Spectrofluorimetric Assay Research Articles

The Fibrinogen Elongated γ-Chain Inhibits Thrombin-Induced Platelet Response, Hindering the Interaction with Different Receptors.

The product of thrombin digestion of fibrinogen, that is fibrin, binds to thrombin with high specificity. The latter interacts with two classes of binding sites on fibrin, one of low affinity in the E domain and the other of high affinity in the D domain of fibrin(ogen) molecules. Binding of thrombin to fibrinogen involves sequences of both Aα and Bβ chain in the fibrinogen E domain. This recognition sites are still able to interact with thrombin after cleavage of fibrinopeptide A and B and form the low affinity binding site for the enzyme. The D domains contain a γ chain variant, termed γ′, arising from an alternative mRNA splicing, resulting in an elongated chain composed of 427 instead of 411 residues. The inserted region at the C-terminus is composed of 20 amino acids (408VRPEHPAETEYDSLYPEDDL427), rich of acidic residues. The γ′ chain, mainly heterodimerizes in the fibrinogen molecule with the more abundant γA chain, thus generating the γA/γ′ dimers (about 10% of the total γ chain content). The different expression of γ′ chain has been variably associated with both venous and arterial thrombosis. Previous genetic studies showed that the fibrinogen γ-H2 haplotype is characterized by a reduced fibrinogen γ′ levels. This haplotype is associated with a significantly increased risk for venous thrombosis. At variance with venous thromboembolism, the effect of altered expression of γ′ chain on arterial thrombosis remains largely elusive and still debated. Biochemical studies showed that γ′ chains bind to α-thrombin with high affinity and that the 408-427 region of γ′ chain binds to the anion binding exosite (ABE)-II of thrombin. We can speculate that the fibrinogen γ′ chain, through its ability to bind to thrombin, might enhance the amount of clot-bound thrombin. The latter may represent a storage pool of the enzyme, facilitating arterial thrombus formation via platelet activation. In this study, we investigated the effect of the fibrinogen γ′ and of its 20-amino acid-insertion peptide on the thrombin interaction with the platelet receptors glycoprotein (Gp) Ibα, and protease-activated receptors PAR-1 and PAR-4, involved in platelet activation. Fragment D was used as the best surrogate to selectively study the high affinity binding site for thrombin in γ chain in a conformation similar to that present in the native fibrinogen molecule. Both synthetic γ′ peptide and fragment D*, containing the elongated γ′ chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC50 values of 42±3.5 μM and 0.47±0.03 μM, respectively. Solid phase binding and titration spectrofluorimetric assays analyzing the tryptophan fluorescence of thrombin showed that both fragment D* and the synthetic γ′ peptide specifically bind to ABE-II of thrombin and competitively inhibit the thrombin interaction with GpIbα with a mean Ki≈0.5 μM and ≈35 μM, respectively. HPLC assays showed that both the γ′ chain allosterically inhibited thrombin cleavage of the synthetic PAR-1 38-60 peptide, with an IC50 value of 45 μM, in good agreement with the Kd value of thrombin binding to γ′ chain derived from fluorimetric titrations. Likewise, flow cytometry studies showed that the hydrolysis of native PAR-1 molecules on intact platelets was progressively inhibited by increasing concentrations of both fragment D* and γ′ peptide, while PAR-4 cleavage was unaffected. In summary, fibrinogen γ′ chain binds with high affinity to thrombin and inhibits with combined mechanisms the thrombin-induced platelet activation. The role of different expression of γ′ chain in circulating fibrinogen may variably influence the thrombotic and hemorrhagic manifestations in different clinical settings or different phases of thrombotic diseases.

Fibrinogen-elongated γ Chain Inhibits Thrombin-induced Platelet Response, Hindering the Interaction with Different Receptors

The expression of the elongated fibrinogen gamma chain, termed gamma', derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how gamma' chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibalpha (GpIbalpha), protease-activated receptor (PAR) 1, and PAR4. Both synthetic gamma' peptide and fibrinogen fragment D*, containing the elongated gamma' chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC(50) values of 42+/-3.5 and 0.47+/-0.03 microm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic gamma' peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbalpha with a mean K(i) approximately 0.5 and approximately 35 microm, respectively. Both these gamma' chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide D-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen gamma' chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation.

Spinal cord degeneration in C57BL/6N mice following induction of experimental parkinsonism with MPTP

We examined neurodegeneration in spinal cord (SC) and role of such extra-nigral degeneration in MPTP-induced experimental parkinsonism in C57BL/6N mice. HPLC-photodiode array analysis confirmed presence of the active neurotoxin MPP+ in SC after single injection of MPTP (25 mg/kg, i.p.). Mitochondrial enzyme monoamine oxidase-B (MAO-B) responsible for in vivo conversion of MPTP to MPP+ was inhibited in SC by pre-treatment with l-deprenyl, a specific inhibitor of MAO-B. Besides in vitro conversion of MPTP to MPP+ occurred by SC mitochondrial preparation, which was inhibited by l-deprenyl implicating SC as a specific target of MPTP-neurotoxicity. Double immunofluorescent labeling and spectrofluorimetric assay via kynuramine oxidation showed MAO-B expression and activity in SC neurons. Localization of dopamine transporter immunoreactivity in SC along with specific uptake of (3)H-MPP+ by SC synaptosomal preparation further confirmed SC as target of MPTP-neurotoxicity. Compared with control, increased neuronal death on the seventh day in SC of mice injected with MPTP (2 x 25 mg/kg, at 6 h interval) strongly suggested SC degeneration in pre-symptomatic phase of MPTP-induced experimental parkinsonism. Such extra-nigral neurodegeneration in Parkinson's disease indicated novel molecular mechanism preceding nigrostriatal degeneration and suggested designing broad therapeutic intervention for this complex movement disorder.

Glucose Modulates Basement Membrane Fibroblast Growth Factor-2 via Alterations in Endothelial Cell Permeability

The effects of glucose extremes on vascular physiology and endothelial cell function have been examined across a range of time scales. Not unexpectedly, chronic glucose exposure induces long term tissue effects. Yet short term exposure can also impose lasting consequences. The persistence of vascular pathology after euglycemic restoration further suggests a glucose exposure memory. Slow turnover reservoirs such as basement membrane are candidates for prolongation of acute events. We hypothesized that glucose-induced vascular dysfunction is related to altered vasoactive compound handling within the endothelial cell-basement membrane co-regulatory unit. Endothelial cell basement membrane-associated fibroblast growth factor-2 increased linearly with culture glucose within days of elevated glucose exposure. Surprisingly, basement membrane fibroblast growth factor-2 binding kinetics remained unchanged. The glucose-induced increase in basement membrane fibroblast growth factor-2 was instead related to enhanced endothelial cell fibroblast growth factor-2 release and permeability. Cellular fibroblast growth factor-2 release occurred concomitant with apoptosis but was not blocked by caspase inhibitors. These data suggest that release was associated with sub-lethal early apoptotic cell membrane damage, perhaps related to reactive oxygen species formation. High glucose basement membrane in turn enhanced endothelial cell proliferation in a fibroblast growth factor-2-dependent manner. We now show that glucose-induced alterations in endothelial cell function promote changes in basement membrane composition, and these changes further affect endothelial cell function. These data highlight the interrelationship of cell and basement membrane in pathological conditions such as hyperglycemia. These phenomena may explain long term effects on the endothelium of short term exposure to glucose extremes.

Recognition and Blocking of HIV-1 gp41 Pre-transmembrane Sequence by Monoclonal 4E10 Antibody in a Raft-like Membrane Environment

The conserved (664)DKWASLWNWFNITNWLWYIK(683) (preTM) sequence preceding the transmembrane anchor of human immunodeficiency virus (HIV-1) gp41 glycoprotein subunit is accessible to the broadly neutralizing 4E10 antibody and, therefore, constitutes a potential target for vaccine design. Recently reported structural data are compatible with preTM insertion into the viral external membrane monolayer in the gp41 pre-fusion state (Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J. D., Grisé, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441, 847-852). Here we demonstrate that the broadly neutralizing 4E10 antibody is able to specifically block the membrane-restructuring activity of a peptide mimic inserted into membranes. Recognition and restructuring blocking occurred in the presence of cholesterol, whereas transmembrane versions as those promoted in 1-palmitoyl-2-oleoylphosphatidylcholine:sphingomyelin mixtures could not be effectively arrested. Spectrofluorimetric assays using rhodamine-labeled peptides revealed that recognition correlated better with pore-formation blocking than with membrane-fusion inhibition. The capacity of the antibody to recognize preTM peptides in a raft-like environment was further corroborated employing planar-supported lipid layers and fluorescence microscopy. These data support that membrane-bound epitope recognition by 4E10 results in clustering reorganization of preTM at the membrane interface. We propose that this process might interfere with the formation of fusion-competent complexes at the low spike densities existing in the HIV-1 membrane. This work comprises the first experimental report on a lipid-modulated antibody capacity to bind a membrane-bound epitope sequence and arrest its restructuring activity.

Interplay between lipoproteins and bee venom phospholipase A2 in relation to their anti-plasmodium toxicity

We previously showed that the in vitro intraerythrocytic development of the malarial agent Plasmodium falciparum is strongly inhibited by secreted phospholipases A(2) (sPLA(2)s) from animal venoms. Inhibition is dependent on enzymatic activity and requires the presence of serum lipoproteins in the parasite culture medium. To evaluate the potential involvement of host lipoproteins and sPLA(2)s in malaria, we investigated the interactions between bee venom phospholipase A(2) (bvPLA(2)), human triglyceride-rich lipoproteins, and infected erythrocytes. Even at high enzyme concentration (100x IC(50)), bvPLA(2) binding to Plasmodium-infected or normal erythrocytes was not detected. On the contrary, tight association with lipoproteins was observed through the formation of buoyant bvPLA(2)/lipoprotein complexes. Direct involvement of the hydrolysis lipid products in toxicity was demonstrated. Arachidonic acid (C20:4), linoleic acid (C18:2), and, to a lesser extent, docosahexaenoic acid (C22:6) appeared as the main actors in toxicity. Minimal oxidation of lipoproteins enhanced toxicity of the lipolyzed particles and induced their interaction with infected or normal erythrocytes. Fresh or oxidized lipolyzed lipoproteins induced the parasite degeneration without host cell membrane disruption, ruling out a possible membranolytic action of fatty acids or peroxidation products in the death process. In conclusion, our data enlighten on the capability of secreted PLA(2)s to exert cytotoxicity via the extracellular generation of toxic lipids, and raise the question of whether such mechanisms could be at play in pathophysiological situations such as malaria.

Study of the polyol pathway in the porcine epididymis

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.

A fluorescence-based assay for the reductase activity of protein disulfide isomerase

We report on a new spectrofluorimetric assay for the measurement of reductase activity of proteins belonging to the superfamily of thioredoxins such as protein disulfide isomerase (PDI). The assay relies on the preparation of a fluorescence-quenched substrate easily accessible in two steps through functional group transformations of the peptide Gly-Cys-Asp. In the first step fluorescein isothiocyanate is linked to the Gly-NH 2 terminus and in the second step the Cys-SH groups are converted into a disulfide bond. Both intermediate and final substrate have been fully characterized by mass spectrometric and nuclear magnetic resonance measurements. Dimethyl sulfoxide is here reported to be a mild oxidizing agent allowing us to obtain in good overall yield the assay substrate in a single synthetic step. A reliable estimation of PDI reductase activity is obtained via the detection of a strong fluorescence enhancement after enzymatic reduction. Moreover, our assay provides further support for the key role played by thioredoxin reductase in enabling disulfide reductase activity of PDI.

Effects of hepatic fibrosis on ofloxacin pharmacokinetics in rats

The purpose of the present work was to study the pharmacokinetics of ofloxacin, a poorly metabolised drug, in experimental hepatic fibrosis. The possible roles of small intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP) in the bioavailability of ofloxacin were also evaluated. Rat hepatic fibrosis model was successfully induced using complex factors including carbon tetrachloride, ethanol and high fat. After rats received a single oral or intravenous dose of ofloxacin (40 mg kg −1), the plasma concentrations of ofloxacin were monitored at the scheduled time using spectrofluorimetric assay. Plasma concentration–time profiles were comodeled using compartmental method. Meanwhile, microsomal CYP isoenzymatic levels and P-gp expression in small intestines were compared between normal and hepatic fibrosis rats. When ofloxacin was administered intravenously, C max and the distribution half-life increased significantly in comparison with normal group, whereas the distribution rate constants, the apparent volume of distribution decreased. Oral ofloxacin bioavailability was significantly altered in hepatic fibrosis rats. AUC and C max were reduced, while the absorption half-life, peak time and elimination half-life significantly were prolonged, suggesting that both the extent and the rate of ofloxacin absorption were decreased. Furthermore, the increases in the levels of microsomal ethoxyresorufin O-deethylase and erythromycin N-demethylase were accompanied with up-regulation of mdr 1a mRNA in the small intestines of hepatic fibrosis rats when compared to those of the normal rats. The Results showed that pharmacokinetics of ofloxacin could be altered in hepatic fibrosis. Up-regulated P-gp expression and increased CYP isoenzymatic activities of small intestines in hepatic fibrosis rats may contribute to the decreased bioavailability and increased elimination of ofloxacin after oral administration.

A New Fluorescent Assay for Enalapril Maleate

A new spectrofluorimetric method for the enalapril maleate monitoring was studied. Enalapril maleate was found to be highly photolabile. This drug was evaluated according to photodegradation assay at pH 2.5 and 6. Enalapril maleate was exposed to UVA-UVB radiations. Under these specific conditions was found as degradation product, the diketopiperazine. The modification of the fluorescent properties of enalapril maleate in solution after exposure UV-radiation and the degradation mechanisms were studied. The photodegradation was followed by the developed spectrofluorimetric assay.

Spectrofluorimetric Assay of Cationic Surfactants by Fluorescence Quenching of 9-Anthracenecarboxylic Acid

A new simple, selective and sensitive fluorescence quenching method was developed to determine cationic surfactants with the 9-anthracenecarboxylic acid (ACA). The fluorescence intensity of ACA was decreased by addition of trace amounts of cationic surfactants. Under optimum conditions, the ratio of fluorescence intensity in the absence and presence of cationic surfactants was proportional to the concentration of cationic surfactants over the range of 0.3–4.5 × 10−5 mol L−1 for cetylpyridinium chloride (CPC) and 0.4–6.0 × 10−5 mol L−1 for cetyl trimethyl-ammonium bromide (CTAB). The detection limits are 1.0 × 10−6 mol L−1 for CPC and 1.2 × 10−6 mol L−1 for CTAB, respectively. Based on this approach, this paper presents a new quantitative method for cationic surfactants assay.

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

A continuous spectrofluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonfluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent K m ( K app) values for protoporphyrinogen IX were measured as 3.8 ± 0.3, 3.6 ± 0.5, and 1.0 ± 0.1 μM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The K i for acifluorfen, a diphenylether herbicide, was measured as 0.53 μM for the human enzyme. Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h −1/mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min −1, representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.

Purification and characterization of the aromatic desulfinase, 2-(2 ′-hydroxyphenyl)benzenesulfinate desulfinase

2-(2 ′-Hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the cleavage of the carbon–sulfur bond of 2-(2 ′-hydroxyphenyl)benzenesulfinate (HPBS) to form hydroxybiphenyl and sulfite. This is the final step in the desulfurization of dibenzothiophene, the organosulfur compound used to study biodesulfurization of petroleum middle distillate. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS–PAGE. The p I of HPBS desulfinase is 5.6, the temperature optimum is 35 °C, and the pH optimum is 7.0. HPBS desulfinase has a K m of 0.90 ± 0.15 μM and a k cat of 1.3 ± 0.07 min −1. Several analogs were tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. HPBS desulfinase activity decreases in the presence of Cu 2+ and Zn 2+, while no metals significantly enhance enzyme activity. HPBS desulfinase is susceptible to tyrosine, tryptophan, and cysteine specific modification agents.

A continuous anaerobic fluorimetric assay for ferrochelatase by monitoring porphyrin disappearance

A continuous spectrofluorimetric assay for determining ferrochelatase activity has been developed using the physiological substrates ferrous iron and protoporphyrin IX under strictly anaerobic conditions. In contrast to heme, the product of the ferrochelatase-catalyzed reaction, protoporphyrin IX is fluorescent, and therefore the progress of the reaction can be monitored by following the decrease in protoporphyrin fluorescence intensity (with excitation and emission wavelengths at 505 and 635 nm, respectively). This continuous fluorimetric assay detects activities as low as 0.01 nmol porphyrin consumed min −1, representing an increase in sensitivity of up to two orders of magnitude over the currently used, discontinuous assays. The determination of the steady-state kinetic parameters of ferrochelatase yielded K m PPIX =1.4±0.2 μM , K m Fe 2+ =1.9±0.3 μM , and k cat =4.0±0.3 min −1 . In addition to its applicability for acquisition of kinetic data to characterize ferrochelatase and recombinant variants, this new method should permit detection of low concentrations of ferrochelatase in biological samples.

Spectrofluorimetric assay for the photodegradation products of alprazolam

A new spectrofluorimetric assay for the photodegradation products of the ansiolytic drug alprazolam is described. Alprazolam was found to be highly photolabile and special care should be taken to avoid light exposure during alprazolam storage and handling. The photostability of alprazolam was evaluated at pH 2.0, 3.6 and 5.0. The drug was exposed to UVA–UVB radiations, the photodegradation of alprazolam was followed by high-performance liquid chromatographic (HPLC) and the developed spectrofluorimetric assay allowed determination of the photodegradation products at very low concentrations (≥10 −5 M). The photoinstability was found to increase with the pH value decreasing, consequently acidic media should be avoided during the drug-development process.

Free radical production and labile iron pool decrease triggered by subtoxic concentration of aclarubicin in human leukemia cell lines

Aclarubicin (ACLA), which belongs to the antracycline class of antineoplasic agents, has been demonstrated as a differentiating agent for a broad range of human solid tumors and leukemia. By using dihydroethidium as a fluorescent probe, we show the ability of subtoxic (i.e. differentiating) concentration of ACLA to generate reactive oxygen species in both K562 and HL-60 leukemia cell lines. Besides, we have used a calcein-based spectrofluorimetric assay to determine the influence of ACLA treatment on the cellular labile iron pool (LIP). In both cell lines, the LIP level was markedly decreased in the presence of ACLA. Nevertheless, whereas ACLA-induced differentiation was obviously ROS-dependent, the LIP decrease was rather ROS-independent.

LVV-hemorphin-4 modulates Ca2+/calmodulin-dependent pathways in the immune system by the same mechanism as in the brain.

The effect of synthetic LVV-hemorphin-4 (LVV-H4) on human blood and tonsils lymphocytes total phosphatase activity was studied by a spectrofluorimetric assay using 4-methylumbelliferyl phosphate (4-MUP) as a substrate. It has been established that LVV-H4 at concentrations of 10(-9) to 10(-7) M induces the inhibition of human blood (12-24%) and tonsils (42-45%) lymphocytes total phosphatase activity as 1 mM EGTA. The same peptide at concentrations of 10(-5) to 10(-4) M induces activation of human blood (48-57%) and tonsils (20-25%) lymphocytes total phosphatase activity. LVV-H4 is able to neutralize the inhibitory effect of calmodulin (CaM) antagonist and calcineurin inhibitor trifluoperazine (TFP) on human blood lymphocyte total phosphatase activity. It is suggested that a dose-dependent activation/inhibition of lymphocytes total phosphatase activity is due to activation/inhibition of lymphocyte calcineurin activity. Using enzyme-linked immunosorbentassay (ELISA) it was found that LVV-H4 neutralized the inhibitory effect of cyclosporin A (CsA) and TFP on interleukin-2 (IL-2) synthesis by activated blood lymphocytes. LVV-H4 also affects the lymphocytes proliferation, suppressed in pathophysiological condition, and restores their function by enhancement of DNA synthesis, as determined by measuring of [3H] thymidine incorporation into lymphocytes. It has been proposed that CaM is an essential component in starting up the molecular mechanism of hemorphins action and that calcineurin is a key enzyme underlying the molecular mechanism of hemorphins action on the brain and immune system.

A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli.

A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1). NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.

Effect of overexpression of human Cu/Zn-SOD on activation-induced lymphocyte proliferation and apoptosis

The process of lymphocyte proliferation and apoptosis is known to be linked to oxidative stress. In the present study, we have used a new transgenic mouse model to investigate the effect of human Cu/Zn superoxide dismutase (Cu/Zn-SOD) overexpression on activation-induced lymphocytes proliferation and apoptosis. Cu/Zn-SOD activity was 3.5-fold higher in the spleen of the transgenic mice overexpressing Cu/Zn-SOD (Tg-Cu/Zn-SOD) compared to the wild-type littermates. Proliferative response of lymphocytes to lipopolysaccharide (LPS), Concanavalin A (Con A), and anti-CD3 was measured by [3H]-thymidine incorporation. Activation-induced apoptosis was determined by incubating the T cells with anti-CD3 (primary stimulus) for 72 h, followed by restimulation with Con A (secondary stimulus) for various times. Apoptosis was assessed by measuring DNA fragmentation using a spectrofluorimetric assay and monitoring the expression of the specific apoptotic markers (Fas/CD95 receptor and Fas/CD95 ligand (Fas-L) using flow cytometry. There was no significant difference in proliferative response of lymphocytes to LPS, Con A, or anti-CD3 in transgenic mice overexpressing human Cu/Zn superoxide dismutase (Tg-Cu/Zn-SOD) compared to wild-type littermates. In addition, no significant difference was observed in lymphocyte populations and subsets between Tg-Cu/Zn-SOD mice and wild-type littermates. However, splenic T cells from Tg-Cu/Zn-SOD mice exhibited a significantly (p < .05) higher level of activation-induced DNA fragmentation than T cells from wild-type littermates. The increase in DNA fragmentation was paralleled with an increase in the proportion of T cells expressing Fas and Fas-L molecules. The possible consequences of Cu/Zn-SOD overproduction on activation-induced apoptosis are discussed.

99/02385 Processes for the sorption of gaseous components at high temperatures (>900°C)

2-(2′-Hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the cleavage of the carbon–sulfur bond of 2-(2′-hydroxyphenyl)benzenesulfinate (HPBS) to form hydroxybiphenyl and sulfite. This is the final step in the desulfurization of dibenzothiophene, the organosulfur compound used to study biodesulfurization of petroleum middle distillate. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS–PAGE. The pI of HPBS desulfinase is 5.6, the temperature optimum is 35 °C, and the pH optimum is 7.0. HPBS desulfinase has a Km of 0.90 ± 0.15 μM and a kcat of 1.3 ± 0.07 min−1. Several analogs were tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. HPBS desulfinase activity decreases in the presence of Cu2+ and Zn2+, while no metals significantly enhance enzyme activity. HPBS desulfinase is susceptible to tyrosine, tryptophan, and cysteine specific modification agents.