Novel chromogenic substrates with metal chelating properties for the histochemical detection of peroxidasic activity, derived from 3-amino-9-ethylcarbazole (AEC) and 3,6-diamino-9-ethylcarbazole.

For staining of peroxidase activity routinely employed 3-amino-9-ethylcarbazole 1 (AEC) was chemically modified in order to obtain chromogenic enzyme substrates with improved staining properties. In conclusion of systematically structure/staining considerations of a series of novel substrates, it can be generalized that the performance of traditional chromogenic peroxidase amine-substrates is accessible an considerably improvement in terms of sensitivity and adaptibility for various application purposes (solubility and color of the reaction product, electron dense and osmiophilic properties, ...) by attachment of chelating N-benzyl-moieties making available highly efficient the well known metal catalytic effect in a proposed intramolecular way. Thus, the model compounds 3(arylmethyl)amino-9-ethyl-carbazole 4 and 3,6-bis-(arylmethyl)amino-9-ethyl-carbazole 7 were synthesized by condensation of 3-amino-9-ethylcarbazole 1 (AEC) or the corresponding 3,6-diamine 5 with aromatic aldehydes 2. The resulting Schiff bases 3 and 6 were subsequently reduced with sodium borohydride. The obtained benzylamines 4 and 7 were examined as chromogenic substrates: 1) qualitatively in test tube experiments concerning color, precipitation behavior and solubility of the precipitates, 2) quantitatively by means of electroblotted dilution series of horseradish peroxidase, and finally in a biological environment for the localization of endogenous peroxidasic activity 3) in native cryotome tissues of Wistar rats. 4) The usefulness of the new approach for electron microscopy was revealed, too. Thus the discrimination between internum and externum of specific granules after osmium tetroxide contrastate was higher if compared with results obtained by the Karnovsky protocol. The wide spread variation of substitution patterns of the novel reagents gave reason for structure-reactivity considerations and ongoing leading structures. The stereochemical and electronic factors as well as competing reaction pathways governing the reaction course are briefly discussed. In addition, the metal associating reagents are highly effective in oxidative side-coupling reactions with aromatic amine or phenol-additives exemplified here by means of 4-amino-N,N-diphenylamine. The reagents 4 and 7 are obtainable in a simple in situ synthesis, too, offering in principle a 'chemical construction unit'. The demonstrated approach is of general interest for bioanalytical applications offering an access to potentially novel chromogens and electron opaque markers for the detection of peroxidasic activity/hydroperoxides or related redox enzyme systems.     

Synthesis of serine-AMC-carbamate: a fluorogenic tryptophanase substrate.

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.     

Collagen of fibrocartilage: a distinctive molecular phenotype in bovine meniscus

A fluorogenic substrate for plasmin, CBZ-Gly-Pro-Arg-AEC, has been synthesized and used to develop a new sensitive photometric and fluorometric assay of plasminogen activator activity. The fluorescence intensity of free AEC at 460 nm is about 3 orders of magnitude higher than that of acyl-AEC. The release of AEC from the peptidyl derivative was monitored fluorometrically after extraction of free AEC in ethylacetate. Under such conditions, the Km was 0.16 mM. This method was used to monitor the activity of plasminogen activator synthetized by fibroblastic cells (BHK 21 C 13) either released in the supernatants or cell-associated.     

Detection and characterization of a selective endopeptidase from Plasmodium berghei by using fluorogenic peptidyl substrates

By using fluorogenic peptidyl-3-amino-9-ethyl-carbazole a highly selective endopeptidase for the Val-Leu-Gly-Arg sequence was demonstrated in endoerythrocytic stages of $\text{Plasmodium berghei}$. Val-Leu-Gly-Arg-endopeptidase showed a maximum activity in pH range 7.0–8.0; it was completely inhibited by 1 mM leupeptin and 1 mM antipain. A complete inhibition was also obtained by 15 mM chloroquine. This trypsin-like activity was negligible in uninfected red blood cells. The high sensitive fluorogenic procedure could be performed on cell fractions, cell lysates as well as supernatants.     

Aminopeptidase activities present in tissues of Helix aspersa.

1. Tissues of Helix aspersa have been examined for the presence of aminopeptidase activities. 2. Enzyme activities were detected using synthetic peptide substrates containing the fluorescent leaving group 7-amino-4-methyl coumarin. The methods used included continuous fluorimetric assay and activity staining of enzymes in non-denaturing polyacrylamide gels. 3. Several types of peptidase activity were detected, including three distinct aminopeptidases which differ in molecular size and substrate preference.     

Hydrolysis of 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine by human pancreatic phospholipase A2

A novel fluorescent phospholipid analogue, 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (C30PHPC) was employed as a substrate for human pancreatic phospholipase A2. C30PHPC has a main endothermic phase transition with Tm at 46 degrees C as determined by differential scanning calorimetry (DSC). For an aqueous dispersion of C30PHPC the ratio of the intensities of pyrene excimer and monomer fluorescence emission, (IE/IM) has a maximum between 32 and 36 degrees C. The excimer emission intensity (at 480 nm) exceeds the monomer emission intensity (at 400 nm) 6.5-fold thus indicating a close packing of the phospholipid pyrene moieties in the lipid phase. C30PHPC has a limiting mean molecular area of 37 A2 at surface pressure 35 dyn cm-1 as judged by the compression isotherm at an air-water interphase. The hydrolysis of C30PHPC by human pancreatic phospholipase A2 was followed by monitoring the increase in the pyrene monomer fluorescence emission intensity occurring as a consequence of transfer of the reaction product, pyren-1-yl hexanoic acid into the aqueous phase. The enzyme reaction exhibited an apparent Km of 2.0 microM substrate. Calcium at a concentration of 0.2 mM activated the enzyme 4-fold. Maximal hydrolytic rates were obtained at 45 degrees C and at pH between 5.5 and 6.5. The enzyme reaction could be inhibited by 5 mM EDTA, confirming the absolute requirement for Ca2+ of this enzyme. The present fluorimetric assay easily detects hydrolysis of C30PHPC in the pmol min-1 range. Accordingly, less than nanogram levels of human pancreatic phospholipase A2 can be detected.     

Synergistic Tissue Counterstaining and Image Segmentation Techniques for Accurate, Quantitative Immunohistochemistry

Quantitative analysis of digitized IHC-stained tissue sections is increasingly used in research studies and clinical practice. Accurate quantification of IHC staining, however, is often complicated by conventional tissue counterstains caused by the color convolution of the IHC chromogen and the counterstain. To overcome this issue, we implemented a new counterstain, Acid Blue 129, which provides homogeneous tissue background staining. Furthermore, we combined this counterstaining technique with a simple, robust, fully automated image segmentation algorithm, which takes advantage of the high degree of color separation between the 3-amino-9-ethyl-carbazole (AEC) chromogen and the Acid Blue 129 counterstain. Rigorous validation of the automated technique against manual segmentation data, using Ki-67 IHC sections from rat C6 glioma and β-amyloid IHC sections from transgenic mice with amyloid precursor protein (APP) mutations, has shown the automated method to produce highly accurate results compared with ground truth estimates based on the manually segmented images. The synergistic combination of the novel tissue counterstaining and image segmentation techniques described in this study will allow for accurate, reproducible, and efficient quantitative IHC studies for a wide range of antibodies and tissues. (J Histochem Cytochem 56:873–880, 2008)     

Fluorogenic substrates for chymotrypsin with 2-quinolinone derivatives as leaving groups

Sensitive and convenient fluorometric assays for the determination of chymotrypsin have been developed by using the substrates Glt-Leu-Phe-NH-Meq, Glt-Phe-NH-FMeq and Glt-Leu-Phe-NH-FMeq, which have been synthesized utilizing the mixed anhydride method. The amidolytic activity of chymotrypsin was measured by the release of the highly fluorescent amine 7-amino-4-methyl-2-quinolinone (AMeq) or 7-amino-4-trifluoromethyl-2-quinolinone (AFMeq). The fluorescence properties of the synthesized substrates and the new fluorescent marker AFMeq were examined. Kinetic constants, as well as the maximum sensitivity for the hydrolysis of the new substrates, were determined. All new substrates permit a rapid and sensitive determination of chymotrypsin in a continuous assay system. As little as 0.7 ng of enzyme can be detected using the substrate Glt-Leu-Phe-NH-Meq, which is the most sensitive fluorogenic substrate thus far reported.     

Fluorimetric assay of renin

A simple fluorimetric assay was set up to test renin within 2 h. N-acetyltetradecapeptide was synthesized and used as substrate. It was demonstrated that N-acetyl-angiotensin I and Leu-Val-Tyr-Ser were the two peptides obtained after hydrolysis by renin. Fluorescamine reaction reacted with the free NH2 of the tetrapeptide generated to induce a fluorimetric reaction detected at 395--405 nm. The Michaelis constant of the reaction was 1.87 . 10(-5) M. With this method as little as one milliGoldblatt Unit (mG.U.) of hog renin could be detected and the generation of tetrapeptide was linear with respect to the renin concentration up to 20 mG.U. The fluorimetric assay was applied to the detection of renin during its purification and to the characterization of renin inhibitors.     

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.     

Simultaneous demonstration of lectin-binding sites and antigens detected by monoclonal antibodies in a parallelized double-staining technique: a highly discriminative and quickly developing technique for frozen sections

A sensitive immunoenzymatic double-staining technique is presented for the simultaneous visualization of lectin-binding sites and antigenic structures detected by monoclonal antibodies. The lectin is demonstrated by an extended unlabeled peroxidase-antiperoxidase (PAP) technique and the monoclonal antibody by an alkaline phosphatase-antialkaline phosphatase (APAAP) method, which corresponds to the standard PAP technique. 3-amino-9-ethylcarbazole (AEC) and fast blue BB salt serve as substrates for the peroxidase and the alkaline phosphatase, respectively. The antisera and the enzyme complexes raised in different animals enable the performance of three parallel incubation steps. The staining procedure requires three and a half hours altogether. This method proved to be highly discriminative and rather insensitive to interference by various artifacts.     

A comparison of the conversion of 1-amino-2-ethylcyclopropane-1-carboxylic acid stereoisomers to 1-butene by pea epicotyls and by a cell-free system

The characteristics of the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by pea (Pisum sativum L.) epicotyls and by pea epicotyl enzyme are compared. Of the four stereoisomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC), only (1R,2S)-AEC is preferentially converted to 1-butene in pea epicotyls. This conversion is inhibited by ACC, indicating that butene production from (1R,2S)-AEC and ethylene production from ACC are catalyzed by the same enzyme. Furthermore, pea epicotyls efficiently convert ACC to ethylene with a low K _m (66 M) for ACC and do not convert 4-methylthio-2-oxo-butanoic acid (KMB) to ethylene, thus demonstrating high specificity for its substrate. In contrast, the reported pea epicotyl enzyme which catalyzes the conversion of ACC to ethylene had a high K _m (389 mM) for ACC and readily converted KMB to ethylene. We show, moreover, that the pea enzyme catalyzes the conversion of AEC isomers to butene without stereodiscrimination. Because of its lack of stereospecificity, its low affinity for ACC and its utilization of KMB as a substrate, we conclude that the reported pea enzyme system is not related to the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Amino cyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - KMB 4-methylthio-2-oxobutanoic acid     

Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24- or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.     

Structural features of luliberin (luteinising hormone-releasing factor) inferred from fluorescence measurements.

The fluorescence and excitation spectra of luliberin (luteinizing hormone-releasing factor) in 0.005 M aqueous ammonium acetate are identical in shape to those of N-acetyltryptophan amide and are related to the indole side chain of Trp3. The change of fluoresecence intensity of luliberin with pH was measured in the range of pH 4-11. The increase of pH from 4 to 7.5 is followed by about 50% increase in fluorescence intensity due to deprotonation of the imidazolium side chain of His2. The fluorimetric titration curve in this pH region reveals a pK value for His2 of 5.95. Increasing of pH from 8 to 11 results in about 40% quenching of the fluorescence due to electronic energy transfer from the excited indole of Trp3 to the phenolate side chain of Tyr5. The pK value of Tyr5, obtained independently from the fluorimetric and photometric titrations indicate that at pH 7-8 luliberin contains only one charged residue, Arg8, which is in close vicinity to both His2 and Tyr5. The side chains of His2, Tyr5 and Arg8 presumably form a combined unit which may play an active role in the hormone action. Trp3 is at a maximal distance from this unit and may thus act as an independent active unit.     

Construction of a more sensitive fluorescence sensing material for the detection of vascular endothelial growth factor, a biomarker for angiogenesis, prepared by combining a fluorescent peptide and a nanopillar substrate

We have established a highly sensitive and selective protein detection technology in combination with the nanofabrication technique. A silica nanopillar chip with a 200-nm pitch and 1000-nm height pillar substrate was fabricated by electron beam lithography and deep reactive ion etching method. Fluorescent peptides, with high affinity towards vascular endothelial growth factor (VEGF), were immobilized on nanopillar chip via a self-assembled monolayer made from 3-aminopropyltrimethoxysilane and glutaraldehyde under optimal conditions. The fluorescence intensity of the fluorescent peptide on the nanopillar substrate increased with increasing VEGF concentrations, as determined by a fluorescence spectrophotometer and fluorescent scanning image analysis. The dissociation constant (K(d) value) calculated by the non-linear least square curve fitting method was 6.0 × 10(-9)M, which contributed to the highly sensitive detection of VEGF. The fluorescence intensity of the fluorescent reagent on the nanopillar substrate upon binding to VEGF was higher than that obtained using the flat substrate because the dense and tall nanopillar array increased the virtual protein binding area. The reproducibility tests and lifetime measurement indicate the fluorescent reagent to be a useful biosensor for the detection of VEGF in this system. These experimental results clearly showed that the combination of a fluorescent reagent and a nanopillar substrate may be widely applicable as a convenient method for the detection of VEGF.     

Association of endonuclease activity with serotypes belonging to the three subgroups of human adenoviruses.

Endonuclease activity has been detected in association with highly purified virions, pentons, and/or dodecons of adenovirus types 2, 3, 5, 9, 12, 15, and 16. Only single-strand scissions in substrate DNA were detected. The nuclease activity was detected by a highly sensitive ethidium bromide fluorimetric assay procedure.     

The Hippo–YAP pathway regulates the proliferation of alveolar epithelial progenitors after acute lung injury

Regeneration of pulmonary epithelial cells plays an important role in the recovery of acute lung injury (ALI), which is defined by pulmonary epithelial cell death. However, the mechanism of the regenerative capacity of alveolar epithelial cells is unknown. Using a lung injury mouse model induced by hemorrhagic shock and lipopolysaccharide, a protein mass spectrometry‐based high‐throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells (AEC2s), we analyzed the mechanism of alveolar epithelial cells proliferation. We demonstrated that the expression of Hippo‐yes‐associated protein 1 (YAP1) key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI. Furthermore, the results showed that YAP1+ cells in lung tissue after ALI were mainly Sftpc lineage‐labeled AEC2s. An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 small interfering RNA and Hippo inhibitor. These findings revealed that YAP functioned as a key regulator to promote AEC2s proliferation, with the Hippo signaling pathway playing a pivotal role in this process.     

Metachromasia of 3-amino-9-ethylcarbazole (AEC) and its prevention in immunoperoxidase techniques

Summary 3-Amino-9-ethylcarbazole (AEC) used as chromogen in immunoperoxidase techniques normally has an intense, red colour. However, as an inconstant phenomenon, a pale yellowish-green reaction product severely impairing the evaluation can be observed. In order to circumvent this undesired effect, factors such as tissue fixative, proteolytic digestion, antibody concentrations and incubation time of the primary antibody were analyzed. The most important factor inducing a change in colour is probably the inadequately high local peroxidase concentration arising as the consequence of high amounts of bound primary antibody. This high enzyme concentration might cause metachromasia of AEC by producing the yellowish-green quinone-di-imine form of the substrate. As could be shown by spectrophotometry in test tube experiments, AEC metachromasia was proven to be enzyme dependent. Thus, the best way to trigger the local enzyme concentration on a tissue section to adequate levels appears to be the dilution of the primary antibody.This study was supported by the Deutsche Forschungsgemeinschaft (DFG: Mo. 384/1-2)     

Simple flow-cell for the fluorimetric study of conformational changes of proteins on agarose matrix

The construction and utilization of a new cylindrical cell for fluorescence measurements on protein-Sepharose 4B conjugates is described. This experimental device proved very convenient for fluorimetry of proteins covalently bound to agarose gels, for measurements on proteins in solution, and finally for monitoring the adsorption of proteins in the course of affinity chromatography. With the aid of this cell, the fluorescence spectra of human α-lactalbumin in solution and in an insoluble state were compared. The α-lactalbumin-Sepharose 4B complex gives a spectrum which closely resembles that of the native protein. Fluorescence spectra were recorded with as little as 50 μliters gel in the cell, which corresponds to approximately 0.015–23 nmoles of chemically bound protein. The fluorescence intensity was within experimental error proportional to protein concentration from 0.03 to 0.20 nmole bound protein/mg dry resin. The application of this fluorimetric method to conformational studies on membrane bound enzymes such as the proteins of the lactose synthetase function is discussed.     

A fiber optic biosensor for fluorimetric detection of triple-helical DNA.

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.