Concurrent Demonstration of Desoxyribose Nucleic Acid and 1,2-Glycols

Sections from rat tissue were fixed in 10% formalin in 90% alcohol and placed in a 1.0% suspension of sodium bismuthate (NaBiO₃) in 40% phosphoric acid for 40 minutes at room temperature. Bismuth phosphate crystals were removed with 2N HCl. The sections were next placed in the Schiff reagent for 20 minutes. By this method the DNA was hydrolyzed by the phosphoric acid and the 1,2-glycols were oxidized by the NaBiO₃. In both cases aldehyde groups were released and subsequently stained by the Schiff reagent. A photomicrograph is included demonstrating the nuclei, goblet cells, striated border and basement membrane stained by this combined method.     

A Triple Stain for Deoxyribonucleic Acid, Polysaccharides and Proteins

Tissues from mammalian, amphibian, insect and plant sources were fixed in formalin or in Carnoy's, Helly's, or Smith's fluid and processed in the usual manner to mounted paraffin sections. These were stained by the sequence: Feulgen reaction (using azure-A-Schiff reagent); periodic acid Schiff (with basic-fuchsin-Schiff reagent); and a 0.02% solution of naphthol yellow S in 1 % acetic acid. Nuclei stained blue to green; polysaccharides, red; and proteins, yellow. Thus, deoxyribonucleic acid, vicinal hydroxy and hydroxy-amino groups, and free basic groups of proteins were demonstrated selectively. Of the tissues tested, stomach, intestine, kidney, thyroid gland and plant root tips were most strikingly stained.     

Comparative Spectroscopy of Basic Fuchsin, Schiff Reagent, and a Formalin-Schiff Derivative in Relation to Dye Structure and Staining

Ultraviolet (UV) absorption (200-330 nm) for 0.5 mg/ml aqueous solutions of basic fuchsia unadjusted and those adjusted to pH 0, 1.5, 8.5, and 11 were determined as well as spectra in the visible range (400-675 nm) for solutions with pH 1.9, 2.8, 3.9, 4.7, 5.5, 5.9, 6.5, 7.5, 9.3, 10.4, and 11. The UV absorbance of degassed Schiff reagent containing 1 or 0.5 mg dye/ml, and that of this reagent adjusted to pH 1.5, 2.3, 3.1, 4.5, 6.0, 7.1, and 8.4 were obtained for comparison. The progressive reaction of formalin with degassed Schiff reagent, followed spectrometrically for 2.5 hr, required 2 hr to reach completion. The degassed Schiff reagent contained only traces of-SO₃H as judged from its minimal absorbance between 280 and 295 nm. The UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical. The absorbance is that of basic fuchsin reduced by the addition of Cl^- or SO₃H^- to the central methane carbon and H to the amino groups, therefore the leuco structure of basic fuchsin so reduced shows the fomation-NH₃ groups. Infrared (IR) spectra of basic fuchsin, Schiff crystals, and a crystalline formalin-Schiff reaction product support these observations and indicate that the final colored product is a methylsulfonic acid derivative of basic fuchsin. Identical IR spectra were obtained for two types of crystals derived from Schiff reagents indicating that both are the same chemically, although only one became colored on exposure to air. When these crystals were redissolved and SO₂ added, a Schiff reagent of appropriate pH was produced. Since it is derived from a crystalline product, this type of reagent should be useful in histochemical studies     

Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.     

Pararosaniline or acriflavine-Schiff staining of epoxy embedded tissue after periodic acid oxidation in ethanol: a method suitable for morphometric and fluorometric analysis of glycogen

A method for preparing tissue sections for automatic image analysis of glycogen is described. Large semithin sections of epoxy embedded tissue fixed in glutaraldehyde-osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydration before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.     

An Improved Lead-Tetraacetate:Schiff Procedure

Sections from tissues fixed in a 10% solution of formalin in 90% alcohol were treated with lead tetraacetate (PbAc₄) in different solvents: glacial acetic acid, dilute acetic acid, methanol, ethanol, toluene and benzene. The excess reagent was removed with a 2% solution of ethylene diamine tetraacetate (EDTA) at pH 8. The sections were then stained with Schiff's reagent for 20 minutes. The chemical stability of PbAc₄ in different solvents, the effect of its concentration and the time of exposure on the intensity of the Schiff reaction, were studied. A 0.023 N solution of PbAc₄ in benzene with a reaction time of 5 minutes is recommended. The stability of PbAc₄ in benzene permitted such solutions to be used for 3-4 days. Satisfactory results were obtained with mammalian tissues, algae, bacteria, fungi and protozoa. Some of the preparations obtained by using the technics described are illustrated in an accompanying plate of photomicrographs.     

Free radical generation by early glycation products: a mechanism for accelerated atherogenesis in diabetes

Non-enzymatic glycation of reactive amino groups in model proteins increased the rate of free radical production at physiologic pH by nearly fifty-fold over non-glycated protein. Superoxide generation was confirmed by electron paramagnetic resonance measurements with the spin-trap phenyl-t-butyl-nitrone. Both Schiff base and Amadori glycation products were found to generate free radicals in a ratio of 1:1.5. Free radicals generated by glycated protein increased peroxidation of membranes of linoleic/arachidonic acid vesicles nearly 2-fold over control, suggesting that the increased glycation of proteins in diabetes may accelerate vascular wall lipid oxidative modification.     

Evaluation of cytotoxic,antifungal, and larvicidal activities of different bis-sulfonamide Schiff base compounds

Schiff bases (imines or azomethines) are versatile ligands synthesized from the condensation of amino compounds with active carbonyl groups and used for many pharmaceutical and medicinal applications. In our study, we aimed to determine the cytotoxic, antifungal and larvicidal activities of biologically potent bis-sulfonamide Schiff base derivatives that were re-synthesized by us. For this aim, 16 compounds were re-synthesized and tested for their cytotoxic, antifungal and larvicidal properties. Among this series, compounds A1B2 , A1B4 , A4B2 , A4B3 , and A4B4 were shown to have cytotoxic activity against tested cancer lung cell line (A549). The most potent antifungal activity was observed in compounds A2B1 and A2B2 against all fungi. A1B1 showed the strongest larvicidal effect at all concentrations at the 72nd h (100% mortality). These obtained results demonstrate that these type of bis-substituted compounds might be used as biologically potent pharmacophores against different types of diseases.     

Periodic acid-Schiff reaction combined with quantitative autoradiography of 3H-thymidine- or 85S-sulfate-Labeled epithelial cells of colon

Summary The influence of periodic acid-Schiff staining on grain counts was examined using autoradiography of guinea pig colon labeled with either ³H-thymidine or ³⁵S-sulfate. Prestaining decreased the grain count, an effect due to the Schiff reagent but not to periodic acid. Poststaining altered the grains which were partly or completely lost, an effect due to periodic acid but not to the Schiff reagent. The suggested procedure is to pretreat the sections with periodic acid, process for autoradiography, and poststain with the Schiff reagent. No silver grain is then lost.     

Evidence for an intercellular covalent reaction essential in antigen-specific T cell activation

The inductive interaction between class II+ APC and Th cell was investigated in a human system at the chemical level. The study set out to test the predictions of a model of Ag presentation in which epsilon-amino groups and carbonyl groups at the surface of APC and T cell react covalently to form reversible intercellular Schiff bases. In the experimental system of oxidative mitogenesis this process results in T cell activation. If oxidative mitogenesis is an experimental amplification of a physiologic process, and intercellular Schiff base formation is essential in Ag presentation, then it should be possible to inhibit Ag presentation by prior formation of Schiff bases on the surface of participating cells. In this situation Ag-induced T cell activation and T cell activation induced by periodate oxidation should invariably behave in the same way. It should also be possible to demonstrate Schiff base formation occurring between accessory cells and lymphocytes directly and definitively by means of specific reduction with sodium cyanoborohydride. Aldehyde treatment of accessory cells should prevent this intercellular Schiff base formation. In this study the following observations were made. 1) Both Ag-specific and periodate-induced T cell activation were inhibited by aldehyde treatment of class II+ accessory cells. 2) Noncross-linking donors of carbonyl groups other than aldehydes inhibited Ag-specific T cell activation. 3) Brief, low-dose treatment of T cells with aldehydes inhibited Ag-dependent T-cell activation. 4) Exogenous amino groups in the form of lysine and other amino acids inhibited both Ag-specific and periodate-induced T-cell activation. 5) The weak reducing agent sodium cyanoborohydride which is specific for Schiff bases at neutral pH inhibited both Ag-induced and periodate-induced T cell activation. Responses to PHA were markedly prolonged by this reagent. 6) Schiff base formation occurring between accessory cells and lymphocytes was detected directly and definitively by means of radiolabeling with NaCNB(3H)3 at neutral pH. These data are consistent with the view that the formation of reversible covalent Schiff bases between ligands on APC and T cell is an essential process in Ag-induced T cell activation.     

Synthesis of new chemical entities from paracetamol and NSAIDs with improved pharmacodynamic profile

It was envisaged to combine high antipyretic activity of paracetamol into commonly used NSAIDs. To achieve this goal new chemical entities were synthesized by chemically combining paracetamol and NSAIDs, and biologically evaluated for their antipyretic, analgesic, anti-inflammatory and ulcerogenic potential. The acid chloride of parent NSAIDs was reacted with excess of p-aminophenol to yield the desired p-amidophenol derivatives (1B–7B). Acetate derivatives (1C–7C) of these phenols (1B–7B) were also prepared by their treatment with acetic anhydride, in order to see the impact of blocking the free phenolic group on the biological activity of the derivatives. All the synthesized p-amidophenol derivatives showed improved antipyretic activity than paracetamol with retention of anti-inflammatory activity of their parent NSAIDs. These compounds elicited no ulcerogenicity unlike their parent drugs.     

Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptides with fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.     

Synthesis of multiresponsive and dynamic chitosan-based hydrogels for controlled release of bioactive molecules

An inexpensive, facile, and environmentally benign method has been developed for the preparation of multiresponsive, dynamic, and self-healing chitosan-based hydrogels. A dibenzaldehyde-terminated telechelic poly(ethylene glycol) (PEG) was synthesized and was allowed to form Schiff base linkages between the aldehyde groups and the amino groups in chitosan. Upon mixing the telechelic PEG with chitosan at 20 °C, hydrogels with solid content of 4-8% by mass were generated rapidly in <60 s. Because of the dynamic equilibrium between the Schiff base linkage and the aldehyde and amine reactants, the hydrogels were found to be self-healable and sensitive to many biochemical-stimuli, such as pH, amino acids, and vitamin B6 derivatives. In addition, chitosan could be digested by enzymes such as papain, leading to the decomposition of the hydrogels. Encapsulation and controlled release of small molecules such as rhodamine B and proteins such as lysozyme have been successfully carried out, demonstrating the potential biomedical applications of these chitosan-based dynamic hydrogels.     

A Simple Procedure for Crystallization of the Schiff Reagent

Formation of crystals in Schiff reagents prepared from SO₂ gas previously has been reported either soon after preparation, using high dye concentrations and heating, or after long periods of storage at room temperature. With the first type of procedure only a low yield of crystals accompanied by dye precipitation was obtained. Crystallization without dye precipitation took place if the reagent, prepared with pararosaniline base or chloride in a saturated SO₂ solution, was stored for a sufficient time at room temperature in partly filled flasks. These crystals remained colorless if washed with acid alcohol after being separated by filtration. Schiff reagents layered with paraffin oil or supplemented with 0.1 M hydroquinone took much longer to crystallize, suggesting that crystallizaticn is promoted by the partial oxidation of sulfurous acid to sulfuric acid. A high yield of crystals can be obtained at room temperature after as little as 24 hr by adding 0.04 M of H₂SO₄ to a Schiff reagent prepared with 2% pararosaniline chloride in a saturated SO₂ solution. A Schiff reagent prepared with only 0.2% of these crystals gives an intense staining in the Feulgen and in the Periodic acid-Schiff reactions.     

Preparation of heteroenzyme conjugates: trypsin-chymotrypsin and trypsin-alkaline phosphatase

Hybrid enzymes which have two different enzyme activities linked together covalently may be useful reagents for various applications, such as the determination of complex biological structures. The present paper describes the preparation and purification of two such enzyme-enzyme conjugates, namely, trypsin-chymotrypsin and trypsin-alkaline phosphatase. Whereas the former has been prepared by using the well-known bifunctional reagent glutaraldehyde, the latter exploited the Schiff base formation between the oxidized carbohydrate moiety of alkaline phosphatase and the free amino groups of trypsin.     

Synthesis and biological properties of polysaccharide-peptide conjugates as potential antigens for a vaccine against meningococci of serogroups A and B

A new approach to the development of a vaccine against meningococci of serogroups A and B was proposed. It involves the synthesis of conjugates of high-molecular capsule polysaccharides of the serogroup A meningococcus (PsA) with earlier synthesized protective fragments of membrane proteins from serogroup B meningococci. The conjugates were synthesized using a method that consists of the generation of aldehyde groups by oxidizing free vicinal hydroxyl groups of PsA and subsequent reaction of these groups with amino groups of the peptide. The reaction proceeds with the intermediate formation of the Schiff base, which is reduced to the stable secondary amine. The main parameters of the reaction were optimized in the synthesis of a PsA conjugate with a model peptide and methods of their characterization were developed. The reproducibility and efficiency of the synthetic procedure were demonstrated by the example of synthesis of PsA conjugates with fragments of protein PorA from the outer membrane of the serogroup B meningococcus. It was shown that, when administered without adjuvant, a conjugate of PsA with a protective peptide, which represents an exposed conserved fragment 306–332 of protein PorA, stimulates the formation of antibodies to the peptide and polysaccharide moieties of the molecule and is also capable of decreasing the degree of bacteremia in animals infected with serogroup A and serogroup B meningococci. The approach can be applied to the development of a complex vaccine for serogroup A and serogroup B meningococci.     

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.     

The substrate activation process in the catalytic reaction of Escherichia coli aromatic amino acid aminotransferase

Aromatic amino acid aminotransferase is active toward both aromatic and dicarboxylic amino acids, and the mechanism for this dual substrate recognition has been an issue in the enzymology of this enzyme. Here we show that, in the reactions with aromatic and dicarboxylic ligands, the pK(a) of the Schiff base formed between the coenzyme pyridoxal 5'-phosphate and Lys258 or the substrate increases successively from 6.6 in the unliganded enzyme to approximately 8.8 in the Michaelis complex and to >10.5 in the external Schiff base complex. Mutations of Arg292 and Arg386 to Leu, which mimic neutralization of the positive charges of the two arginine residues by the ligand carboxylate groups, increased the Schiff base pK(a) by 0.1 and 0.7 unit, respectively. In contrast to these moderate effects of the Arg mutations, the cleavage of the Lys258 side chain of the Schiff base, which was brought about by preparing a mutant enzyme in which Lys258 was changed to Ala and the Schiff base was reconstituted with methylamine, produced the Schiff base pK(a) value of 10.2, that being 3.6 units higher than that of the wild-type enzyme. The observation indicates that the Schiff base pK(a) in the enzyme is lowered by the torsion around the C4-C4' axis of the Schiff base and suggests that the pK(a) is mainly controlled by changing the torsion angle during the course of catalysis. This mechanism, first observed for the reaction of aspartate aminotransferase with aspartate [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085], does not require the electrostatic contribution from the omega-carboxylate group of the substrate, and can explain why in aromatic amino acid aminotransferase the aromatic substrates can increase the Schiff base pK(a) during catalysis to the same extent as the dicarboxylic substrates. This is the first example in which the torsion pK(a) coupling of the pyridoxal 5'-phosphate Schiff base has been demonstrated in pyridoxal enzymes other than aspartate aminotransferase, and suggests the generality of the mechanism in the catalysis of aminotransferases related to aspartate aminotransferase.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.