Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.     

Synthesis of unique cagelike thiacalix[4]arene derivatives in a 1,3-alternate conformation

A novel type of doubly bridged thiacalix[4]arenes in the 1,3-alternate conformation has been prepared by direct aminolysis reaction of easily accessible thiacalix[4]arene tetraacetates with alpha,omega-diamines. Despite the high excess of diamine, both sites of a 1,3-alternate conformer can be intramolecularly bridged to form the cagelike structures in high yields. Optimum results were obtained using 1,2-ethanediamine as bridging units. X-ray analysis of the novel cagelike molecules revealed a highly preorganized array of -C(O)-NH- bonds pointing to the interior of the cavity.     

Screening for drugs in serum by electrospray ionization/collision-induced dissociation and library searching

Factors influencing in-source collision-induced dissociation (ESI/CID) of organic molecules in a Perkin-Elmer/SCIEX ionspray source have been investigated. Breakdown curves of four drugs and organic compounds were acquired by monitoring the intensities of MH+ and specific fragment ions while ramping the orifice voltage. Haloperidol, diazepam, 1,4-acetamido-acetoxybenzene and diacetamido-1,2-benzene were found to be substances with characteristic breakdown curves, with maximums and points of intersection at orifice voltages between 20 and 70 V. The breakdown curves of haloperidol were used for comparison of ESI/CID with ionspray and turboionspray sources on three PE/SCIEX-API instruments. Using standardized source parameters and mass resolution, very similar fragmentation graphs were obtained for haloperidol with all instruments. Infusion of varying concentrations of haloperidol (0.1 to 10 micrograms/mL with ionspray, and 0.01 to 1 microgram/mL with turboionspray) yielded comparable breakdown curves. With turboionspray, a preconcentration of the aerosol occurred, yielding higher ion abundances. Solvent pH and the ratio of aqueous ammonium formate/acetonitrile had minor effects on the degree of fragmentation of haloperidol in a wide range. With these preconditions, a currently expanding mass spectral library of 400 drugs was set up by liquid chromatography/mass spectrometry with alternating orifice voltages (20, 50, and 80 V, respectively) in a looped experiment. An example of drug identification in a patient's serum with library search is shown.     

Raman spectroscopy at different excitation wavelengths (1064, 785 and 532 nm) as a tool for diagnosis of colon cancer

Raman spectroscopy is structure sensitive non‐destructive method that allows observing the status of biological tissues with minimal impact. This method has a great potential in the diagnosis of various types of degenerative diseases including cancer damages. Near‐infrared Fourier transform (NIR‐FT)‐Raman (λ_ex ~1064 nm), NIR‐visible (Vis)‐Raman (λ_ex ~785 nm) and Vis‐Raman (λ_ex ~532 nm) spectra of normal and colorectal carcinoma colon tissue samples were recorded in macroscopic mode at 10–20 randomly chosen independent sites. In the cases of NIR‐Vis‐ and Vis‐Raman spectra, enhanced resonance effects were observed for tissue chromophores absorbing in the visible area. Evident spectral differences were noticed for Raman spectra of normal colon tissue samples in comparison with abnormal samples. The average Raman spectra of colon tissue samples were analysed by principal component analysis (PCA) to discriminate normal and abnormal tissues. PCA of combined dataset containing Raman intensities of chosen NIR‐FT, NIR‐Vis or Vis‐Raman bands led to discrimination of normal and abnormal colon tissue samples. Therefore, combination of these three Raman methods can be helpful for recognizing cancer lesions in colon for diagnostic purposes. Copyright © 2014 John Wiley & Sons, Ltd.     

Use of a kinetic energy orifice as a probe of metastable dissociation in Fourier transform ion cyclotron resonance mass spectrometry

Although Fourier transform ion cyclotron resonance mass spectrometry is a powerful tool in the qualitative observation of gas phase reactions, ion detection is on the millisecond time scale, orders of magnitude longer than typically found when using a sector instrument. Observations of short-lived species such as chemically activated adduct ions can be accomplished using selective ion excitation as a probe of intermediate lifetime. Whereas ion elimination has been shown to be effective in monitoring ion lifetimes on the microsecond time scale, problems associated with detecting ions produced with high kinetic energies limits the technique. Use of a kinetic energy orifice as an ion skimmer effectively eliminates ions near the center of the ion cell at relatively low kinetic energies. By modifying a single section cell to include a kinetic energy orifice, the lifetimes of chemically activated adduct ions have been investigated.     

Dendritic Metalloporphyrin-Fullerene Conjugates: Changing the Microenvironment around Redox-Active Centers and its Impact on Charge-Transfer Reactions

Photophysical investigations on a series of (2,4,6)-tris-substituted metalloporphyrin-fullerene conjugates revealed the effects of an electron-rich microenvironment surrounding the electron-donating porphyrin as a function of the metal center. On one hand, for all conjugates-water-soluble and non-water-soluble-ultrafast charge separation was observed upon photoexcitation. On the other hand, when examining the charge recombination dynamics for the non-water-soluble conjugates it becomes obvious that the (2,4,6)-tris-substitution stabilizes the radical-ion-pair state relative to the mono-substitution in the ortho-, meta-, and para-position. The more efficient protection of the electron-donating porphyrin from solvation is thought to be the major cause for this impact. Nevertheless, the situation is slightly different for the water-soluble conjugates. At first glance, the radical-ion-pair state lifetimes are, also in the case of the (2,4,6)-tris-substitution, longer than for the mono-substituted ortho-, meta- and para-conjugates. Upon closer inspection, they fail, however, to exhibit any metal dependence. Competing with the protection from solvation of the dendrons, dipole-charge interactions impact the stabilization in the polar aqueous environment and, in turn, become the dominant force governing the electron-transfer dynamics.     

In silico mutagenesis and docking study of Ralstonia solanacearum RSL lectin: performance of docking software to predict saccharide binding

In this study, in silico mutagenesis and docking in Ralstonia solanacearum lectin (RSL) were carried out, and the ability of several docking software programs to calculate binding affinity was evaluated. In silico mutation of six amino acid residues (Agr17, Glu28, Gly39, Ala40, Trp76, and Trp81) was done, and a total of 114 in silico mutants of RSL were docked with Me-α-L-fucoside. Our results show that polar residues Arg17 and Glu28, as well as nonpolar amino acids Trp76 and Trp81, are crucial for binding. Gly39 may also influence ligand binding because any mutations at this position lead to a change in the binding pocket shape. The Ala40 residue was found to be the most interesting residue for mutagenesis and can affect the selectivity and/or affinity. In general, the docking software used performs better for high affinity binders and fails to place the binding affinities in the correct order.     

Synthesis and Optical Properties of Diaza‐ and Tetraazatetracenes

A series of functionalized diaza‐ and tetraazatetracenes was synthesized, either by condensation of an aromatic diamine with an ortho‐quinone/diethyloxalate followed by chlorination with POCl₃ to give diazatetracenes or by palladium‐catalyzed coupling of a phenylenediamine with various 2,3‐dichloroquinoxalines to give tetraazatetracenes (after oxidation with MnO₂). Representative examples included halogenated and nitrated derivatives. The optical properties of these azatetracenes were discussed with respect to their molecular structures and substitution patterns. The diazatetracenes and tetraazatetracenes formed two different groups that had significantly different electronic structures and properties. Furthermore, 1,2,3,4‐tetrafluoro‐6,11‐bis((triisopropylsilyl)ethynyl)benzo[b]phenazine was synthesized, which is the first reported fluorinated diazatetracene. Single‐crystal X‐ray analysis of this compound is reported.     

A charge-transfer challenge: combining fullerenes and metalloporphyrins in aqueous environments

A series of truly water-soluble C(60)/porphyrin electron donor-acceptor conjugates has been synthesized to serve as powerful mimics of photosynthetic reaction centers. To this end, the overall water-solubility of the conjugates was achieved by adding hydrophilic dendrimers of different generations to the porphyrin moiety. An important variable is the metal center of the porphyrin; we examined zinc(II), copper(II), cobalt(II), nickel(II), iron(III), and manganese(III). The first insights into electronic communication between the electron donors and the electron acceptors came from electrochemical assays, which clearly indicate that the redox processes centered either on C(60) or the porphyrins are mutually affected. Absorption measurements, however, revealed that the electronic communication in terms of, for example, charge-transfer features, remains spectroscopically invisible. The polar environment that water provides is likely to be a cause of the lack of detection. Despite this, transient absorption measurements confirm that intramolecular charge separation processes in the excited state lead to rapid deactivation of the excited states and, in turn, afford the formation of radical ion pair states in all of the investigated cases. Most importantly, the lifetimes of the radical ion pairs were found to depend strongly on several aspects. The nature of the coordinated metal center and the type of dendrimer have a profound impact on the lifetime. It has been revealed that the nature/electronic configuration of the metal centers is decisive in powering a charge recombination that either reinstates the ground state or any given multiplet excited state. Conversely, the equilibrium of two opposing forces in the dendrimers, that is, the interactions between their hydrophilic regions and the solvent and the electronic communication between their hydrophobic regions and the porphyrin and/or fullerene, is the key to tuning the lifetimes.