Biomedical subjects

Q Chen

Publications and source records attributed to Q Chen.

At least 451 records · Page 25Linked to original sources

PubMed1998

Genome analysis of Thinopyrum intermedium and Thinopyrum ponticum using genomic in situ hybridization.

Genomic in situ hybridization (GISH) using genomic DNA probes from Thinopyrum elongatum (Host) D.R. Dewey (genome E, 2n = 14), Thinopyrum bessarabicum (Savul. & Rayss) A. Löve (genome J, 2n = 14), and Pseudoroegneria strigosa (M. Bieb.) A. Löve (genome S, 2n = 14), was used to examine the genomic constitution of Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (2n = 6x = 42) and Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey (2n = 10x = 70). Evidence from GISH indicated that hexaploid Th, intermedium contained the J, Js, and S genomes, in which the J genome was related to the E genome of Th. elongatum and the J genome of Th. bessarabicum. The S genome was homologous to the S genome of Ps. strigosa, while the Js genome referred to modified J- or E-type chromosomes distinguished by the presence of S genome specific sequences close to the centromere. Decaploid Th. ponticum had only the two basic genomes J and Js. The Js genome present in Th. intermedium and Th. ponticum was homologous with E or J genomes, but was quite distinct at centromeric regions, which can strongly hybridize with the S genome DNA probe. Based on GISH results, the genomic formula of Th. intermedium was redesignated JJsS and that of Th. ponticum was redesignated JJJJsJs. The finding of a close relationship among S, J, and Js genomes provides valuable markers for molecular cytogenetic analyses using S genome DNA probes to monitor the transfer of useful traits from Th. intermedium and Th. ponticum to wheat.

Centromere↗

PubMed1998

Ionizing radiation stimulates mitochondrial gene expression and activity.

Radiation-induced gene expression was examined in cells of a radioresistant human glioblastoma cell line, T98G, using RNA fingerprinting by arbitrary primer polymerase chain reaction. Three of the differentially induced transcripts were cloned and identified as the mitochondrially encoded cytochrome c oxidase (MTCO) subunits 1 and 2, and NADH dehydrogenase subunit 4. The levels of all three mRNAs were increased 1 h after irradiation, with elevated expression persisting for at least 24 h. Similar to radiation, other oxidants lead to induction of MTCO1, an effect which could be prevented by the antioxidant pyrrolidine dithiocarbamate. These results indicate that the increase in mitochondrial gene expression is mediated by oxidative stress. Mitochondria could be a target of signaling by ionizing radiation and oxidants since it is known to be at the site of cellular oxidative stress. The proteins encoded by MTCO1 and other mitochondrial mRNAs characterized are part of the mitochondrial respiratory chain which produces adenosine triphosphate through the process of oxidative phosphorylation. Adenosine triphosphate levels and the mitochondrial membrane potential were found to be increased significantly after irradiation, while mitochondrial mass and mitochondrial DNA levels were unaffected. These data demonstrate the specificity of changes in mitochondrial activity and gene expression after irradiation.

Adenosine Triphosphate↗

PubMed1997

Structure-based discovery of ligands targeted to the RNA double helix.

Ligands capable of specific recognition of RNA structures are of interest in terms of the principles of molecular recognition as well as potential chemotherapeutic applications. We have approached the problem of identifying small molecules with binding specificity for the RNA double helix through application of the DOCK program [Kuntz, I. D., Meng, E. C., and Shoichet, B. K. (1994) Acc. Chem. Res. 27, 117-123], a structure-based method for drug discovery. A series of lead compounds was generated through a database search for ligands with shape complementarity to the RNA deep major groove. Compounds were then evaluated with regard to their fit into the minor groove of B DNA. Those compounds predicted to have an optimal fit to the RNA groove and strong discrimination against DNA were examined experimentally. Of the 11 compounds tested, 3, all aminoglycosides, exhibited pronounced stabilization of RNA duplexes against thermal denaturation with only marginal effects on DNA duplexes. One compound, lividomycin, was examined further, and shown to facilitate the ethanol-induced B to A transition in calf thymus DNA. Fluorine NMR solvent isotope shift measurements on RNA duplexes containing 5-fluorouracil provided evidence that lividomycin binds in the RNA major groove. Taken together, these results indicate that lividomycin recognizes the general features of the A conformation of nucleic acids through deep groove binding, confirming the predictions of our DOCK analysis. This approach may be of general utility for identifying ligands possessing specificity for additional RNA structures as well as other nucleic acid structural motifs.

Aminoglycosides↗

PubMed1997

Regulation of hepatic cytochrome P450 2C11 by glucocorticoids.

Previous studies have shown that the expression of cytochrome P450 2C11 is increased in primary hepatocyte culture in the presence of 10(-8) M dexamethasone (DEX). We and others have demonstrated that injection of rats with DEX resulted in 2C11 repression. In the present study, we show that regulation of 2C11 expression in hepatocytes by glucocorticoids is biphasic. Low concentrations (<10(-8) M) of DEX activate 2C11 expression, while higher concentrations of (>10(-7) M) suppress it. Corticosterone has a similar biphasic effect, although this physiological glucocorticoid was less potent than DEX. The transition between activation and suppression of 2C11 expression happens at glucocorticoid concentrations relevant to the transition between normal and stress levels of the hormones. Both the inductive and suppressive effects of glucocorticoids are blocked by RU486, a glucocorticoid receptor antagonist. We postulate that the biphasic nature of the 2C11 response to glucocorticoids may result in a high sensitivity of this P450 to stressful stimuli.

Animals↗

PubMed1997

Distribution and regulation of ornithine decarboxylase activity along the villus-crypt axis in the small intestine.

Distribution of ornithine decarboxylase activity in rat intestinal villi and crypts was determined by serially sectioning frozen mucosa and measuring enzyme activity in pools of sections composed of villi or crypts. Contents of the pools was determined by histological examination of representative sections, and simultaneous measurement of sucrase as a marker of villus samples demonstrated excellent separation of villi and crypts. In fasted and ad lib fed rats, enzyme activity was highest in the villus-crypt junctional area and in crypts (P < 0.05). Refeeding after a fast increased enzyme activity 15-fold, with greatest activity in villus tips and the villus-crypt junctional area. Luminal 0.4 M glycine stimulated enzyme activity only in villus and villus-crypt junctional samples, while luminal 10 mM putrescine stimulated activity only in crypts. Parenteral epidermal growth factor caused increased enzyme activity in all mucosal areas, but the 18-28-fold increase in the three villus samples (top, middle and bottom) was significantly greater (P < 0.05) than the 7-9-fold increase in crypt and junctional samples. In rats refed after a fast, parenteral putrescine (2 mmol/kg) depressed enzyme activity in all mucosal areas. Ornithine decarboxylase activity is usually greatest in junctional and crypt cells, and villus and crypt cells respond differently to luminal and systemic stimuli.

Animals↗

PubMed1997

Ligand-independent activation of fibroblast growth factor receptor-2 by carboxyl terminal alterations.

To assess the effect(s) of the C-terminal domain on FGFR2 function, we engineered a series of mutant FGFR2 cDNAs encoding deletions in the C-terminus of the receptor and compared their growth properties in NIH3T3 fibroblasts. In contrast to FGFR2-WT, receptors with C-terminal truncations induced ligand-independent transformation of NIH3T3 cells and transfectants expressing these mutant receptors efficiently formed colonies in semisolid medium. Introduction of point mutations (Y to F) into the C-terminus of FGFR2 at positions 813, 784 or 780 revealed that these mutant receptors also displayed activities similar to that of C-terminally truncated receptors. C-terminally altered FGF receptors did not show an increase in the basal level of receptor phosphorylation compared to that of FGFR2-WT suggesting that elevated receptor phosphorylation does not underlie the transforming activity of these receptors. Interestingly, expression of transforming FGFR2 derivatives, unlike H-Ras transformed cells, did not result in the activation of the mitogen-activated protein kinases (MAPKs), p42/ERK2 and p44/ERK1, indicating that this pathway is not constitutively active in FGFR2-transformed cells. Finally, we report the overexpression of FGFR2 mRNA and protein in several human tumor cell lines suggesting activation of the receptor in these tumors.

3T3 Cells↗

PubMed1997

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken.

Two unique cDNA clones containing chicken slow myosin heavy chain (MyHC) inserts have been isolated from an expression library. Immunochemical analyses of the expressed proteins using different slow MyHC specific monoclonal antibodies were consistent with the two clones encoding slow MyHC 1 (SM1) and slow MyHC 2 (SM2) protein sequences. Northern blot analyses showed that the clones hybridized with 6-kb mRNAs that are differentially expressed in developing and adult slow muscles, further supporting the conclusion that these two clones represent SM1 and SM2 cDNAs. Sequence analyses show that both clones encode the highly conserved light meromyosin portion of the sarcomeric myosin rod and are 78-81% homologous to a mammalian slow/cardiac beta-MyHC cDNA. Hybridization using PCR generated probes specific for SM1 and SM2 sequences demonstrated that the genes encoding these two slow MyHCs colocalized to an 80-kb BssHII genomic fragment. We further show that a probe specific to a third slow MyHC gene also hybridized with the same 80-kb genomic fragment. We conclude that in the chicken genome there is a slow MyHC locus containing at least three distinct slow MyHC genes.

Amino Acid Sequence↗

PubMed1997

BglF, the sensor of the E. coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein.

The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). It catalyses transport and phosphorylation of beta-glucosides. In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to beta-glucoside availability. By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and thus its ability to bind RNA and antiterminate transcription of the bgl operon. BglF has two phosphorylation sites. The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar. We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed.

Bacterial Proteins↗

PubMed1997

The localization of the phosphorylation site of BglG, the response regulator of the Escherichia coli bgl sensory system.

BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue. We report here the localization of the phosphorylation site to histidine 208. Localization of the phosphorylated histidine was carried out in two steps. We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate. This allowed the localization of the phosphorylation site to the last 111 residues. Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor. Histidine 208 was the only histidine which failed both tests. The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins. The bgl system represents a new family of sensory systems. Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family.

Bacterial Proteins↗

PubMed1997

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors.

In this study, we have localized an enhancer element in the 5'-flanking region of the HGF gene promoter and have identified its functional transcriptional factors. Through transient transfection of NIH3T3 fibroblast cells and gel mobility shift assays, the functional binding site was localized to a short region (-318 to -303 bp from the transcription start site) which has a CTCCC sequence. This motif is also conserved in the human HGF promoter and acts as a binding site for the Sp family of transcription factors. In the presence of NIH3T3 nuclear protein extracts, this enhancer region formed specific DNA-protein complexes which were totally abrogated by excess amounts of consensus Sp1 oligonucleotide. In gel mobility supershift assays, anti-Sp1 and anti-Sp3 antibodies specifically recognized these complexes as was evident by their slower migration. Site-specific mutation of this binding motif resulted in total loss of Sp1 and Sp3 binding and a concomitant loss of enhancer function within the context of the HGF promoter. Furthermore, in transient cotransfection assays, overexpression of Sp1 and/or Sp3 stimulated HGF promoter activity independently and additively through binding to the Sp1 binding site in the HGF gene promoter region. DNaseI hypersensitive analysis of freshly isolated nuclei from NIH3T3 cells revealed that five hypersensitive sites (HSS) are present within the 2 kb region immediately upstream of the HGF promoter. One of these sites was mapped to position -0.3 kb from the transcription start site. These data show that both Sp1 and Sp3 transcription factors upregulate HGF promoter activity by binding to the Sp1 binding site at position -318 to -303 bp in the HGF gene promoter.

3T3 Cells↗

PubMed1997

Organelle-specific targeting of protein kinase AII (PKAII). Molecular and in situ characterization of murine A kinase anchor proteins that recruit regulatory subunits of PKAII to the cytoplasmic surface of mitochondria.

Experiments were designed to test the idea that A kinase anchor proteins (AKAPs) tether regulatory subunits (RII) of protein kinase AII (PKAII) isoforms to surfaces of organelles that are bounded by phospholipid bilayers. S-AKAP84, one of three RII-binding proteins encoded by a single-copy murine gene, was studied as a prototypic organelle-associated AKAP. When S-AKAP84 was expressed in HEK293 cells, the anchor protein was targeted to mitochondria and excluded from other cell compartments. The RII tethering site is located in the cytoplasm adjacent to the mitochondrial surface. Endogenous RII subunits are not associated with mitochondria isolated from control cells. Expression of S-AKAP84 in transfected HEK293 cells triggered a redistribution of 15% of total RII to mitochondria. Thus, the tethering region of the organelle-inserted anchor protein is properly oriented and avidly binds RII (PKAII) isoforms in intact cells. Two critical domains in S-AKAP84 were mapped. Residues 1 to 30 govern insertion of the polypeptide into the outer mitochondrial membrane; amino acids 306-325 constitute the RII-binding site. Properties established for S-AKAP84 in vitro and in situ strongly suggest that a physiological function of this protein is to concentrate and immobilize RII (PKAII) isoforms at the cytoplasmic face of a phospholipid bilayer.

A Kinase Anchor Proteins↗

PubMed1997

Cytotoxicity and apoptosis produced by arachidonic acid in Hep G2 cells overexpressing human cytochrome P4502E1.

The goal of the current study was to evaluate the effects of arachidonic acid, as a representative polyunsaturated fatty acid, on the viability of a Hep G2 cell line, which has been transduced to express human cytochrome P4502E1 (CYP2E1). Arachidonic acid produced a concentration- and time-dependent toxicity to Hep G2-MV2E1-9 cells, which express CYP2E1, but little or no toxicity was found with control Hep G2-MV-5 cells, which were infected with retrovirus lacking human CYP2E1 cDNA. In contrast to arachidonic acid, oleic acid was not toxic to the Hep G2-MV2E1-9 cells. The cytotoxicity of arachidonic acid appeared to involve a lipid peroxidation type of mechanism since toxicity was enhanced after depletion of cellular glutathione; formation of malondialdehyde and 4-hydroxy-2-nonenal was markedly elevated in the cells expressing CYP2E1, and toxicity was prevented by antioxidants such as alpha-tocopherol phosphate, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), propylgallate, ascorbate, and diphenylphenylenediamine, and the iron chelator desferrioxamine. Transfection of the Hep G2-MV2E1-9 cells with plasmid containing CYP2E1 in the sense orientation enhanced the arachidonic acid toxicity, whereas transfection with plasmid containing CYP2E1 in the antisense orientation decreased toxicity. The CYP2E1-dependent arachidonic acid toxicity appeared to involve apoptosis, as demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA laddering experiments. Trolox, which prevented toxicity of arachidonic acid, also prevented the apoptosis. Transfection with a plasmid containing bcl-2 resulted in complete protection against the CYP2E1-dependent arachidonic acid toxicity. It is proposed that elevated production of reactive oxygen intermediates by cells expressing CYP2E1 can cause lipid peroxidation, which subsequently promotes apoptosis and cell toxicity when the cells are enriched with polyunsaturated fatty acids such as arachidonic acid. The Hep G2-MV2E1-9 cells appear to be a valuable model to study interaction between CYP2E1, polyunsaturated fatty acids, reactive radicals, and the consequence of these interactions on cell viability and to reproduce several of the key features associated with ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment.

Antioxidants↗

PubMed1997

Metabolites produced during the peroxisomal beta-oxidation of linoleate and arachidonate move to microsomes for conversion back to linoleate.

When [1-(14)C]4,7,10-16:3, a product produced after two cycles of arachidonate beta-oxidation, was incubated with rat liver peroxisomes and microsomes it was metabolized to 2-trans-4,7,10-16:4, a catabolic product; 6,9,12-18:3 and 8,11,14-20:3, anabolic products made via microsomal chain elongation of the substrate; and 7,10-16:2 and 9,12-18:2. Analysis of the acyl-CoAs produced when 6,9,12-18:3 and its catabolic product, 4,7,10-16:3, where incubated under the above conditions showed that the acyl-CoAs of all of the above compounds, as well as 5,8-14:2-CoA and 6:0-CoA accumulated. Our results show that when 5,8-14:2 and 4,7,10-16:3 are produced by peroxisomal beta-oxidation they can be further degraded to hexanoyl-CoA or move to microsomes for conversion back to linoleate, which is a precursor of arachidonate.

Adenosine Triphosphate↗

PubMed1997

Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin.

Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.

Amino Acid Sequence↗

PubMed1997

Cell anchorage permits efficient signal transduction between ras and its downstream kinases.

Cell anchorage strongly affects the signal transduction cascade initiated by peptide mitogens. For both epidermal growth factor and platelet-derived growth factor, activation of the consensus mitogen-activated protein kinase cascade is impaired when cells are held in suspension as compared with cells anchored to a fibronectin substratum. Upstream events in the signaling cascade, including tyrosine phosphorylation of the mitogen receptor and GTP loading of Ras, are similar in anchored and suspended cells. However, propagation of the signal to Raf and subsequently to the downstream kinases MEK and mitogen-activated protein kinase is markedly attenuated in suspended cells. Thus, there seems to be a distinct anchorage-dependent step between Ras and Raf in the signaling cascade initiated by peptide mitogens. These observations may have important implications for understanding the anchorage dependence of cell growth.

3T3 Cells↗

PubMed1997

Remarkable sequence conservation of transcripts encoding amphibian and mammalian homologues of quaking, a KH domain RNA-binding protein.

Mutations in the mouse quaking locus can result in two different types of developmental phenotypes: (1) a deficiency of myelin in the central nervous system that is accompanied by a characteristic tremor, or (2) embryonic lethality around day 9 of gestation. A quaking candidate gene (qkI) that encodes a KH motif protein has recently been identified. We have isolated and characterized cDNAs encoding the Xenopus quaking homologue (Xqua) and also assembled an almost complete human quaking sequence from expressed sequence tags. Sequence comparisons show that the amphibian and mammalian quaking transcripts exhibit striking conservation, both within the coding region and, unexpectedly, in the 3' UTR. Two Xqua transcripts 5 kb and 5.5 kb in length are differentially expressed in the Xenopus embryo, with the 5 kb transcript being detected as early as the gastrula stage of development. Using an in vitro assay, we have demonstrated RNA-binding activity for quaking protein encoded by the 5 kb transcript. Overall, the high sequence conservation of quaking sequences suggests an important conserved function in vertebrate development, probably in the regulation of RNA metabolism.

Amino Acid Sequence↗

PubMed1997

Integrin-mediated activation of MAP kinase is independent of FAK: evidence for dual integrin signaling pathways in fibroblasts.

Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2-Sos complex. Since Grb2-Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a beta1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.

3T3 Cells↗

PubMed1997

Electrochemical glucose and lactate sensors based on "wired" thermostable soybean peroxidase operating continuously and stably at 37 degrees C.

Following a recent report from our laboratory on a thermostable amperometric H2O2 sensor based on "wiring" soybean peroxidase, glucose and lactate sensors maintaining stable output under continuous operation at 37 degrees C for 12 and 8 days, respectively, were built. The vitreous carbon base of the sensor was coated with four polymer layers. The first was made by cross-linking thermostable soybean peroxidase and the redox polymer formed through complexing part of the rings of poly-(vinylpyridine) with [Os(bpy)2Cl]+/2+ (bpy = bipyridine) and quaternizing part of the rings with bromoethylamine. The second was an insulating and H2O2 transport controlling cellulose acetate layer. The third was an immobilized glucose oxidase or lactate oxidase layer. The fourth was a substrate transport controlling cellulose acetate layer In the case of the glucose sensor, the current output was independent of potential between -0.2 and +0.3 V (vs SCE), and the response time (t10/90) was < 2 min when the concentration was raised from 0 to 5 mM glucose. The current was independent of the O2 partial pressure above 15 Torr. The sensor was relatively insensitive to motion and to interferants. Changing the rotation speed of the electrode from 50 to 2500 rpm increased the current by < 10%. At a glucose concentration of 4 mM, the addition of 0.1 mM ascorbate decreased the current by < 1%. The operational stability was glucose oxidase loading dependent. Though the current decreased by 85% after 100 h of operation at 37 degrees C when the 3-mm-diameter electrode was loaded with only 1.3 micrograms of glucose oxidase, it decreased by < 1% after such operation when loaded with 52 micrograms of the enzyme. Similar results were obtained for the lactate sensor, with the exception of a more noticeable oxygen concentration dependence of the lactate response at low oxygen concentrations.

Biosensing Techniques↗