Fluorescent Adducts Research Articles

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Formation of Pentosidine Cross-Linking in Myoglobin by Glyoxal: Detection of Fluorescent Advanced Glycation End Product.

Glyoxal, a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significance of protein modification by glyoxal-derived AGEs, we investigated the in vitro effect of glyoxal (200μM) on the monomeric heme protein myoglobin (Mb) (100μM) after incubation for one week at 25°C. Glyoxal-treated Mb exhibited increased absorbance around the Soret region, decreased α-helicity and thermal stability compared to control Mb. Intrinsic fluorescence spectrum of the treated Mb showed an additional signal in the 400-500nm region on excitation at 280nm that was absent in control Mb. When excited at 335nm, the glyoxal-treated sample gave a strong fluorescence indicating AGE formation. Mass spectrometric studies revealed formation of glyoxal-derived fluorescent AGE adduct pentosidine between Lys-145 and Arg-139 residues of Mb. Other than pentosidine, additional AGE adducts, namely, carboxymethyllysine at Lys-133, hydroimidazolone at Arg-31 and pyrrolidone-carboxymethyllysine at Lys-145 were also detected. Lys-145 was thus found to contain two different types of AGE adducts, indicating the heterogeneous nature of in vitro glycation reaction. AGE-induced protein modifications might be associated with complications in disease conditions.

Characterization of the selective alkylation site in hemoglobin A by dihydroartemisinin with tandem mass spectrometry

The reaction between the antimalarial drug dihydroartemisinin (DHA) and hemoglobin A (HbA) was investigated in vitro. A fluorescein-tagged artemisinin analog reacted with HbA and fluorescent HbA-drug adducts could be visualized on SDS-PAGE to confirm stable covalent reaction adducts and necessity of the endoperoxide moiety and Fe(II). Mass spectrometric analyses revealed that DHA favourably alkylated protein part rather than heme and the modification site was identified to be at Tyr35 of the beta globin chain.

Highly sensitive detection of cysteine over glutathione and homo-cysteine: New insight into the Michael addition of mercapto group to maleimide

A fluorescence “off–on” probe CMP for thiols was designed with coumarin as the fluorophore and maleimide as the receptor. The fluorescence of the coumarin was quenched through photoinduced electron transfer (PET) from the fluorophore to maleimide group. The Michael addition of the mercapto group toward maleimide formed a thioether with relatively weak fluorescence. The intramolecular nucleophilic substitution of amino group in cysteine (Cys) to alkylthio produced a much stronger fluorescent amino adduct, which was supported by UPLC-MS and NMR titration. The above sensing mechanism ensured CMP a highly sensitive probe toward Cys over GSH and Hcy. The fluorescence intensity at 495nm was linear with Cys concentration over the range of 0–10μM with a detection limit of 14nM and a rapid response time of 20min. High selectivity and good competition of the probe toward thiols over other biologically relevant species enabled us to monitor mercapto-containing proteins as well as fluorescence imaging Cys in living cells.

Photoinduced Tetrazole‐Based Functionalization of Off‐Stoichiometric Clickable Microparticles

We report the preparation of tetrazole‐containing step‐growth microparticles and the subsequent use of photoinduced nitrile imine‐mediated tetrazole‐ene cycloaddition (NITEC) reactions on the particles with spatiotemporal control. Microparticles with an average diameter of 4.1 µm and with inherent tetrazole‐ene dual functionality are prepared by a one‐pot off‐stoichiometric thiol‐Michael addition dispersion polymerization. The NITEC reaction is performed efficiently in the solid phase by UV irradiation, leading to the formation of fluorescent pyrozoline adducts, with an estimated quantum yield of 0.7. Particle concentration‐independent reaction kinetics are observed and full conversion is reached within 10 min of UV exposure at an intensity of 8 mW cm−2. Temporal control is demonstrated with either UV or rooftop sunlight irradiation of variable duration. By using two‐photon writing with a laser centered around 700 nm wavelength, spatial control is demonstrated with micrometer‐scale resolution via surface patterning of the microparticles. Further, microparticles with exclusive tetrazole functionality are prepared by a one‐pot, two‐step thiol‐Michael addition dispersion polymerization. The NITEC reaction between tetrazole‐functional particles and acrylates in solution is examined at various tetrazole/alkene molar ratios, and a 10:1 excess of alkenes in solution is found necessary for efficient functionalization.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298nm and λ em 550nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

Cellular glutathione content in the organ of Corti and its role during ototoxicity.

Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation.

Вплив біоактивних альдегідів на властивості желатини

Bioactive aldehydes are among main factors of proteins postsynthetic modifications, which are the cause and consequence of many diseases. Comparative study of some aldehydes modifying action on gelatin was carried out in vitro. Gelatin samples (20 mM) were incubated with: ribose, deoxyribose, glyoxal, methylglyoxal, formaldehyde, acrolein (20 mM each) and their combinations in 0.1 M Naphosphate buffer (pH 7.4) containing 0.02% sodium azide at 37 °C in the dark for 30 days. We investigated the fluorescent properties of these samples and their molecular weight distribution by electrophoresis. It has been revealed that formed adducts had different fluorescence spectra. According to fluorescence intensity these aldehydes were put in order: formaldehyde < methylglyoxal < acrolein <ribose < deoxyribose < glyoxal. The electrophoresis results showed fragments of gelatin molecular weight redistribution. By this index, the aldehydes rating was as follows: ribose < deoxyribose < acrolein < glyoxal < formaldehyde < methylglyoxal. Comparison of these two ratings indicates that aldehydes with a lower ability to form fluorescent adducts have higher ability to form intermolecular crosslinks. Therefore, the traditional clinical fluorescent test of a patients' skin surface for collagen crosslinks determination has to be verified by other tests for proteins postsynthetic modifications.

Experimental and computational evidence for hydrogen bonding interaction between 2′-deoxyadenosine conjugate adduct and amino-terminated organic film on Si(001)

A simple method for immobilization of malonaldehyde-acetaldehyde conjugate adduct with DNA base onto an amino-terminated surface of silicon from water solution is proposed. The Si(001) surface which contains OH groups was modified with 3-aminopropyltrimethoxysilane (APTMS) to serve as a linker between the silica surface and the organic adduct. The 2′-deoxyadenosine adduct was adsorbed on the APTMS/Si surface from acetonitrile/water solution. This nucleoside derivative is stable under laboratory conditions and emits a natural fluorescence, allowing for its adsorption on the APTMS/Si surface to be easily verified by fluorescence microscopy, non-contact atomic force microscopy and attenuated total reflectance Fourier transform infrared spectroscopy. The degree of surface coverage by the adduct was evaluated by X-ray photoelectron spectroscopy (XPS). Analysis of the XPS spectra revealed bands at 400.2eV and 533.1eV which are characteristic of a hydrogen bonded –NH2 and –OH group. This observation implies that the free electron donating –NH2 groups from the APTMS layer makes hydrogen bonds with the fluorescent adduct and immobilize it on the surface. The wetting angle of the APTMS/Si surface before and after adsorption of the nucleoside derivative does not differ significantly, which points to the involvement of an – OH group from 2′-deoxyadenosine to be involved in hydrogen bonding. These experimental results were further supported using quantum chemical calculations to demonstrate that the 2′deoxyadenosine adduct makes hydrogen bonds with the APTMS molecule. Furthermore, this hydrogen bond involves the –NH2 group from APTMS and –OH group at carbon atoms C3 and C6 from the deoxyribose ring of 2′deoxyadenosine.

Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE-dual-LIF.

Pt-based anticancer drugs, such as cisplatin, are known to undergo several (bio-)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA-Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60h at nanomolar concentrations of CFDA-Pt by using CE-LIF. CE-MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA-Pt are detectable in sensitive and cisplatin-resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA-Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE-LIF detection within 5min.

Unveiling the triplet state of a 4-amino-7-nitrobenzofurazan derivative in cyclohexane.

The nitrobenzofurazan (NBD) moiety has gained tremendous popularity over the last decades due to its fluorogenic nature. Indeed, upon interaction with aliphatic amines, it generates a stable fluorescent adduct, which has been used for protein and lipid labeling. In fact the 4-amino substituted NBD belongs to the broad family of intramolecular charge transfer molecules, with the amino group acting as an electron donor upon photoexcitation, and the nitro group as an electron acceptor. Although the singlet excited state of 4-amino NBD derivatives has been abundantly studied, investigation of its triplet manifold is scarce and even the absence of intersystem crossing for this type of molecules has been suggested. However, intramolecular charge transfer molecules are known to undergo intersystem crossing and high phosphorescence quantum yields have been reported in a nonpolar solvent. In the present paper, we have investigated the photophysical and photochemical properties of N-hexyl-7-nitrobenzo[c][1,2,5]xadiazole-4-amine. We have shown the existence of a triplet state for this molecule in cyclohexane via nanosecond laser flash photolysis. Interestingly, deactivation of the triplet state leads to photoproducts formation, which are only present in the absence of oxygen.

Rapid and thiol-specific high-throughput assay for simultaneous relative quantification of total thiols, protein thiols, and nonprotein thiols in cells.

Thiol groups in biological molecules play a significant role in various physiological functions and pathological conditions. Thiols are divided into two major groups: protein thiols and nonprotein thiols. Numerous methods have been reported for thiol assays. Most of these methods have been developed for glutathione, the principal nonprotein thiol, despite the fact that cellular protein thiols are more abundant than glutathione. Further, these methods usually involve a process of biological sample preparation followed by a separation method, and they are time-consuming. We reported previously a series of thiol-specific fluorogenic benzofurazan sulfides. These nonfluorescent benzofurazan sulfides react rapidly and specifically with a thiol to form a strong fluorescent thiol adduct. The rapid reaction, thiol-specific and fluorogenic nature of the sulfides successfully yielded an application of one of the sulfides for relative quantitation of total thiols in live cells through fluorescence microscopy. In this work, we employed the same compound to develop the first high-throughput method for simultaneous monitoring of protein thiols, nonprotein thiols, and total thiols in cells in a 96-well plate on a fluorescence microplate reader at λex = 430 nm and λem = 520 nm, respectively. The method is rapid and sensitive, and has been validated by an HPLC thiol assay method. The method can detect thiols with cell concentrations as low as 500 cells/well. We also demonstrated that the method can readily monitor changes in cellular thiol levels. Although the method cannot provide an absolute quantification for thiols because fluorescence intensity of different thiol adducts varies, it provides an accurate measurement of relative quantification, relative to the control. The method will be a valuable tool in thiol-related biomedical/pharmaceutical research.

Reactive carbonyl-scavenging ability of 2-aminoimidazoles: 2-amino-1-methylbenzimidazole and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The carbonyl-scavenging ability of 2-amino-1-methylbenzimidazole (AMBI) and the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was investigated in an attempt to identify new routes that can modify the carbonyl content of foods. The reaction of both AMBI and PhIP with 2-alkenals, 2,4-alkadienals, 4-oxo-2-alkenals, 4,5-epoxy-2-alkenals, and 4-hydroxy-2-nonenal produced fluorescent adducts, whose structure was determined for the adduct produced between AMBI and 2-pentenal. This adduct was isolated and identified by one- and two-dimensional nuclear magnetic resonance and high-resolution mass spectrometry as 2,10-dihydro-2-ethyl-10-methylpyrimido[1,2-a]benzimidazole. The formation of these adducts was parallel to the elimination of AMBI and PhIP. The Ea of the reaction between PhIP and 4-oxo-2-nonenal was 27.4 kJ/mol. All these results suggest that 2-aminoimidazoles can be employed to modify the carbonyl content of foods. At the same time and because the reaction produces the disappearance of the amino compound, lipid-derived carbonyl compounds can be employed to eliminate 2-aminoimidazoles, which suggests a new strategy for the elimination of heterocyclic aromatic amines in foods.

Atheroprotective immunization with malondialdehyde-modified LDL is hapten specific and dependent on advanced MDA adducts: implications for development of an atheroprotective vaccine

Immunization with homologous malondialdehyde (MDA)-modified LDL (MDA-LDL) leads to atheroprotection in experimental models supporting the concept that a vaccine to oxidation-specific epitopes (OSEs) of oxidized LDL could limit atherogenesis. However, modification of human LDL with OSE to use as an immunogen would be impractical for generalized use. Furthermore, when MDA is used to modify LDL, a wide variety of related MDA adducts are formed, both simple and more complex. To define the relevant epitopes that would reproduce the atheroprotective effects of immunization with MDA-LDL, we sought to determine the responsible immunodominant and atheroprotective adducts. We now demonstrate that fluorescent adducts of MDA involving the condensation of two or more MDA molecules with lysine to form malondialdehyde-acetaldehyde (MAA)-type adducts generate immunodominant epitopes that lead to atheroprotective responses. We further demonstrate that a T helper (Th) 2-biased hapten-specific humoral and cellular response is sufficient, and thus, MAA-modified homologous albumin is an equally effective immunogen. We further show that such Th2-biased humoral responses per se are not atheroprotective if they do not target relevant antigens. These data demonstrate the feasibility of development of a small-molecule immunogen that could stimulate MAA-specific immune responses, which could be used to develop a vaccine approach to retard or prevent atherogenesis.

WE‐E‐BRE‐03: Biological Validation of a Novel High‐Throughput Irradiator for Predictive Radiation Sensitivity Bioassays

Purpose:The large dose‐response variation in both tumor and normal cells between individual patients has led to the recent implementation of predictive bioassays of patient‐specific radiation sensitivity in order to personalize radiation therapy. This exciting new clinical paradigm has led us to develop a novel high‐throughput, variable dose‐rate irradiator to accompany these efforts. Here we present the biological validation of this irradiator through the use of human cells as a relative dosimeter assessed by two metrics, DNA double‐strand break repair pathway modulation and intercellular reactive oxygen species production.Methods:Immortalized human tonsilar epithelial cells were cultured in 96‐well micro titer plates and irradiated in groups of eight wells to absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy. High‐throughput immunofluorescent microscopy was used to detect γH2AX, a DNA double‐strand break repair mechanism recruiter. The same analysis was performed with the cells stained with CM‐H2DCFDA that produces a fluorescent adduct when exposed to reactive oxygen species during the irradiation cycle.Results:Irradiations of the immortalized human tonsilar epithelial cells at absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy produced excellent linearity in γH2AX and CM‐H2DCFDA with R2 values of 0.9939 and 0.9595 respectively. Single cell gel electrophoresis experimentation for the detection of physical DNA double‐strand breaks in ongoing.Conclusions:This work indicates significant potential for our high‐throughput variable dose rate irradiator for patient‐specific predictive radiation sensitivity bioassays. This irradiator provides a powerful tool by increasing the efficiency and number of assay techniques available to help personalize radiation therapy.

Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection.

Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6M HCl (110°C for 24h), 4.2M NaOH (105°C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60°C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25°C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20-25°C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal).

A General Method for Site Specific Fluorescent Labeling of Recombinant Chemokines

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11–12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4°C, and co-internalization with their receptors at 37°C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.

A rapid test for toxins from Amanita phalloides mushrooms

A rapid test for amanitins is proposed on the basis of their complexation with ethydium bromide leading to an intensively fluorescent adduct; the fluorescence band of the product has a shorter wavelength than that of ethydium bromide, which ensures the high specificity of the analysis. The fluorescence intensity in the system of ethydium bromide — amanitin grows linearly with the concentration of amanitin in the range 0.2–1.7 μM at the constant concentration of ethydium bromide, 0.6 μM. The detection limit is 1 μM, the time of the development of the analytical signal is up to 5 s.

Determination of Sulfite in Beer Based on Fluorescent Derivatives and Liquid Chromatographic Separation

A method was developed for quantification of sulfite in beer based on derivatization with the maleimide-derived probe ThioGlo I followed by separation of fluorescent adducts by reversed-phase high- ...

Effect of Hyperoxia on Resuscitation of Experimental Combined Traumatic Brain Injury and Hemorrhagic Shock in Mice

Hypotension and hypoxemia worsen traumatic brain injury outcomes. Hyperoxic resuscitation is controversial. The authors proposed that hyperoxia would improve hemodynamics and neuronal survival by augmenting oxygen delivery despite increased oxidative stress and neuroinflammation in experimental combined controlled cortical impact plus hemorrhagic shock in mice. Adult C57BL6 mice received controlled cortical impact followed by 35 min of hemorrhagic shock (mean arterial pressure, 25-27 mmHg). The resuscitation phase consisted of lactated Ringer's boluses titrated to mean arterial pressure greater than 70 mmHg. Definitive care included returning shed blood. Either oxygen or room air was administered during the resuscitation phases. Brain tissue levels of oxidative stress and inflammatory markers were measured at 24 h and hippocampal neuronal survival was quantified at 7 days. Hyperoxia markedly increased brain tissue oxygen tension approximately four- to fivefold (n = 8) and reduced resuscitation fluid requirements approximately 15% (n = 53; both P < 0.05). Systemic and cerebral physiologic variables were not significantly affected by hyperoxia. Hippocampal neuron survival was approximately 40% greater with oxygen versus room air (n = 18, P = 0.03). However, ascorbate depletion doubled with oxygen versus room air (n = 11, P < 0.05). Brain tissue cytokines and chemokines were increased approximately 2- to 20-fold (n = 10) after combined controlled cortical impact injury plus hemorrhagic shock, whereas hyperoxia shifted cytokines toward a proinflammatory profile. Hyperoxic resuscitation of cortical impact plus hemorrhagic shock reduced fluid requirements and increased brain tissue oxygen tension and hippocampal neuronal survival but exacerbated ascorbate depletion and neuroinflammation. The benefits of enhanced oxygen delivery during resuscitation of traumatic brain injury may outweigh detrimental increases in oxidative stress and neuroinflammation.