PubMed1987

Residual alveolar ridge maintenance with a new endosseous implant material.

In an effort to maintain the height of the residual alveolar ridge, Bioglass cones were placed in fresh sockets after tooth extraction. Today the emphasis is on maintaining the residual alveolar ridge instead of letting it resorb and subsequently resorting to heroic efforts for augmentation. To date, 242 cone implants have been placed in 29 patients. Implant follow-up observation time ranged from 12 to 32 months with an average of 19.9 months. Twenty-seven patients have been fitted with dentures. To date seven implants (2.9%) have been lost. The Bioglass implants appear to be highly biocompatible as evidenced by absence of infection, normal mucosal healing, and radiographic evidence of bone healing around the implants. Evaluation of several implants reexposed surgically revealed new bone in direct contact with the immobile implants circumferentially. The problem of implant dehiscence that started to occur after approximately 10 months in other investigations had not manifested itself in the Bioglass implant patients who have been wearing their dentures for at least 12 months.

Alveolar Process↗
PubMed1987

An unusual presentation of temporal arteritis.

Serious ocular and systemic complications may result from inflammatory processes of the orbit. Temporal arteritis is one such process that can affect the eye. The patient described herein is unusual because she presented initially with signs and symptoms similar to orbital cellulitis. As her disease evolved, she displayed marked, hemorrhagic chemosis with some limitation of abduction and resistance to retropulsion on the involved side. Laboratory and radiologic findings eliminated orbital cellulitis, limited Wegener's granulomatosis, and other systemic inflammatory disorders and led to the diagnosis of temporal arteritis. To our knowledge, this complex of presenting signs has not been previously reported.

Aged↗
PubMed1985

DNA repeat length in chromatin from murine bone marrow and L1210 leukaemia cells.

Previous studies have suggested that 1-(4-amino-2-methylpyrimidine-5-yl)-methyl-3-(2-chloroethyl) -3-nitrosoureahydrochloride (ACNU) and 1,(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) bind specifically to the nucleosomal DNA of murine bone marrow and L1210 leukaemia cells whereas the glucose nitrosoureas, 2-(3-(2-chloroethyl)-3-nitrosoureido)-2-deoxy-D-glucopyranose, (chlorozotocin, CLZ) and 1-(2-chloroethyl)-3-(-D-glucopyranosyl)-1-nitrosourea (GANU), bind preferentially to the linker DNA of bone marrow but not tumour cell chromatin. In order to provide an explanation for this differential, the DNA repeat and linker lengths in murine bone marrow and L1210 leukaemia cells were measured using electrophoresis of micrococcal nuclease-digested DNA. The linker length of bone marrow chromatin was approximately 22% longer than that in L1210 leukaemia cells from mouse ascites. The linker length of L1210 cells maintained in suspension culture was 27% less than in those from ascites fluid. The tissue-specific toxicity of sugar nitrosoureas and the differential binding of these drugs to chromatin does not appear to correlate quantitatively with differences in DNA linker length.

Animals↗