Dithioerythritol Research Articles

The Mycotoxin Patulin Inhibits the Mitochondrial Carnitine/Acylcarnitine Carrier (SLC25A20) by Interaction with Cys136 Implications for Human Health.

The effect of mycotoxin patulin (4-hydroxy-4H-furo [3,2c] pyran-2 [6H] -one) on the mitochondrial carnitine/acylcarnitine carrier (CAC, SLC25A20) was investigated. Transport function was measured as [3H]-carnitineex/carnitinein antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Patulin (PAT) inhibited both the mitochondrial native and recombinant transporters. The inhibition was not reversed by physiological and sulfhydryl-reducing reagents, such as glutathione (GSH) or dithioerythritol (DTE). The IC50 derived from the dose-response analysis indicated that PAT inhibition was in the range of 50 µM both on the native and on rat and human recombinant protein. The kinetics process revealed a competitive type of inhibition. A substrate protection experiment confirmed that the interaction of PAT with the protein occurred within a protein region, including the substrate-binding area. The mechanism of inhibition was identified using the site-directed mutagenesis of CAC. No inhibition was observed on Cys mutants in which only the C136 residue was mutated. Mass spectrometry studies and in silico molecular modeling analysis corroborated the outcomes derived from the biochemical assays.

A fluorescence signal amplification and specific energy transfer strategy for sensitive detection of β-galactosidase based on the effects of AIE and host-guest recognition

Benefit from the efficient energy transfer, aggregation-induced emission (AIE) and host-guest recognition as strategies of signal amplification and specific binding have been applied to establish the sensing system; however, the application of these two attractive strategies in one system is rare. Herein, we propose a “turn-on” to “turn-off” fluorescent strategy for sensitive detection of β-galactosidase (β-Gal) based on the application of AIE and host-guest recognition. In this work, a novel red-emitted (635 nm) copper nanoclusters (CuNCs) protected by dithioerythritol (DTE) and β-cyclodextrin (β-CD) has been found to possess AIE property induced by aluminum cations to achieve the “turn-on” process, and the coordinated behavior between aluminum cations and DTE/β-CD CuNCs is also discussed. As the hydrolysis product of β-Gal and 4-nitrophenyl-β-D-galactopyranoside, p-nitrophenol can combine with β-CD in DTE/β-CD CuNCs by the host-guest recognition to realize the efficient photoelectron transfer to “turn-off” the fluorescence. This strategy performs the exciting linear range of 0.0–50.0 U/L and detection limit of 0.56 U/L (S/N = 3) for sensitive detection of β-Gal and further applies in biologic samples successfully. As far as we know, this is the first work to combine AIE and host-guest recognition in one system to construct the sensing platform, which can achieve efficient and specific energy transfer to improve the sensitivity of the system.

Artificial Oral Processing of Extruded Pea Flour Snacks

The structure of extruded pea flour can affect chewing performances. Our objective was to relate the bolus properties (fragmentation, moisture content and viscosity) of chewed extruded pea snacks to their structure. In order to have control over oral physiological parameters, we opted for an in vitro approach using a chewing simulator, the variables of which were the flow rate of artificial salivary fluid and chewing time. The structure of the extruded pea snacks was characterized by its density and protein solubility in dithioerythritol (DTE), which reflected the amount of protein aggregates cross-linked by disulphide bonds. The particle size distribution and median width (D50) of the bolus fragments were determined by imaging technique. Bolus viscosity and saliva uptake (ΔWC) were respectively determined by capillary rheometry and gravimetric method. Extruded snacks were persistently reduced to small particles (~ 2 mm) during chewing process following a unique curve of D50 = f (chewing time) fitted by a power function. ΔWC evolution during chewing process was expressed as a function of the structure index, defined as the product of the snacks’ relative density by protein solubility in DTE. The boluses’ viscosity exhibited a shear thinning behaviour the consistency index of which was negatively correlated to the saliva uptake (ΔWC) through a plasticization coefficient (α). α values (< 2) were much smaller than those reported for cereal products (15–30) and depended on protein solubility in DTE.

Effect of Copper on the Mitochondrial Carnitine/Acylcarnitine Carrier Via Interaction with Cys136 and Cys155. Possible Implications in Pathophysiology.

The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [3H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in E. coli. Cu2+ (as well as Cu+) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC50 of 1.6 µM for Cu2+ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu2+ on the recombinant protein, an IC50 of 0.28 µM was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu2+. However, a substrate protection experiment indicated that the interaction of Cu2+ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC50 values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu2+. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu2+ with the two Cys residues, which blocks the conformational changes required for transport cycle.

Successful renal transplantation in a patient with perinuclear antineutrophil cytoplasmic antibody-associated vasculitis with chronic kidney disease with complement-dependent cytotoxicity crossmatch positivity (autoantibody induced) and donor-specific antibodies and flow cytometry crossmatch negative

The positive complement-dependent cytotoxicity crossmatch (CDCXM) is considered as a contraindication for renal transplantation (RT) since long in an effort to avoid immediate rejection. There has been tremendous development in our understanding of transplant immunology today, and more sensitive and specific methods such as flow cytometry crossmatch (FCXM) and solid-phase antibody screening are used for detailed immunological assessment. We report successful RT in a 23-year-old girl with end-stage renal disease due to vasculitis on dialysis for 2 years. Before transplantation, the patient had Anti Human Globulin CDCXM positive while Dithioerythritol (DTT)-CDCXM was negative. The patient had no donor-specific antibodies examined by Luminex single-antigen beads and her FCMXM was negative. In the posttransplant period, there was no evidence of immune injury. Her serum creatinine was 0.7 mg/dl on the 3rd posttransplant day at the time of discharge. Induction immunosuppression was rabbit thymoglobulin (1.5 mb/kg) and methylprednisolone (500 mg, 3 doses) and maintenance immunosuppression was tacrolimus + prednisolone + mycophenolic acid. We found that even though CDCXM positivity has been traditionally considered as a contraindication for renal transplantation, in few carefully selected patients, we can still proceed to renal transplantation even if CDCXM is positive if more advanced and robust immunological tools such as FCMXM and donor-specific antibodies are negative. In our patient, AHG-CDCXM was positive while DTT-CDCXM was negative making it clear that antibodies of IgM origin were probably responsible for CDCXM positivity.

Colorimetric determination of mercury(II) using gold nanoparticles and double ligand exchange.

A colorimetric assay is described for highly selective and sensitive determination of Hg(II) ions by using gold nanoparticles (AuNPs) functionalized with dithioerythritol (DETL). This method relies on the unique optical properties of DETL-functionalized AuNPs as well as the thiophilicity of both AuNPs and Hg(II). In the presence of DETL, the AuNPs aggregate due to ligand exchange between thiol groups of DETL and the citrate ions on the surface of AuNPs. This induces a color change from red to blue. On addition of Hg(II), the thiol groups preferably interact with Hg(II) rather than with AuNPs. Thus, the DETL is released from the surface of the AuNPs and binds to Hg(II). This triggers the redispersion of the AuNPs. The ratio of absorbances at 650 and 525nm drops linearly in two Hg(II) concentration ranges (viz. from 0.1 to 0.5μM, and from 0.5 to 5μM). The ions Cu(II), Pb(II), and Cd(II) do not interfere even in the absence of masking agents. The detection limit is as low as 24nM. Graphical abstract A highly selective colorimetric method based on gold nanoparticles via double ligand exchange reaction is described for determination of Hg2+. This assay can selective detect Hg2+ with no response to major interfering metal ions such as Cu2+, Pb2+, and Cd2+ in the absence of masking agents compared with previous works.

Preparation of core-crosslinked linear-dendritic copolymer micelles with enhanced stability and their application for drug solubilisation

In this study we explore the preparation of core-crosslinked micelles of linear-dendritic methoxy-poly(ethylene glycol) (MPEG)-co-poly(ester-sulfide) (PES) polymers to improve the stability of such polymeric micelle systems against premature disintegration and drug release. A series of MPEG-PES copolymers were synthesised via stepwise reactions of acetylation and thiol-ene photoreaction. Surface tension measurement showed that the copolymers with ethenyl surface groups could self-associate in dilute aqueous solutions to form micelles. Crosslinking within the micelle cores in the presence of dithioerythritol (DTT) linker was initiated under UV radiation. The formation of core-crosslinked micelles was confirmed by HPLC in combination with charged aerosol detection (CAD). The copolymers were found to readily hydrolyse under acidic conditions due to the ester-containing dendrons. Drug solubilisation capacities of the micellar solutions were determined using griseofulvin as a poorly water-soluble model drug. The solubility of griseofulvin showed a 10-fold enhancement in 1% w/v micelle solution and increased with the concentration of the copolymers. Drug release studies indicated that a more sustained release of griseofulvin was achieved for the core-crosslinked micelles compared to the non-crosslinked micelles, attributable to greater stability of the crosslinked core structure. The findings of this study present a new pathway towards developing biodegradable polymeric nanocarriers.

The reaction of dithioerythritol and dithiothreitol with As(III), Sb(III), and Bi(III) compounds

Arsenic trichloride and As2O3 reacted in methanol with dithioerythritol (DTE) and dithiothreitol (DTT) giving the known bicyclic adducts DTE-As and DTT-As, while Sb2O3 (in DMSO) and Bi2O3 (in MeOH) did not react. The oligo- or polymeric phenylarsine oxide reacted with DTE/DTT giving the cyclic adduct DTE-AsPh, while the structure of the DTT-AsPh is not certain. Under a 1:1 stoichiometry, antimony(III) and bismuth(III) chlorides as well as bismuth(III) nitrate pentahydrate afforded the respective monomeric, cyclic adducts with DTE/DTT in methanol, but in acetonitrile were also spectroscopically detected NH4 + and acetamide (AcNH2) occluded in the adducts. The 1H and 13C NMR spectra of Sb(III) and Bi(III) complexes showed broad to very broad signals. The exchange reaction between complexed DTE/DTT and 2,3-dithio-1-propanol (British Anti-Lewisite) was also studied. .

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics

BackgroundThe macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases. MethodsThe inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction. ResultsWhen individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I1 and I2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI1I2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT≥1μM. The TBT-driven enzyme desensitization was reversed by DTE. ConclusionsThe macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT. General significanceThe findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.

Dithiols as more effective than monothiols in protecting biomacromolecules from free-radical-mediated damage: in vitro oxidative degradation of high-molar-mass hyaluronan

AbstractOxidative stress and the resulting damage to cellular and extracellular components has been observed in a variety of degenerative processes, including degenerative joint disorders, where high-molar-mass hyaluronan (HA) is often found to be massively degraded. The present study sought to test the hypothesis that dithiols are more effective in protecting biomacromolecules from free-radicalmediated damage than monothiols. The materials/thiols tested included bucillamine (BUC), dithioerythritol (DTE), dithiothreitol (DTT) and glutathione (GSH), as a reference, for their effectiveness in protecting HA from oxidative degradation induced in vitro. Since HA degradation results in a decrease in its dynamic viscosity, rotational viscometry was applied to follow HA oxidative degradation. The free-radical-scavenging activities of the thiols tested were determined by 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) assays. It was found that all the dithiols in the concentration range tested protected HA from the oxidative degradation. On the other hand, monothiol GSH exerted protection only at high concentrations (10 μmol L−1 and 100 μmol L−1) and 1 μmol L−1 of GSH even exhibited a prodegradative effect. The ABTS assay revealed free-radical scavenging activities in the following order: BUC, DTT, DTE, GSH, and that of the DPPH assay: BUC, DTE, DTT, GSH. In conclusion, it was demonstrated that dithiols may be more effective than monothiols in affording biomacromolecule protection from oxidative degradation.

Glutathione controls the redox state of the mitochondrial carnitine/acylcarnitine carrier Cys residues by glutathionylation

BackgroundThe mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches. MethodsThe effect of GSH and GSSG on the [3H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody. ResultsGSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37°C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC. ConclusionsCAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation. General significanceCAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations.

High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity methods: Implications for in vitro diagnostics

Determination of serum thymidine kinase 1 (STK1) activity has been used as a proliferation marker for neoplastic diseases in both human and veterinary medicine. The purpose of this study was to determine STK1 activity and enzyme levels in different dog tumours. Serum samples from three dogs with leukaemia, five with lymphoma, 21 with solid tumours and 18 healthy dogs were analyzed for STK1 activity, using an optimized [3H]-deoxythymidine (dThd) phosphorylation assay, and for STK1 protein levels using an immunoaffinity/western blot assay.STK1 activity in dogs with haematological tumours was significantly higher than in the solid tumour and healthy dog groups (mean±standard deviation [SD]=65±79, 1.1±0.5, and 1.0±0.4pmol/min/mL, respectively). Serum samples were analyzed after immunoaffinity isolation by western blot and the TK1 26kDa band intensities quantified revealing that concentrations were significantly higher in dogs with haematological tumours and solid tumours compared to healthy dogs (mean±SD=33±12, 30±13, and 10±5ng/mL, respectively). Pre-incubation with the reducing agent dithioerythritol (DTE) showed a decrease in STK1 activity and protein levels in most samples, but an increase of about 20% in sera from healthy dogs and from those with haematological malignancies. Compared to animals with solid tumours, the specific STK1 activity (nmol [3H]-deoxythymidine monophosphate (dTMP)/min/mg of TK1 protein of 26kDa) was 30-fold higher in haematological malignancies and 2.5-fold higher in healthy dogs, respectively. The results demonstrate that there is a large fraction of inactive TK1 protein, particularly in sera from dogs with solid tumours. The findings are important in the use of STK1 as a biomarker.

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

Mechanisms of Heat-Mediated Aggregation of Wheat Gluten Protein upon Pasta Processing

During pasta processing, structural changes of protein occur, due to changes in water content, mechanical energy input, and high temperature treatments. The present paper investigates the impact of successive and intense thermal treatments (high temperature drying, cooking, and overcooking) on aggregation of gluten protein in pasta. Protein aggregation was evaluated by the measurement of sensitivity of disulfide bonds toward reduction with dithioerythritol (DTE), at different reactions times. In addition to the loss in protein extractability in sodium dodecyl sulfate buffer, heat treatments induced a drastic change in disulfide bonds sensitivity toward DTE reduction and in size-exclusion high-performance liquid chromatography profiles of fully reduced protein. The protein solubility loss was assumed to derive from the increasing connectivity of protein upon heat treatments. The increasing degree of protein upon aggregation would be due to the formation of additional interchain disulfide bonds.

Inhibition Of Scramblase-1 Attenuates Endothelial Fluid Loss During Ischemia Reperfusion Injury

Introduction: Scramblase 1(PLSCR-1) is perceived to trigger apoptosis in damaged cells by translocating phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the lipid bilayer. The exposed PS is thought to target damaged cells for apoptosis. Though originally described in red blood cells, the existence of this system in endothelial cells is unknown. We hypothesized that PLSCR-1 plays a critical role during ischemia-reperfusion injury (IRI) by increasing endothelial cell surface PS leading to apoptosis and contributing to increased microvascular permeability. Our specific aims were: 1) to document inducible PS exposure on endothelial cell membranes, 2) to evaluate the impact of PLSCR-1 inhibition on microvascular permeability during IRI, and 3) to confirm protein knockdown of PLSCR-1 by RNA interference via Western Blot. Methods: Cultured bovine pulmonary artery endothelial cells (BPAECs) were exposed to calcium ionophore (A23187) to induce translocation of PS. PS exposure was assessed on BPAEC membranes by flow cytometry using the PS-binding molecule, Alexa 488 Di-annexin. Next, mesenteric venular microvascular permeability (Lp) during IRI was examined using an intra-vital micro-occlusion technique in rats. Prior to IRI, rats were pretreated with: 1) Dithioerythritol (DTE) an inhibitor of the cysteine residues essential for PLSCR-1 translocation (n=3), 2) 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the anion exchange mechanism necessary for PLSCR-1 function (n=3), 3) 2-bromopalmitate, an inhibitor of PLSCR-1 trafficking to the plasma membrane which renders PLSCR-1 nonfunctional (n=3), and 4) PLSCR-1 specific siRNA (n=3). Lp was measured throughout IRI for each group. PLSCR-1 knockdown was confirmed by Western blot (n=3). Results: Treatment of BPAECs with calcium ionophore increased Di-annexin binding in treated cells (N = 3) compared to controls (N = 3) (mean absorbance controls vs. treated = 109.4±42.1nm vs. 2765.5±848.5nm, p=0.04). In vivo, IRI increased Lp approximately 7-fold. This increase was attenuated 40% (p<0.01), 37% (p<0.01), and 55% (p< 0.01) by DTE, DIDS, and 2-BP respectively. The attenuation due to scramblase siRNA was most robust and attenuated Lp 70% during IRI (p<0.01). Western blots confirmed knockdown of PLSCR-1 as measured by mean peak band optical intensity units (OIU): PLSCR-1 siRNA = 2600±170 OIU, dsDNA control = 3100±66 OIU, p=0.02). Conclusions: The data demonstrates that endothelial cells have the capacity to translocate PS to the cell surface. The data also suggests that both non-specific (DTE, DIDS, and 2-BP) and specific (siRNA) inhibition of PLSCR-1 attenuate increases in microvascular venular permeability observed during IRI. Methods of targeting PLSCR-1 function and resultant PS exposure may be of potential therapeutic value against the development of microvascular permeability observed during IRI.

The effect of in vitro dithioerythritol exposure on human metaphase chromosome structure.

When human lymphoid cells in G2-phase are treated with Dithioerythritol (DTE), a widely used agent which both maintains sulphydryl groups in the reduced state, and reduces disulphides, the frequencies of banded and uncoiled metaphase chromosomes, observed on traditional chromosome slides are increased. In agreement with SUMNERS deduction (1973) it is suggested that sulphydryl-disulphide transition during mitosis is involved in chromosome contraction and that disulphides maintain chromosomes in the condensed structurally stable state during metaphase.

Simultaneous determination of several natural steroids in blood plasma from perch (Perca fluviatilis) by GC-HRMS

A gas chromatography-high resolution mass spectrometry (GC-HRMS) procedure for the simultaneous determination of 18 endogenous steroid hormones in blood plasma from teleost fish has been developed. Proteins were removed by precipitation in methanol and lipids were removed by a liquid–liquid extraction. The protein and lipid free extract was further purified by using two successive solid phase extraction (SPE) methods (C18 and NH2). The isolated steroid hormones were silylated with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA): iodotrimethylsilane (TMSI):dithioerythritol (DTE) prior to determination with GC-HRMS working in selective ion recording mode. A volume of 200 µL blood plasma was sufficient for accurate determination of the steroid hormone levels, which enabled determination in individual fish. The detection limits ranged from 0.0003 to 0.2 ng g−1 blood plasma from perch (Perca fluviatilis), approximately 10 to 100 times lower then previously reported in this field. The recoveries for the entire procedure were in the range 58 to 150% with a variation, expressed as standard deviation (SD), below 10% with some exceptions. Despite the multi-step clean-up procedure, the intra-assay coefficient of variation, i.e. the within-day variation, for most steroid hormones was well below 14%. Finally, the procedure has been successfully applied to the determination of steroid hormones in blood plasma from female perch caught in two Swedish lakes.

Routine method for the simultaneous quantification of 17α-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography–mass spectrometry

Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic–mass spectrometric method for the simultaneous quantification of 17α-hydroxyprogesterone (17αOHP), testosterone ( T), dehydroepiandrosterone (DHEA), androstenedione (Δ4-A), cortisol ( F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5 mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl- N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1 ng/mL. The limit of quantification was 0.1 ng/mL for all steroids except cortisol which was at 0.25 ng/mL. d 3-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10 ng/mL ( n = 10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10 ng/mL and ranged from −9.2% to 10.6% ( n = 10). Linear calibration equations were obtained for all five steroids (0.125–31.25 ng/mL) and for cortisol (0.125–200 ng/mL). Relative recoveries at concentrations 1.0 and 12.5 ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels.

Sputum eosinophils from asthmatics express ICAM-1 and HLA-DR

Sputum from symptomatic asthmatics is a rich source of eosinophils from the respiratory tract. Following liquefaction of sputum with dithioerythritol (DTE), a cell suspension for indirect double immunofluorescence with flow cytometry was obtained. Eosinophils were identified using anti-CD9 fluorescein conjugate, and particular surface markers measured with the relevant mouse MoAb followed by goat anti-mouse immunoglobulin phycoerythrin conjugate. Blood and sputum eosinophil surface markers were determined in parallel from asthmatics not receiving steroid therapy. Sputum eosinophils were found to have considerably elevated levels of CD11b, a reflection of eosinophil activation. Sputum but not blood eosinophils were found to express ICAM-1 (nine out of 11 cases) and HLA-DR (eight out of 11 cases). Furthermore, following culture of normal blood eosinophils with pooled T cell supernatants, ICAM-1 and HLA-DR could be induced in vitro. The induction of eosinophil adhesion molecules such as ICAM-1 and HLA-DR may influence eosinophil localization and function in asthma.

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells ( Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca 2+ dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential ( Δ Ψ m ) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells. To cite this article: S.K. Panda et al., C. R. Biologies 331 (2008).