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PubMed1996

In vitro interaction between a chloroplast transit peptide and chloroplast outer envelope lipids is sequence-specific and lipid class-dependent.

Interaction of artificial lipid bilayers (liposomes) with the purified transit peptide (SS-tp) of the precursor form of the small subunit for ribulose-2,5-bisphosphate carboxylase/oxygenase (prSSU) has been studied using a vesicle-disruption assay (calcein dye release) and electron microscopy. Employing purified forms of Escherichia coli-expressed prSSU, mature small subunit, glutathione S-transferase-transit peptide fusion protein, and SS-tp in dye release studies demonstrated that lipid interaction is mediated primarily through the transit peptide. Using chemically synthesized peptides (20-mers), the lipid-interacting domain of the transit peptide was partially mapped to the C-terminal 20 amino acids of the transit peptide. Peptides corresponding to other regions of the transit peptide and control peptides promoted significantly less calcein release. Interaction between the transit peptide and the bilayer was very rapid and could not be resolved by stopped-flow fluorometry with a mixing time of <50 ms. Interaction between the peptides and bilayer was also lipid class-dependent. Disruption occurred only when the bilayer contained the galactolipid monogalactosyldiacylglycerol (MGDG). The extent of bilayer disruption directly correlated with the relative concentration of MGDG in the liposome, with maximum calcein release occurring in 20 mol % MGDG liposomes. Lipid bilayers with greater than 20 mol % MGDG could not be achieved as determined by calcein entrapment. Electron microscopy of the liposomes before and after addition of the transit peptide suggested that the transit peptide induced a dramatic reorganization of lipids. These results are discussed in light of a possible mechanism for the early steps in protein transport that may involve polymorphic changes in the envelope membrane organization to include localized non-bilayer HII structures.

Amino Acid Sequence↗
PubMed1997

Organic anion transporting polypeptide mediates organic anion/HCO3- exchange.

Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem. 270, 25591-25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (Km = 19.4 +/- 3.3 microM; Vmax = 62.2 +/- 1.4 pmol/min/mg protein; n = 3). To test whether organic anion transport is coupled to HCO3- extrusion, we compared the rates of taurocholate-dependent HCO3- efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi ( approximately 7.7) by transient exposure to a 50 mM HCO3-, Cl--free solution. In the absence of extracellular Cl- and taurocholate, isohydric reduction of superfusate HCO3- concentration from 50 to 25 mM resulted in an insignificant change in pHi over time (dpHi/dt) in both groups. Addition of 25 microM taurocholate to the superfusate led to a rapid fall in pHi in induced (-0.037 +/- 0.011 pH units/min to pHi of 7.41 +/- 0.14) but not in noninduced (0.003 +/- 0.006 pH units/min to pHi of 7.61 +/- 0.08) cells (p < 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3- exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function.

Anion Transport Proteins↗
PubMed1998

Acyl coenzyme A binding protein. Conformational sensitivity to long chain fatty acyl-CoA.

Cellular unbound long chain fatty acyl-CoAs (>14 carbon) are potent regulators of gene transcription and intracellular signaling. Although the cytosolic acyl-CoA binding protein (ACBP) has high affinity for medium chain fatty acyl-CoAs, direct interaction of ACBP with >14-carbon fatty acyl-CoAs has not been established. Steady state, photon counting fluorescence spectroscopy directly established that rat liver ACBP bound 18-carbon cis- and trans-parinaroyl-CoA, Kd = 7.03 +/- 0.95 and 4.40 +/- 0.43 nM. Time-resolved fluorometry revealed that ACBP-bound parinaroyl-CoAs had high rotational freedom within the single, relatively hydrophobic (epsilon <32), binding site. Tyr and Trp fluorescence dynamics demonstrated that apo-ACBP was an ellipsoidal protein (axes of 15 and 9 A) whose conformation was altered by oleoyl-CoA in the holo-ACBP as shown by a 2-A decrease of ACBP hydrodynamic diameter and increased Trp segmental motions. Thus, native liver ACBP binds >14-carbon fatty acyl-CoAs with nanomolar affinity at a single binding site. Acyl-CoA-induced conformational alterations in ACBP may be significant to its putative functions in lipid metabolism and regulation of processes sensitive to unbound long chain fatty acyl-CoAs.

Acyl Coenzyme A↗
PubMed2000

Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway.

Adenine deoxynucleosides, such as 2-chlorodeoxyadenosine (2CdA) and fludarabine, induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. We previously demonstrated that that the 5'-triphosphate metabolite of 2CdA (2CdATP), similar to dATP, can cooperate with cytochrome c and apoptosis protein-activating factor-1 (APAF-1) to trigger a caspase pathway in a HeLa cell-free system. We used a fluorometry-based assay of caspase activation to extend the analysis to several other clinically relevant adenine deoxynucleotides in B-chronic lymphocytic leukemia extracts. The nucleotide-induced caspase activation displayed typical Michaelis-Menten kinetics. As estimated by the V(max)/K(m) ratios, the relative efficiencies of different nucleotides were Ara-ATP > 9-fluoro-9-beta-D-arabinofuranosyladenine 5'-triphosphate > dATP > 2CdATP > 9-beta-D-arabinofuranosylguanine 5'-triphosphate > dADP > ATP. In contrast to dADP, both ADP and its nonhydrolyzable alpha, beta-methylphosphonate analog were strong inhibitors of APAF-1-dependent caspase activation. The hierarchy of nucleotide activation was confirmed in a fully reconstituted system using recombinant APAF-1 and recombinant procaspase-9. These results suggest that the potency of adenine deoxynucleotides as co-factors for APAF-1-dependent caspase activation is due both to stimulation by the 5'-triphosphates and lack of inhibition by the 5'-diphosphates. The capacity of adenine deoxynucleoside metabolites to activate the apoptosome pathway may be an additional biochemical mechanism that plays a role in the chemotherapy of indolent lymphoproliferative diseases.

Apoptosis↗
PubMed2000

Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer.

We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.

Animals↗
PubMed2001

Mechanism of cyclosporin-induced inhibition of intracellular collagen degradation.

The immunosuppressant cyclosporin A (CsA) markedly inhibits collagen degradation by an intracellular phagocytic pathway in fibroblasts, an effect that can lead to massive gingival overgrowth. We used a collagen bead model of collagen phagocytosis to determine whether CsA inhibits internalization by blocking efflux of calcium from endoplasmic reticulum (ER) and mitochondrial calcium stores. CsA caused dose-dependent inhibition of phagocytosis of collagen-coated (but not bovine serum albumin-coated) beads. Chelation of intracellular Ca(2+) with BAPTA/AM or inhibition of Ca(2+)-ATPase of ER stores with thapsigargin reduced collagen bead phagocytosis. Measurement of intracellular calcium by ratio fluorometry showed increases in response to collagen-coated beads. Preincubation with CsA or thapsigargin caused a >3-fold decrease in intracellular calcium elevations in response to stimulation with collagen beads. Direct measurements of Ca(2+) in mitochondrial and ER stores showed that CsA only slightly inhibited collagen bead-induced discharge of calcium from mitochondria, but almost completely blocked discharge from ER stores. We reduced the numbers of mitochondria with chronic ethidium bromide treatment to test for the importance of ER/mitochondrial interactions. In these cells, CsA delayed collagen bead-induced calcium discharge from mitochondria. Collectively, these data indicate that CsA inhibits collagen phagocytosis by blocking calcium release from ER stores and may perturb functional interactions between the ER and mitochondria that regulate calcium stores.

Animals↗
PubMed2002

Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism.

Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.

Binding Sites↗
PubMed2002

Illumination of the malaria parasite Plasmodium falciparum alters intracellular pH. Implications for live cell imaging.

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasite Plasmodium falciparum, including pH homeostasis, Ca(2+) signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by light-induced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.

Animals↗
PubMed2003

The specific interaction of dinitrosyl-diglutathionyl-iron complex, a natural NO carrier, with the glutathione transferase superfamily: suggestion for an evolutionary pressure in the direction of the storage of nitric oxide.

The interaction of dinitrosyl-diglutathionyl-iron complex (DNDGIC), a natural carrier of nitric oxide, with representative members of the human glutathione transferase (GST) superfamily, i.e. GSTA1-1, GSTM2-2, GSTP1-1, and GSTT2-2, has been investigated by means of pre-steady and steady state kinetics, fluorometry, electron paramagnetic resonance, and radiometric experiments. This complex binds with extraordinary affinity to the active site of all these dimeric enzymes; GSTA1-1 shows the strongest interaction (KD congruent with 10-10 m), whereas GSTM2-2 and GSTP1-1 display similar and slightly lower affinities (KD congruent with 10-9 m). Binding of the complex to GSTA1-1 triggers structural intersubunit communication, which lowers the affinity for DNDGIC in the vacant subunit and also causes a drastic loss of enzyme activity. Negative cooperativity is also found in GSTM2-2 and GSTP1-1, but it does not affect the catalytic competence of the second subunit. Stopped-flow and fluorescence data fit well to a common minimal binding mechanism, which includes an initial interaction with GSH and a slower bimolecular interaction of DNDGIC with one high and one low affinity binding site. Interestingly, the Theta class GSTT2-2, close to the ancestral precursor of GSTs, shows very slow binding kinetics and hundred times lowered affinity (KD congruent with 10-7 m), whereas the bacterial GSTB1-1 is not inhibited by DNDGIC. Molecular modeling and EPR data reveal structural details that may explain the observed kinetic data. The optimized interaction with this NO carrier, developed in the more recently evolved GSTs, may be related to the acquired capacity to utilize NO as a signal messenger.

Binding Sites↗
PubMed2003

Kinetic studies of cAMP-induced allosteric changes in mutants T127I, S128A, and T127I/S128A of the cAMP receptor protein from Escherichia coli.

The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry measurements of CRP mutants bearing amino acid substitutions T127I, S128A, and T127I/S128A to study the kinetics of conformational changes in the protein induced by cAMP binding. Amino acid substitutions at positions 127 and 128 were chosen because these residues play a crucial role in interdomain and intersubunit communication during allosteric transition. Using N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid-labeled Cys178, localized in the protein helix-turn helix motif, we observed conformational changes in the helix-turn helix, localized in the C-terminal domain, upon binding of cAMP to high affinity sites (CRP.cAMP2) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: kc = 3.62 s-1 and k-c = 3.13s-1 for CRP T127I and kc = 0.42 s-1 and k-c = 0.78 s-1 for CRP S128A. These values can be compared with kc = 9.7 s-1 and k-c = 0.31 s-1 for wild-type CRP. The observed conformational changes can be described by the sequential model of allostery, with the amino acid substitutions influencing the allosteric changes. In the case of the double mutant, the observed rate constant of cAMP binding supports the suggestion that this unligated mutant possesses the structure that is close to the allosteric conformation necessary for promoter binding. The results of intrinsic fluorescence measurements suggest that the formation of the CRP.cAMP4 complex results from displacement of equilibrium between the two forms of the CRP.cAMP2 complex in the mutants studied, similar to wild-type CRP. The observed conformational changes occur according to a concerted model of allostery, and isomerization equilibrium between the two CRP states depends on the amino acid substitution. The data presented in this study indicate that Ser128 and Thr127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP.

Allosteric Site↗
PubMed2004

Stimulation of capacitative calcium entry in HL-60 cells by nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (approximately 100 nM), while the uptake of propidium iodide was absent. This suggests that increases in intracellular calcium were not because of plasma membrane electroporation. nsPEF and the purinergic agonist UTP induced calcium mobilization in the presence and absence of extracellular calcium with similar kinetics and appeared to target the same inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pools in the endoplasmic reticulum. For cells exposed to either nsPEF or UTP in the absence of extracellular calcium, there was an electric field-dependent or UTP dose-dependent increase in capacitative calcium entry when calcium was added to the extracellular media. These findings suggest that nsPEFs, like ligand-mediated responses, release calcium from similar internal calcium pools and thus activate plasma membrane calcium influx channels or capacitative calcium entry.

Calcium↗
PubMed2004

Protonation state of a single histidine residue contributes significantly to the kinetics of the reaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator.

Stopped-flow fluorometry was used to study the kinetics of the reactive center loop insertion occurring during the reaction of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 plasminogen activator inhibitor-1 (PAI-1) with tissue-(tPA) and urokinase (uPA)-type plasminogen activators and human pancreatic elastase at pH 5.5-8.5. The limiting rate constants of reactive center loop insertion (k(lim)) and concentrations of proteinase at half-saturation (K(0.5)) for tPA and uPA and the specificity constants (k(lim)/K(0.5)) for elastase were determined. The pH dependences of k(lim)/K(0.5) reflected inactivation of each enzyme due to protonation of His57 of the catalytic triad. However, the specificity of the inhibitory reaction with tPA and uPA was notably higher than that for the substrate reaction catalyzed by elastase. pH dependences of k(lim) and K(0.5) obtained for tPA revealed an additional ionizable group (pKa, 6.0-6.2) affecting the reaction. Protonation of this group resulted in a significant increase in both k(lim) and K(0.5) and a 4.6-fold decrease in the specificity of the reaction of tPA with NBD P9 PAI-1. Binding of monoclonal antibody MA-55F4C12 to PAI-1 induced a decrease in k(lim) and K(0.5) at any pH but did not affect either the pKa of the group or an observed decrease in k(lim)/K(0.5) due to protonation of the group. In contrast to tPA, the k(lim) and K(0.5) for the reactions of uPA with NBD P9 PAI-1 or its complex with the monoclonal antibody were independent of pH in the 6.5-8.5 range. Since slightly acidic pH is a feature of a number of malignant tumors, alterations in PAI-1/tPA kinetics could play a role in the cancerogenesis. Changes in the protonation state of His(188), which is placed closely to the S1 site and is unique for tPA, has been proposed to contribute to the observed pH dependences of k(lim) and K(0.5).

Binding Sites↗
PubMed2003

Fluorescein transport through the blood-aqueous and blood-retinal barriers in diabetic macular edema.

PURPOSE: To investigate the integrity of the blood-aqueous barrier (BAB) and the blood-retinal barrier (BRB) in diabetic patients with clinically significant macular edema (CSME). METHODS: The rate constant (Kd(F)) of the BAB and the permeability of the blood-retinal barrier for non-protein bound fluorescein were determined simultaneously by fluorometry. Results were analysed pairwise in diabetic patients (n = 25) with CSME in one eye and without CSME in the other eye. RESULTS: Kd(F) for the eyes with CSME was significantly increased compared to eyes without CSME (444. 10(-6) min( -1) and 387. 10(-6) min(-1) respectively, p = 0.01). The passive permeability of the BRB was also significantly increased in CSME (5.7 nm/sec and 3.5 nm/sec respectively, p = 0.009, n = 19). CONCLUSIONS: Both the rate constant of the BAB and the BRB permeability were significantly increased in CSME indicating that eye-specific factors are common for both barriers in diabetic patients with CSME.

Adult↗
PubMed1998

Selenium depletion in patients with gastrointestinal diseases: are there any predictive factors?

BACKGROUND: Patients with intestinal disease are at risk of developing selenium deficiency due to impaired intestinal absorption. The aim of the present study was to evaluate selenium status and to identify predictive factors of selenium depletion in patients with gastrointestinal disease. METHODS: The concentration of selenium and the activity of glutathione peroxidase in plasma and erythrocytes were measured by fluorometry and by spectrophotometry. Eighty-six patients with Crohn's disease, 40 patients with ulcerative colitis, and 39 patients with various other gastrointestinal diseases were studied. Twenty-seven patients (16%) received home parenteral nutrition. Stool mass, faecal fat, and vitamin B12 absorption were analysed in 100 patients. RESULTS: The plasma selenium concentration was decreased in 85% of the patients receiving supplementary parenteral nutrition and in 20% of the patients receiving oral nutrition, among them in 26% of the patients with Crohn's disease. Almost all patients with ulcerative colitis had normal selenium levels. A statistically significant correlation was found between plasma selenium and vitamin B12 absorption, stool mass, faecal fat excretion, body mass index, P-albumin, P-zinc, and the length of the remaining small bowel. Stepwise regression analyses showed that the strongest predictors of selenium deficiency were stool mass, vitamin B12 absorption, and the length of the small-bowel resection. CONCLUSION: Selenium deficiency is common in patients with severe gastrointestinal disorders. The deficiency is mainly related to malabsorption, and a low selenium level was almost invariably present in patients who needed parenteral supplementation due to gut failure.

Adult↗
PubMed1998

Effect of ethanol on the metabolism of trichloroethylene in the perfused rat liver.

The effect of 2 mM ethanol, a concentration indicative of daily alcohol consumption, was investigated on trichloroethylene (TRI) metabolism in perfused Wistar rat liver. The study consisted of two parts: The first part studied TRI administration with or without ethanol. In the second study chloral hydrate (CH), an intermediate in TRI metabolism, was administered in the absence or presence of ethanol to phenobarbital (PB) treated or non-PB-treated rats. The concentrations of the metabolites, total trichloroethanol (TCE), and trichloroacetic acid (TCA) were measured by gas chromatography and intracellular reduced pyridine nucleotides by surface fluorometry. In the first study, ethanol infusion significantly increased the TCE/TCA ratio, TCE production rate, and percentage of reduced pyridine nucleotides, and decreased TCA production rate without an associated change in the sum of TCE and TCA formation rates. In the second study, ethanol infusion in the absence or presence of PB produced similar significant increases in the TCE/TCA ratio, TCE production rate, and percentage of reduced pyridine nucleotides, accompanied by a decrease in TCA formation. The observed shift in TRI metabolism in the presence of ethanol, from oxidation to TCA to reduction to TCE, suggests that alcohol exerts alterations in hepatic intracellular oxidation-reduction (redox) states.

Animals↗
PubMed1998

Environmental particulate-mediated cytokine production in lung epithelial cells (A549): role of preexisting inflammation and oxidant stress.

Epidemiologic data show that air pollution particulates cause adverse pulmonary health effects, especially in individuals with preexisting lung disease. We sought to model in vitro preexisting lung inflammation in order to investigate the hypothesis that "primed" lung epithelial cells will exhibit enhanced phlogistic responses [e.g., interleukin-8 (IL-8) production] to particulate air pollution. Exposure of tumor necrosis factor alpha (TNF-alpha) primed or control A549 cells to the air pollution particulates, residual oil fly ash (ROFA), and the known pathogenic dust alpha-quartz, but not inert TiO2, caused increased IL-8 production in primed cells compared to normal cells in a concentration-dependent manner (particle concentration range 0-200 microg/ml). We hypothesized that oxidant mechanisms may be involved in the cellular response to particulates. Addition of the antioxidant N-acetylcysteine (NAC, 1.0 mM) decreased ROFA and alpha-quartz-mediated IL-8 production by approximately 50% in normal and TNF-alpha-primed A549 cells. In addition, exposure of A549 cells to ROFA caused a substantial (and NAC inhibitable) increase in oxidant levels as measured by fluorometry (DCFH oxidation). These data suggest that (1) lung epithelial cells primed by inflammatory mediators can show enhanced cytokine production after exposure to air pollution particulates, and (2) oxidant stress is a key mechanism for this response.

Acetylcysteine↗
PubMed1985

Mitochondrial redox responses to cerebral ischaemia produced by four-vessel occlusion in the rat.

Changes in reduction/oxidation (redox) ratios of the initial and terminal members of the mitochondrial respiratory chain (nicotine adenine dinucleotide and cytochrome a,a3) were measured by fluorometry and reflection spectrophotometry in rat cerebral cortex. Three patterns of responses were observed during the transition to global ischaemia produced by electrocoagulation of the vertebral arteries and carotid artery ligation. Most often, arterial occlusion was accompanied by shifts towards reduction of the mitochondrial components followed by partial reoxidation and then further reduction. Less frequently, ischaemia produced reductive shifts of the respiratory chain reactants that rapidly reached maximal values. In some rats, however, arterial, occlusion produced mitochondrial reduction followed by partial or complete reoxidation. ECoG remained suppressed, despite mitochondrial reoxidation, in approximately 1/3 of the latter animals. This study demonstrates that responses to 4-vessel occlusion can vary greatly among animals and suggests that monitoring of signals of ischaemic insult, such as mitochondrial redox status, may offer advantages for assessment of the consequences of ischaemic insult.

Anesthesia, General↗
PubMed1990

Simultaneous immunoenzymometric assay for antibodies against human interleukin-2 and human serum albumin in rat serum.

A simultaneous immunoenzymometric assay for anti-human interleukin-2 antibody and anti-human serum albumin antibody in rat serum was developed. Two antigen-immobilized polystyrene balls were immersed in a diluted serum sample in an assay tube and then the antibodies on the balls were made to react with a horseradish peroxidase labelled anti-rat IgG antibody in the same tube after washing. The enzyme activity of each ball was measured by fluorometry. Not only were the sensitivity (70 ng/ml each), assay recovery (100-101%), and precision (C.V. = 5-13%) comparable to those of conventional immunoenzymometric assays using one antigen-immobilized ball but the assay was also much more feasible for mass routine assays. Thus, conventional immunoassays can be replaced by this convenient simultaneous method.

Animals↗