Water-stable fluorophores, produced by reaction with aldehyde solutions, for the histochemical localization of catechol- and indolethylamines.

The properties of a new fluorescence histochemical method for arylethylamines based on reaction with a mixture of 4% formaldehyde and 0.5% glutaraldehyde in aqueous solution are described. At room temperature the aldehyde mixture produced a well-localized fluorescence reaction in tissues, which, when examined microscopically in aqueous solution, was sufficiently intense for fine terminal noradrenergic axons to be seen. If the tissue was subsequently dried, the fluorescence intensity increased. At the same time as inducing the fluorophores, the aldehyde mixture fixed the tissue to a standard well suited for electron microscopy. It thus proved possible to locate amine containing cells in the fluorescence microscope and subsequently examine their ultrastructure. In aqueous models, the aldehyde mixture formed fluorescent products with adrenaline, noradrenaline, dopamine, dopa, 5-hydroxytryptamine and 5-hydroxytryptophan, but not with histamine or octopamine. The fluorescence induced in the aldehyde mixture remained stable if the tissue was subsequently transferred to saline or distilled water and when it was dehydrated in ethanol and cleared with xylene, benzene, chloroform or acetone.     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue

Summary Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5–1%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.     

Microspectrofluorometric characterization of the fluorescent derivatives of biogenic amines produced by aqueous aldehyde (Faglu) fixation

Summary The fluorescent derivatives of the reaction between an aqueous aldehyde (Faglu) solution and the biogenic amines (5-hydroxytryptamine, dopamine and noradrenaline) have been examined in order to determine the conditions required for maximal fluorescence yield. The fluorescence intensity and spectra of the final reaction products have been characterized and found to be highly dependent on the pH of the reaction mixture. Fluorophores derived from catecholamines have maximal yield and are most easily characterized when the reaction is performed at pH 7.3, whilst those derived from 5-hydroxytryptamine have maximal yield and are most readily characterized when the reaction is performed at pH 10.0. The addition of potassium ferricyanide to the Faglu further enhances the fluorescence yield of 5-hydroxytryptamine-containing models and tissues at both pH 10.0 and pH 7.3. Using the modified Faglu reaction mixture, it has been possible to demonstrate 5-hydroxytryptamine in the central nervous system without the need for pharmacological manipulation.     

Dimedone as an Aldehyde Blocking Reagent to Facilitate the Histochemical Demonstration of Glycogen

The demonstration of glycogen by the periodic acid-Schiff technique can be clarified by the interposition of a short dimedone blockade after the periodic acid oxidation. This blocks the PAS reaction of the vast majority of materials, but a very much longer blockade is required to abolish the reaction of glycogen. The dimedone-PAS method is valuable in situations where the demonstration of glycogen is otherwise difficult because of the proximity of diastase-fast PAS positive materials. For this purpose dimedone is best used in alcoholic solution (5% in absolute alcohol for about 3 hr at 60°C), since an aqueous solution permits diffusion of the aldehydes produced from the oxidation of glycogen. A saturated aqueous solution, or a 5% solution in 80% alcohol, is much more rapid in its blocking action, however, and may be more satisfactory when dimedone is used simply as an aldehyde blocking reagent.     

Automated precolumn fluorescence labelling by carbodiimide activation of N-acetylaspartate and N-acetylaspartylglutamate applied to an HPLC brain tissue analysis.

An automated method is described to couple carboxyl-containing metabolites to the fluorophore 2-aminoanthracene in aqueous solution (containing 75% methanol) in the presence of N,N-dicyclohexylcarbodiimide. The reaction was optimized for N-acetylaspartate (N-Ac-Asp) and N-acetylaspartylglutamate (N-Ac-Asp-Glu). The reactions occurred within 5 min at room temperature in the presence of 0.5-2 mM HCl. At concentrations of electrolytes exceeding 10 mM the coupling reaction became suboptimal. Derivatization was performed in a commercial precolumn derivatization unit. Additional tubing was needed to provide the reagents prior to reversed-phase HPLC and fluorescence detection. The assay is linear over at least three orders of magnitude; as little as 1 pmol could reproducibly be assayed in 100 micrograms wet weight brain tissue extracted with a mixture of methanol and 4 mM HCl (9:1, v/v). N-Ac-Asp and N-Ac-Asp-Glu levels in several brain regions and spinal cord were similar to those so far reported. The compounds could not be detected in peripheral tissue. The advantages, prospects and limitations of the present approach over existing methods to estimate water-soluble carboxylic acids is discussed.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

Using liquid crystals for the label-free detection of catalase at aqueous-LC interfaces

In this study, we developed a simple label-free method for the detection of catalase (CAT) using liquid crystals (LCs). The optical appearance of LCs changed from bright to dark when hydrogen peroxide was in contact with the dodecanal-doped nematic LC, 4-cyano-4′-pentylbiphenyl (5CB). Since hydrogen peroxide can oxidize aldehyde into carboxylic acid, an orientational transition of the LC from the planar to homeotropic state was induced by the self-assembled carboxylate monolayer formed at the aqueous/LC interface. The optical response of LCs exhibited a higher sensitivity to the presence of hydrogen peroxide in an alkaline solution. A new type of LC-based sensor was developed to monitor the presence of CAT in the aqueous phase. Due to the enzymatically catalytic hydrolysis of hydrogen peroxide, the bright-to-dark shift in the optical signal did not take place in the aqueous mixture of hydrogen peroxide and catalase. In contrast, the optical response changed from bright to dark when the mixture in the optical cell was replaced with an aqueous solution of hydrogen peroxide. Considering the optical response of LCs related to the absence and presence of hydrogen peroxide, the aldehyde-doped 5CB might have potential utility in real-time recognition and detection of chemical and biological events associated with hydrogen peroxide.     

Direct Staining of Reticular Fibers with Gold Chloride

Reticular fibers are selectively stained in paraffin sections of formalin-fixed or Bouin's-fixed tissue as follows: 1% aqueous solution of gold chloride for 20 min, followed by a 10 min immersion in an aqueous solution containing 5% Na₂CO₃ and 0.5% KOH. The sections then are placed in a 5% aqueous solution of KI for 2 min. Counterstaining with a 0.25% aqueous solution of methylene blue chloride is optional. The reticular fibers stain dark pink; the collagen bundles are a light pink to straw color without the counterstain, or a light blue color when the methylene blue is used.     

Evaluation of the intramolecular stacking of the fluorosulfonylbenzoyl derivatives of 1,N6-ethenoadenosine, adenosine, and guanosine

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (? = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (? = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.     

Determination of cycloserine in human plasma by high-performance liquid chromatography with fluorescence detection,using derivatization with p-benzoquinone

A new method for determining cycloserine in plasma samples is described. This method is based on the derivatization of cycloserine with p-benzoquinone, a reaction that takes place at the same time as the process of plasma deproteinization due to the presence of ethanol as solvent in the solution of the derivatization reagent. Four derivatives are obtained from this reaction. The main derivative is well correlated with the cycloserine concentration. The ratio between the volumes of the plasma sample and the reagent solution is 1:2 for a p-benzoquinone concentration of 1000 μg/mL. Elution from a C₁₈ column was isocratic, using a mobile phase containing (v/v) 85% aqueous 0.1% formic acid solution, and 15% (v/v) of a mixture of methanol and acetonitrile (1:1), with a flow-rate of 1 mL/min, at 25°C. Determinations by fluorescence detection were achieved with excitation at 381 nm and emission at 450 nm, with a detection limit of 10 ng/mL for an injection volume of 5 μL. This method was validated and applied to the determination of cycloserine in blood plasma samples of several healthy volunteers.     

Quantitative aspects of the Ninhydrin-Schiff reaction on amino cellulose films and tissue sections

Summary In the ninhydrin-Schiff reaction primary amino groups are converted by oxidation with ninhydrin to aldehyde groups which are subsequently stained with pararosanilin. Amino cellulose films, used as a model, and sections of muscle tissue were submitted to this reaction. The amino groups were stained before and after the ninhydrin reaction with dinitrofluorobenzene and the generated aldehyde groups were stained with dinitrophenyl-hydrazine. The molar extinction coefficients used for the calculation of the molar amounts of chromophores from extinction values, and the conditions for maximal staining intensity were determined on the amino cellulose model. With these data the yields of the different steps in the reaction sequence could be calculated in molar amounts from the extinction measurements. The results showed that from the amino groups originally present in the tissue sections less than 40% were converted by ninhydrin. About 90% of the converted amino groups were found as aldehyde groups and from these only 7% reacted with pararosanilin. On amino cellulose similar data were obtained. Attempts were made by modification of the conditions in the ninhydrin oxidation step to increase the overall yield of the reaction. These were only partially successful, but indicate that further quantitative study of other reaction conditions and different aldehyde generating agents could be promising.     

Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

用于寡核苷酸芯片制备载体的醛基载玻片的制备方法优化及性质研究

优化了醛基载玻片的制备方法 ,探讨了醛基修饰载玻片固定寡核苷酸探针的性质。研究发现氨基硅烷试剂的浓度是影响载玻片荧光背景的主要因素 ;2 %氨基化试剂处理 16min、戊二醛处理 30min可以得到荧光背景较低、固定效果较好的醛基载玻片。寡核苷酸固定过程中 ,末端氨基修饰没有明显的特异性 ,但是可以提高被固定探针的杂交容量。在较低的浓度 (小于 10 μmol L)时 ,探针的浓度与杂交信号趋近线性关系 ,浓度为 2 0 μmol L时杂交信号达到饱和     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

An efficient conversion of carboxylic acids to one-carbon degraded aldehydes via 2-hydroperoxy acids

After the formation of dianions of a carboxylic acid with lithium diisopropylamide, oxygen was bubbled into the solution to produce 2-hydroperoxy acid. Then the reaction mixture was acidified with a 2 N HCl solution and subsequently elevated to 50 degrees C to afford the aldehyde with the loss of one carbon atom. Even saturated (C(10)-C(20)) and unsaturated (C(18:1)) carboxylic acids were converted into the odd aldehydes (C(9)-C(19), C(17:1)) in high yields. This conversion was found to be an efficient method for the preparation of carboxylic acids (Cn) to one-carbon degraded aldehydes (Cn-1) via 2-hydroperoxy acids.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver

The further metabolism of N-desisopropylpropranolol (NDP), a side-chain metabolite of propranolol (PL), was investigated in isolated rat hepatocytes. Propranolol glycol (PGL) was generated from NDP as a major metabolite. Naphtetrazole (NTE), a potent inhibitor of monoamine oxidase (MAO), significantly retarded the disappearance of NDP from the incubation medium, suggesting the involvement of MAO in the deamination of NDP to an aldehyde intermediate. In a reaction mixture of rat liver mitochondria and cytosol with NADPH, phenobarbital, a specific inhibitor of aldehyde reductase, and 4-nitrobenzaldehyde (4-NBA), a substrate inhibitor of aldehyde reductase, decreased the formation of PGL from NDP. 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation. The optimal pH for the formation of PGL from NDP in the reaction mixture was approximately 8.0. Based on these results, we propose the possibility that, in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. Furthermore, the enantioselective metabolism of NDP to PGL was examined. In isolated rat hepatocytes, the amount of PGL formed from S-NDP [S(-)-form of NDP] was larger than that of PGL formed from R-NDP [R(+)-form of NDP].     

Hydrolysis and rearrangement reactions of riboflavin phosphates. An explicit kinetic study

Four isomeric monophosphates and five isomeric bisphosphates of riboflavin were isolated from commercial FMN or were prepared by acid-catalyzed isomerization. The reaction of riboflavin monophosphates in aqueous solution was studied in the pH range between 0 and 9 under various conditions. The predominant reaction at pH values below 2 is the acid-catalyzed migration of the phosphate group. A detailed kinetic study of this reaction was performed by high pressure liquid chromatography. Experimental data were fitted with computer-generated curves based on an algorithm for a network of first-order reactions. Rate constants and activation parameters were obtained for the temperature range of 50-80 degrees C at pH 1. At thermodynamic equilibrium, the reaction mixture contains about 66% 5'-phosphate, 11% 4'-phosphate, 8% 3'-phosphate, and 15% 2'-phosphate (pH 1.0, 50 degrees C). In the pH range between 3 and 7, the hydrolysis of FMN is the prevailing reaction with a rate maximum at about pH 4. The same experimental approach was used in a subsequent kinetic study on the isomerization of riboflavin bisphosphates. The formation of five out of six possible isomers was studied quantitatively and rate constants for each partial reaction were obtained.     

Formal intramolecular photoredox chemistry of anthraquinones in aqueous solution: photodeprotection for alcohols, aldehydes and ketones.

The formal intramolecular photoredox reaction initially discovered for the parent 2-(hydroxymethyl)anthraquinone (1) in aqueous solution has been extended to a variety of anthraquinones derivatives 6-13, to explore the generality of the reaction, and to investigate its potential utility as a photodeprotecting chromophore. In addition, the related diketone 14 was studied to investigate the need for the anthraquinone chromophore in these formal intramolecular reactions. All the anthraquinones studied (except for 9) undergo formal unimolecular photoredox reaction with a range of quantum yields (Phi = 0.02-0.7). Anthraquinones 7, 8, 10 and 11 photoreleased the corresponding alcohol, aldehyde, or ketone with good yields (80-90%), making it potentially useful for photocaging in aqueous solution. Diketone 14 undergoes an analogous photoredox reaction but only in acid (Phi = 0.003, pH < 1), to give the formal redox product diphenylisobenzofuran 32 thereby demonstrating that other aromatic diketones can react in an analogous fashion. The ionic photochemistry exhibited by these aromatic ketones is fully compatible with the recent discovery of the surprising acid-catalyzed photochemical hydration of benzophenone reported by Jacob Wirz and coworkers (M. Ramseier, P. Senn and J. Wirz, J. Phys. Chem. A, 2003, 107, 3305-3315).     

Spectroscopic investigations of the potential-sensitive membrane probe RH421.

The absorbance spectra, fluorescence emission and excitation spectra, and fluorescence anisotropy of the potential-sensitive styryl dye RH421 have been investigated in aqueous solution and bound to the lipid membrane. The potential-sensitive response of the dye has been studied using a preparation of membrane fragments containing a high density of Na+, K(+)-ATPase molecules. In aqueous solution the dye is sensitive both to changes in pH and ionic strength. Evidence has been found that the dye readily aggregates in aqueous solution. Aggregation is enhanced by an increase in ionic strength. The aggregates formed display a low fluorescence intensity. At high pH values (above approx. 8) changes in the dye's fluorescence spectra are observed, which may be due to a reaction of the dye with hydroxide ions. When bound to the membrane the dye also exhibits concentration-dependent fluorescence changes. The potential-sensitive response of the dye in Na(+),K(+)-ATPase membrane fragments after addition of MgATP in the presence of Na+ ions cannot be explained by a purely electrochromic mechanism. The results are consistent with either a potential-dependent equilibrium between membrane-bound dye monomers and membrane-bound dimers, similar to that previously proposed for the dye merocyanine 540, or with a field-induced structural change of the membrane.