Synthesis of a fluorescent microgonotropen (FMGT-1) and its interactions with the dodecamer d(CCGGAATTCCGG)

A new type of microgonotropen that fluoresces upon binding to dsDNA has been synthesized. FMGT-1, an analogue of the minor groove binder Hoechst 33258, is functionalized with a polyamine chain capable of interacting with the phosphate backbone of DNA. Binding studies indicate that FMGT-1 binds more tightly to dsDNA than the parent compound Hoechst 33258.     

Synthesis of beta-substituted cationic porphyrins and their interactions with DNA

The beta-substituted cationic porphyrins (7, 8 and 10) have been synthesized and their interactions with plasmid DNA investigated. We found that substituents at the beta-position of porphyrins (7 and 8) have apparently affected their interactions with DNA compared with non-beta-substituted porphyrins (10).     

Study of Ni(II) ion-DNA interactions with methylene blue as fluorescent probe

Spectroscopic studies of the interactions of Ni(II) ion with herring sperm DNA have been performed in this contribution using methylene blue (MB) as the fluorescent probe molecule. MB binds to double helical DNA via the intercalative mode, and its fluorescence is efficiently quenched by the DNA nucleobases. The fluorescence intensity of the probe molecule increases distinctly when Ni(II) ion is added to the MB-DNA solution system. These results indicate that the Ni(II) ions may be complexed with the DNA helix and probably bind at N-7 of adenine or guanine; consequently, some intercalated MB molecules are released due to the binding of Ni(II) ions to N-7 nitrogen of the purines. The Ni(II) ion-DNA interactions are further investigated by performing the photobleaching experiments of the MB-DNA complex in the presence and absence of Ni(II) ion. The bi-exponential decay functions of the fluorescence intensity have been observed in both cases and the shortening of the slow decay component when added Ni(II) ion also agrees with the release of MB from the DNA duplex.     

Synthesis of the chromophores of fluorescent proteins and their analogs

Members of the green fluorescent protein (GFP) family are widely used in experimental biology as genetically encoded fluorescent tags. Chromophores of GFP-like proteins share a common structural core: 3,5-dihydro-4H-imidazol-4-one. This review covers synthetic approaches to 3,5-dihydro-4H-imida-zol-4-ones, substituted at different positions. General, as well as specific methods, represented by single examples are considered. The most popular synthetic route to substituted 3,5-dihydro-4H-imidazol-4-ones includes synthesis of azlactones, followed by transformation into N-acyldehydroamino acids and, finally, cyclization into target heterocycles. Accordingly, the review is divided into three parts: the first part covers syntheses of azlactones, the second part covers main approaches to N-acyldehydroamino acids, and in the third part we summarize cyclizations of N-acyldehydroamino acids, as well as all other approaches to 3,5-dihydro-4H-imidazol-4-ones.     

Sterol and squalene carrier protein interactions with fluorescent delta 5,7,9(11)-cholestatrien-3 beta-ol

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.     

Green fluorescent protein (GFP) transgenic fish and their applications

The coupling of the GFP reporter system with the optical clarity of embryogenesis in model fish such as zebrafish and medaka is beginning to change the picture of transgenic fish study. Since the advent of first GFP transgenic fish in 1995, GFP transgenic fish technology have been quickly employed in many areas such as analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, cell lineage and axonal pathfinding, cellular localization of protein products, chimeric embryo and nuclear transplantation, cell sorting, etc. The GFP transgenic fish also have the potentials in analysis of upstream regulatory factors, mutagenesis screening and characterization, and promoter/enhancer trap. Our own studies indicate that GFP transgenic fish may become a new source of novel variety of ornamental fish. Efforts are also being made in our laboratory to turn GFP transgenic fish into biomonitoring organisms for surveillance of environmental pollution.     

Monotheophylline and monotheophyllinato complexes of palladium(II) and their interactions with nucleosides

The interaction of potassium tetrachloropalladate(II) with theophylline in a 1:1 molar ratio resulted in the formation of the monotheophylline (K[Pd(ThH)Cl₃]) or monotheophyllinato ([Pd(Th)Cl]₂) complexes, depending on the solvent and the acidity conditions. In the first complex, theophylline coordinates to Pd(II) as a neutral molecule through its N9 atom, while in the second as a monoanion through both its N7 and O6 atoms. Both complexes react with nucleosides, giving the complexes [Pd(Nucl)(ThH)Cl₂] and [Pd(Nucl)(Th)Cl], respectively. Those complexes with one N(1)H ionizable imino-proton undergo deprotonation and two new series of mixed ligand complexes, [Pd(Nucl − H⁺)(ThH)Cl] and [Pd(Nucl − H⁺(Th)] are formed. In the mixed ligand complexes, theophylline maintains its coordination modes. The nucleosides, on the other hand, exhibit their usual coordination sites; i.e. in the nondeprotonated complexes they coordinate only through their N7 atoms, while in the deprotonated they act as bidentate through both their N7 and O6 atoms. All complexes were characterized with elemental analyses, conductivity measurements and various spectroscopic techniques.     

Mono-vitamin B6 complexes of palladium(II) and their interactions with nucleosides

The interactions of potassium tetrachloropalladate(II) with the B6 vitamins pyridoxal, pyridoxine, and pyridoxamine in 1:1 molar ratio have been studied. From DMF solutions, the ionic trichloro (pyridoxal or pyridoxine) palladates(II) were isolated. Pyridoxamine, on the other hand, in aqueous solutions gave the dimeric complex bis [mu-chloro-pyridoxaminato-palladium(II)]. In the first two complexes, the ligands coordinated to palladium through their pyridine nitrogen while, in the last one, pyridoxamine acted as a chelating ligand through its phenolic oxygen and aminomethyl nitrogen. All three complexes reacted with nucleosides, yielding the complexes [Pd(PL)(Nucl)Cl2], [Pd(PN)(Nucl)Cl2], and [Pd(PM-H+)(Nucl)Cl], respectively. Those complexes with one ionizable N(1)H imino proton underwent deprotonation, and the new mixed ligand complexes [Pd(PL)(Nucl-H+)Cl], [Pd(PN)(Nucl-H+)], and [Pd(PM-H+)(Nucl-H+)] were formed. In all mixed ligand complexes, the B6 vitamins maintained their coordination modes. The nucleosides, on the other hand, exhibited their usual coordination sites, i.e., in the nondeprotonated complexes, purine nucleosides coordinated only through their N7 atom. In the deprotonated complexes, they acted as bidentate ligands and coordinated through their N7 and O6 atoms. All complexes were characterized with elemental analyses, conductivity measurements, and various spectroscopic techniques.     

New fluorescent macrolide derivatives for studying interactions of antibiotics and their analogs with the ribosomal exit tunnel

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.     

Synthesis and fluorescent properties of oligonucleotides, containing a new fluorescent marker--n-(2-benzoxazolyl)tolane]

A functional (dihydroxybutyl) derivative of p-(2-benzoxazolyl)tolane, a fluorescent label novel for biopolymers, was synthesized. The functionalized solid support obtained on its basis was employed in the synthesis of oligodeoxynucleotides 3-terminally labeled with benzoxazolyltolane (these oligonucleotides also contained 1-phenylethynylpyrene residues). This fluorophore within its dihydroxybutyl derivative and the oligonucleotides modified with it displays an intensive fluorescence characterized by a high Stokes shift. The oligonucleotides labeled with this fluorophore are potential probes sensitive to the biopolymer microenvironment.     

Synthesis,characterization and fluorescent properties of cerium(III) glutathione complex

The cerium (III) glutathione complex was synthesized by the redox reaction of cerium (IV) with glutathione reduced (GSH) in aqueous solution. The Job‐plots indicate an ML (L = GSSG) stoichiometry of the complex. The fluorescent properties of the compound were investigated. The as‐prepared complex showed the characteristic maximum emission spectra of Ce(III) at 350 nm (λ_ex = 255 nm). The fluorescence results show that the Ce(IV) ions are first reduced to Ce(III), and then form Ce(III) complex after reacting with GSH. The complex was characterized by element analysis and FT‐IR spectra; the stability of the complex was analyzed by cyclic voltammeters and DSC‐TG as well. Finally, Ce(IV) was successfully employed to determine the concentrations of GSH in the presence of GSSG, in which the fluorescence intensities are proportional to the concentrations of GSH in the range of 1–100 nM with the detection limit of 0.05 nM of GSH, without interference from the presence of GSSG. Copyright © 2009 John Wiley & Sons, Ltd.     

Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions

The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues, gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.     

Characterization of an oligonucleotide-binding fluorescent ligand for application in affinity purification of dsDNA

The fluorescent probe PO-PRO-3 was investigated as a potential ligand for the affinity immobilization and purification of genomic or plasmid DNA fragments. Affinities and mechanisms for PO-PRO-3 binding to superhelical and linearized pUC 18 plasmid DNA were examined through measurement of binding isotherms, continuous-variation analysis, and DNA titrations. In addition, the effects of DNA conformation, protein and RNA contaminants, solvent polarity, and ionic strength are examined with the aim of optimizing binding and elution conditions and of assisgning limits to the range of applicability of the affinity purification. (c) 1995 John Wiley & Sons, Inc.     

Peripheral substituents of di(pyridiumyl)porphyrins affected on their interactions with DNA

meso- and beta-Substituted di(pyridiumyl)porphyrins 3, 4, and 7 have been synthesized and their interactions with DNA have been investigated. meso-Substituted porphyrins showed the stronger effect on DNA than that of beta-substituted porphyrin. Cytotoxicity of compound 3 (IC(50)) to THP-1 tumor cell was up to 0.11 nM.     

Visualization and sensing of intermolecular interactions with two-color fluorescent probes

We developed a new generic fluorescence sensing technology based on the change of relative intensities between two well-separated emission bands of the novel functional 3-hydroxychromone (3HC) dyes. A greatly enhanced self-calibrating wavelength-ratiometric response is obtained to all major types of non-covalent interactions that can be used in sensing--to polarity, hydrogen bonding ability and to local electrostatic fields. This technology may find a broad range of applications--from homogeneous assays in solutions to microarrays, microfluidic devices, nanosensors and whole cell imaging systems. It allows transforming micelles or phospholipid vesicles into nanosensor devices. In cellular research a high sensitivity to membrane potentials can be obtained and the membrane changes during apoptosis detected.     

Synthesis of novel multivalent fluorescent inhibitors with high affinity to prostate cancer and their biological evaluation

Prostate-specific membrane antigen (PSMA) is an important biological target for therapy and diagnosis of prostate cancer. In this study, novel multivalent PSMA inhibitors with glutamate-urea-lysine structures were designed to improve inhibition characteristics. Precursors of the novel inhibitors were prepared from glutamic acid with di-tert-butyl ester. A near-infrared molecular dye, sulfo-Cy5.5, was introduced into the precursors to generate the final PSMA fluorescent inhibitors, compounds 12–14, to visualize prostate cancer. Biological behaviors of the inhibitors were evaluated using in vitro inhibition assays, in vivo fluorescent imaging, and ex vivo biodistribution assays. K_i values from inhibition studies indicated that dimeric inhibitor 13 with a glutamine linker showed approximately 3-fold more inhibitory activity than monomeric inhibitor 12. According to other biological studies using a mouse model of prostate cancer, dimeric inhibitor compounds 13 and 14 had higher tumor accumulation than the monomer. However, glutamine-based dimeric inhibitor 13 showed lower liver uptake than dimeric inhibitor 14, which had a benzene structure. Thus, these studies suggest that glutamine-based dimeric inhibitor 13 can be a promising optical inhibitor of prostate cancer.     

Model investigations for vanadium-protein interactions: vanadium(III) compounds with dipeptides and their oxovanadium(IV) analogues

The reaction of VCl(3) with 1,10-phenanthroline and a series of dipeptides (H(2)dip), having aliphatic as well as aromatic side chains, in methyl alcohol and in the presence of triethylamine affords vanadium(III) compounds of the general formula [V(III)(dip)(MeOH)(phen)]Cl. Aerial oxidation/hydrolysis of the vanadium(III) species gives their oxovanadium(IV) analogues of the general formula [V(IV)O(dip)(phen)]. X-ray crystallographic characterization of the [V(IV)O(dip)(phen)] compounds (where dip(2-)=Gly- L-Ala, Gly- L-Val and Gly- L-Phe) revealed that the vanadium atom possesses a severely distorted octahedral coordination and is ligated to a tridentate dip(2-) ligand at the N(amine) atom, the deprotonated N(peptide) atom and one of the O(carboxylate) atoms, as well as an oxo group and two phenanthroline nitrogen atoms. Circular dichroism characterization of the V(III)/V(IV)O(2+)-dipeptide compounds revealed a strong signal for the V(IV)O(2+) species in the visible range of the spectrum, with a characteristic pattern which may be exploited to identify the N(am), N(pep) and O(car) ligation of a peptide or a protein to V(IV)O(2+) center, and a weak Cotton effect of opposite sign to their vanadium(III) analogues. The visible spectra of the V(III)-dipeptide compounds revealed two d-d bands with high intensity, thus indicating that the covalency of the metal-donor atoms is significant, i.e. the vanadium d orbitals are significantly mixed with the ligand orbitals, and this is confirmed by the low values of their Racah B parameters. The high-intensity band of the V(IV)O(2+)-dipeptide compounds at approximately 460 nm implies also a strong covalency of the metal with the equatorial donor atoms and this was supported by the EPR spectra of these compounds. Moreover, the V(III)/V(IV)O(2+)-dipeptide complexes were characterized by EPR and IR spectroscopies as well as conductivity and magnetic susceptibility measurements.     

Preferential interactions of fluorescent probe Prodan with cholesterol.

The fluorescent probe Prodan has been widely used as a probe of model and biological membranes. Its fluorescent maxima in phospholipid bilayers vary as a function of phase state, with maxima at 485 for the liquid crystal Lalpha, 435 nm for the gel L'beta, and 507 nm for the interdigitated gel LbetaI phase, with excitation at 359 nm. These spectral changes have been used for the detection of phase changes among these phases. In the present study, the fluorescent properties and partition coefficients of Prodan in model membranes of phosphatidylcholines and phosphatidylethanols have been studied as a function of lipid phase state and cholesterol content. It is shown that the Prodan spectrum in the presence of cholesterol no longer reflects the known phase state of the lipid; in each phase state, the presence of cholesterol leads to a spectrum with the maximum at 435 nm, characteristic of the noninterdigitated gel phase. The partition coefficient of Prodan into these lipids also varies with the phase state, giving values of 0.35 x 10(4) in the interdigitated gel, 1.8 x 10(4) in the noninterdigitated gel, and 7. 6 x 10(4) in the liquid crystal phase. In the presence of cholesterol these partition coefficients are increased to 13 x 10(4) for the liquid crystal and the gel phase, and 5.1 x 10(4) in the presence of 100 mg/ml ethanol. These results suggest that Prodan has preferential interactions with cholesterol, and is thus not a randomly distributed fluorescent reporter probe in membranes containing cholesterol. These results suggest that Prodan should be used only with great caution in complex lipid mixtures, particularly biological membranes.     

Phytoplasmas and their interactions with hosts

Phytoplasmas are bacteria without cell walls and are responsible for plant diseases that have large economic impacts. Knowledge of their biology is limited because they are uncultivable and experimentally inaccessible in their hosts. It is a mystery how these bacteria use the sugar-rich phloem sap in which they live and how they interact with the host. This makes it difficult to develop means to control them. Recently, the full genomes of two phytoplasmas have been sequenced, allowing new insights into their requirements. Phytoplasmas contain a minimal genome and lack genes coding for ATP synthases and sugar uptake and use, making them dependent on their host. This dependency can be exploited to elucidate the particular physiology of the phloem.