Cryptofluorescent analogs of cobalamin coenzymes: synthesis and characterization.

Upper-axial (beta-position) ligand analogs of the B12 coenzymes 5'-deoxy-5'-adenosylcob(III)alamin and methylcob(III)alamin have been synthesized by reaction of the 5'-chloro-5'-deoxy derivatives of fluorescent nucleosides (1,N6-ethenoadenosine, formycin, 2-amino-nebularine, and 2,6-diaminonebularine) and a fluorescent alkyl halide (dansylamidopropyl chloride) with cob(I)alamin. These analogs were nonfluorescent, but fluorescent products could be generated by photolysis or cyanolysis of the carbon-cobalt bonds. Under anaerobic and aerobic conditions, the major fluorescent photolysis products of 1,N6-ethenoadenosylcob(III)alamin were 1,N6-etheno-5',8-cyclic-5'-deoxyadenosine, and the 5'-aldehyde of 1,N6-ethenoadenosine, respectively. The cryptofluorescent property of these analogs was utilized to follow the kinetics of aerobic photolysis. First-order rate constants determined by this method were comparable to those obtained spectrophotometrically [via appearance of of aquacob(III)alamin]. Pseudo-first-order rate constants determined fluorometrically for the cyanolysis (at 25 degrees C) of 1,N6-ethenoadenosylcob(III)alamin, 2,6-diaminonebularinylcob(III)alamin, 2-aminonebularinylcob(III)alamin and formycinylcob(III)alamin were 5.8 X 10(-2), 2 X 10(-2), 1.8 X 10(-2), and 3 X 10(-5) min-1, respectively; values in good agreement were obtained spectrophotometrically (via appearance of dicyanocobalamin). Dansylamidopropylcob(III)alamin was stable in the presence of cyanide. The nucleoside alpha-ribazole is fluorescent in the free state but nonfluorescent when present as the lower axial (alpha-position) ligand in cobalamin coenzymes. Thus fluorescence of ligands in both the alpha- and beta-positions of cobalamins is quenched, probably as a result of intramolecular energy transfer between the ligands and the nonfluorescent corrinoid.     

Structural mapping of rabbit muscle phosphofructokinase. Distance between the adenosine cyclic 3',5'-phosphate binding site and a reactive sulfhydryl group

The cAMP binding site of rabbit muscle phosphofructokinase has been labeled with the fluorescent molecule 5'-(p-fluorosulfonylbenzoyl)-2-aza-1,N6-ethenoadenosine. The most reactive sulfhydry- group of this modified enzyme, which is catalytically active, has been labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and with N-]4-(dimethylamino)-3,5-dinitrophenyl]maleimide. The calculated distances between the cAMP binding site and the most reactive sulfhydryl group, as determined by resonance energy transfer measurements, are 31 and 26 A, respectively, for the two sulfhydryl group labels. Both steady-state and fluorescent -ifetime techniques were used to measure the energy transfer efficiencies in 50 mM potassium phosphate (pH 8.0) and 1 mM ethylenediaminetetraacetic acid, and a value of 2/3 was assumed for the donor-acceptor orientation factor. If the difference in calculated distances is attributed to a difference in the orientation factor for the two donor-acceptor ,airs, the actual distance between the cAMP ligand binding site and the most reactive sulfhydryl group on phosphofructokinase is shown to be 28 +/- 6 A.     

Fluorescent rotors and their applications to the study of G-F transformation of actin.

New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.     

The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate.

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state.     

The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site.

1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.     

Intrinsically fluorescent PAMAM dendrimer as gene carrier and nanoprobe for nucleic acids delivery: bioimaging and transfection study

This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.     

Design, synthesis, and evaluation of 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate (8MDP-fluor), as a novel equilibrative nucleoside transporter probe

Nucleoside transporters are integral membrane glycoproteins that play critical roles in physiological nucleoside and nucleobase fluxes, and influence the efficacy of many nucleoside chemotherapy drugs. Fluorescent reporter ligands/substrates have been shown to be useful in the analysis of nucleoside transporter (NT) protein expression and discovery of new NT inhibitors. In this study, we have developed a novel dipyridamole (DP)-based equilibrative nucleoside transporter 1 (ENT1) fluorescent probe. The potent ENT1 and ENT2 inhibitor analogue of dipyridamole, 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (4, 8MDP), was modified to replace one β-hydroxyethyl group of the amino substituent at the 2-position with a β-aminoethyl group and then conjugated through the amino group to 6-(fluorescein-5-carboxamido)hexanoyl moiety to obtain a new fluorescent molecule, 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate, designated 8MDP-fluorescein (8MDP-fluor, 6). The binding affinities of 8MDP-fluor at ENT1 and ENT2 are reflected by the uridine uptake inhibitory K(i) values of 52.1 nM and 285 nM, respectively. 8MDP-fluor was successfully demonstrated to be a flow cytometric probe for ENT1 comparable to the nitrobenzylmercaptopurine riboside (NBMPR) analogue ENT1 fluorescent probe SAENTA-X8-fluorescein (SAENTA-fluor, 1). This is the first reported dipyridamole-based ENT1 fluorescent probe, which adds a novel tool for probing ENT1, and possibly ENT2.     

1,N6-Etheno-2-aza-adenosine 3', 5'-cyclic phosphate: human erythrocyte membrane binding and activation of membrane protein kinase.

The relative efficiency of 1,N6-etheno-2aza-adenosine 3', 5'-monophosphate (cyclic 2-aza-epsilon AMP), 1,N6-etenoadenosine 3', 5'-monophosphate (cyclic epsilon AMP) and cyclic AMP in activation of membrane protein kinase and binding to membrane was examined using isolated membranes from human erythrocytes. Cyclic 2-aza-epsilon AMP was 81% as active as cyclic AMP in erythrocyte membrane binding and activation of membrane protein kinase. On the other hand, cyclic epsilon AMP was 37% as active toward membrane protein kinase and 29% toward membrane cyclic AMP binding. Since we have previously shown that the fluorescence of cyclic 2-aza-epsilon AMP is highly sensitive to the polarity of solvents, the high efficiency of cyclic 2-aza-epsilon AMP to substitute for cyclic amp suggests that it may be a suitable microenvironmental fluorescent probe for cyclic AMP binding sites.     

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.     

Covalent binding of 1,N(6)-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP-synthase

The catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of the fluorescent adenosine diphosphate analogue 1,N(6)-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated chloroplast thylakoid membranes. The influence of light-induced transmembrane proton gradient and of phosphate ions on the fluorescence of 1,N(6)-ethenoadenosine diphosphate covalently bound to catalytic sites of reconstituted CF1 (ATP-synthase) was studied. Upon illumination of thylakoid membranes with saturating white light, the quenching of fluorescence of covalently bound 1,N(6)-ethenoadenosine diphosphate was observed. The quenching was reversed by the addition of inorganic phosphate to the reaction mixture in the dark. Repeated illumination induced the quenching once again: however, the addition of phosphate ions did not affect the fluorescence intensity now. When 1,N(6)-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP-synthase, no similar fluorescent changes were observed. The interrelation of the observed changes of 1,N(6)-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent changes in the structure of the catalytic site of ATP-synthase is discussed.     

Fluorescent 2''(3'')-O-aminoacylnucleosides-acceptor substrates for ribosomal peptidyltransferase+.

2'(3')-O-L-Phenylalanylderivatives of fluorescent 1,N6-ethenoadenosine and 3,N4-ethenocytidine were prepared by chemical synthesis. Both compounds are good acceptor substrates in ribosomal peptidyltransferase reactions. Since these compounds cannot form Watson-Crick base pairs, the results indicate that the terminal aminoacyladenosine unit of AA-tRNA is bound to ribosomal protein on the acceptor site of peptidyltransferase and not to rRNA.     

Tricyclic nitrogen base 1,N6-ethenoadenine and its ribosides as substrates for purine-nucleoside phosphorylases: Spectroscopic and kinetic studies

The title compound is an excellent substrate for E. coli PNP, as well as for its D204N mutant. The main product of the synthetic reaction is N9-riboside, but some amount of N7-riboside is also present. Surprisingly, 1,N⁶-ethenoadenine is also ribosylated by both wild-type and mutated (N243D) forms of calf PNP, which catalyze the synthesis of a different riboside, tentatively identified as N6-β-D-ribosyl-1,N⁶-ethenoadenine. All ribosides are susceptible to phosphorolysis by the E. coli PNP (wild type). All the ribosides are fluorescent and can be utilized as analytical probes.     

Application of BODIPY-trimethylmelamine conjugate for DNA cross-linking in vitro

Conjugate the fluorescent dye 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indatsen-8-propionic acid (BODIPY) and N2,N4,N6-trimethylmelamine was obtained. It was shown that this compound in the presence of formaldehyde generates covalent cross-links of DNA strands in vitro.     

Differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen, SAG1 and SRS2

Neospora hughesi is a newly recognised parasite that is closely related to Neospora caninum, and is a cause of equine protozoal myeloencephalitis. We have characterised two N. hughesi immunodominant tachyzoite antigens which exhibit antigenic and molecular differences from the homologous tachyzoite antigens on N. caninum. These antigens on N. hughesi are referred to as NhSAG1 and NhSRS2, using the same mnemonics as used for the N. caninum antigens (NcSAG1 and NcSRS2), and are homologous to Toxoplasma gondii surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2). The NcSAG1 and NcSRS2 were antigenically conserved in six different N. caninum isolates from cattle and dogs. The two equine-derived Neospora isolates, one designated as N. hughesi, were similar to each other but different from N. caninum. There was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. The polymorphism of these genes and their corresponding proteins provide additional markers which can be used to distinguish N. caninum from N. hughesi.     

Site-specific incorporation of the 1-hexanol-1,N6-etheno-2'-deoxyadenosine adduct into oligodeoxyribonucleotides

Modified oligonucleotides that contain the hydrophobic 1-hexanol-1,N(6)-etheno-2'-deoxyadenosine adduct have been synthesized using a mild solid phase phosphoramidite chemistry. The presence and the integrity of the modified nucleoside in the synthetic oligomers were confirmed by electrospray ionization and MALDI mass spectrometry measurements together with analysis of the complete enzymatic hydrolysate by high performance liquid chromatography coupled to UV and fluorescent detection techniques.     

The H,K-ATPase beta subunit as a model to study the role of N-glycosylation in membrane trafficking and apical sorting

The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting.     

Visualization of APP dimerization and APP-Notch2 heterodimerization in living cells using bimolecular fluorescence complementation

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over-expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C-terminally truncated form of N2. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full-length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co-expression of the C-terminal fragment of YFP fused to APP and the N-terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.     

Effects of movement protein mutations on the formation of tubules in plant protoplasts expressing a fusion between the green fluorescent protein and Cauliflower mosaic virus movement protein

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MP(ER2A), a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MP(N6) and GFP-MP(N13), two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MP(WT) and GFP-MP(ER2A) form tubules containing both MP fusions, indicating that although the GFP-MP(ER2A) cannot induce tubule formation, GFP-MP(ER2A) can coassemble or colocalize with CFP-MP(WT) in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.     

Prevalence of Neospora caninum antibodies in dogs from dairy cattle farms in Parana,Brazil

Serum samples from 134 dogs from 22 cattle dairy farms in the northern region of Parana State, Brazil, were tested for antibodies to Neospora caninum in an indirect fluorescent antibody test. Antibodies (> or = 1:50) to N. caninum were found in 29 (21.6%) of the 134 dogs, and seropositive dogs were found on 14 (63.6%) of the 22 dairy cattle farrms. The antibody titers of dogs were 1:50 (3 dogs), 1:100 (7 dogs), 1:200 (7 dogs), 1:400 (6 dogs), and > or = 1:800 (6 dogs). The low prevalence (9%) in < 1-yr-old dogs compared with the 2- to 3-fold higher prevalence in older dogs (17-29%) suggests postnatal exposure to N. caninum infection.