FLUORESCENCE SPECTRAL CHANGES OF HEMATOPORPHYRIN DERIVATIVE UPON BINDING TO LIPID VESICLES, Staphylococcus aureus AND Escherichia coli CELLS

Abstract— The binding of hematoporphyrin derivated (Hpd) to lipid vesicles and bacterial membranes was determined by fluorescence spectroscopy. The fluorescence measurements of Hpd in aqueous solutions showed two bands at 613 and 677 nm. In lipid environments of lecithin vesicles the fluorescence spectrum was shifted to 631 and 692 nm, respectively. Hpd was rapidly bound to the cell membrane of Staphylococcus aureus while much less binding occurred in the presence of Escherichia coli. At the same time, spheroplasts of both bacteria were shown to bind Hpd to a similar extent. These results are well correlated with the photoinactivation of the gram positive bacteria with Hpd while the gram negative cells were shown to be resistant. The pH dependence of both Hpd binding to S. aureus as well as the photodynamic inhibitory effect of the same bacteria are similar. It is concluded that the segregation of Hpd to the cell membrane is a prerequisite for its photodynamic effect.     

Mass spectrometric analysis of ceramide perturbations in brain and fibroblasts of mice and human patients with peroxisomal disorders

In this study, the levels and composition of ceramides in brains of newborn mice lacking peroxisomes (Pex5-/-, Zellweger mice) were analyzed using normal-phase high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (HPLC/APCI-MS). Total ceramide compositions were found to be comparable to that of control animals. However, a minor ceramide species, containing hexacosanoic/hexacosenoic acid as the amide fatty acid, was 9-fold increased. Also, in the sphingomyelin-derived ceramides this species was elevated. Subsequent analysis of extracts from fibroblasts of Pex5-/- mice and mice with a defective peroxisomal beta-oxidation (lacking D-specific multifunctional protein 2 (MFP2)), revealed, again, a similar rise in this particular ceramide. Further, this ceramide was elevated in human X-ALD fibroblasts as well. Whether C26:1/0-ceramide is linked to some of the pathology seen in Zellweger syndrome remains to be investigated. However, an increase in this sphingolipid can be considered as a diagnostic criterion for diseases caused by defects in peroxisome biogenesis or peroxisomal beta-oxidation.     

Discovery of O-GlcNAc transferase inhibitors

O-GlcNAcylation of serine and threonine residues is a dynamic and essential post-translational modification involved in signaling pathways in eukaryotes. Studies of O-GlcNAcylation would be aided by small-molecule inhibitors of O-GlcNAc transferase (OGT), the sole enzyme know to mediate this modification, but discovery of such molecules has been hampered by poor expression of cloned OGT and lack of suitable high-throughput screens. This Communication describes the development an expression system to access large amounts of the catalytic domain of OGT and the implementation of a fluorescence-based substrate analogue displacement assay that has led to the discovery of a set of OGT inhibitors. This work lays the foundation for both structural and functional analysis of the catalytic domain of OGT.     

Intermolecular multiple quantum coherences at high magnetic field: the nonlinear regime

Experiments have been carried at magnetic-field strengths of 9.4, 14.1, and 17.6 T to explore the evolution of intermolecular multiple quantum coherences in the nonlinear regime where the system evolves for times that are much greater than the characteristic time of action of the long-range dipolar field, tau(d). The results show the expected Bessel function form of the recorded signal as a function of time of evolution, with evident zeros and sign changes. As expected, the rate of signal evolution increases at higher-field strengths as a result of the increased equilibrium magnetization. A numerical method for calculating the evolution of magnetization under the action of the distant dipolar field, relaxation, and diffusion that is based on Fourier analysis of the magnetization distribution has been applied to the correlated two-dimensional spectroscopy revamped by asymmetric z-gradient echo detection sequence in the nonlinear regime and shown to produce results that are in good agreement with experimental data acquired at different magnetic fields and rates of spatial modulation. Experiments and simulations have also been used to explore the evolution of magnetization in a mixture of two interacting spin species in the nonlinear regime.     

Mirror image nanostructures

Chiral molecules that self-assemble to form chiral supramolecular structures exhibit interesting structural features reminiscent of tertiary and quaternary structures of proteins and have applications in catalysis and nonlinear optics. Often, these structures are hierarchical, with their chiral structure difficult to interpret on the molecular scale. In this communication, we observe chiral assembling molecules that form well-defined helices with a pitch of 28 nm. We observe the behavior in both R- and S-enantiomers of the molecule, forming mirror image nanostructures. The molecular chirality is determined by the dimethyloctyl alkyl coil of the molecule and is located more than 4 nm from the hydrogen-bonding segment. The nanostructures observed are not hierarchical, which could be a result of the significant separation between the stereocenter and hydrogen-bonding dendron. The subtle structural modification at the periphery of the molecule biases the supramolecular assembly, which is driven primarily by strong hydrogen-bonding and pi-pi stacking interactions.     

Time-dependent pump-probe spectra of NeBr2

Time- and frequency-resolved pump-probe measurements on NeBr2 have been performed to better characterize its fragmentation dynamics on the B electronic state for vibrational levels in the energy region of the transition from direct vibrational predissociation to intramolecular vibrational relaxation dynamics. Above nu'=20 of the Br2 stretching mode, it was observed that the dependence of lifetime on the vibrational quantum number deviates from the energy-gap law by leveling off in the range of 10 psE transitions of the complex. These transitions are shifted 20 cm(-1) to lower energy from the free Br2 resonances, indicating an E state Ne-Br2 bond energy of 82 cm(-1). Measurements of NeBr2 vibrational predissociation via the delta nu=-2 channel were also performed for nu'=27, 28, and 29. The closing of the delta nu=-1 channel leads to an increase in the lifetimes of these vibrational levels. A new Nd:yttrium aluminum garnet pumped dual optical parametric oscillator/optical parametric amplifier system is described that allows us to conveniently record time-delayed pump-probe spectra with 2-cm(-1) spectral resolution and 15-ps time resolution.     

Kinetic modeling of the adsorption rate of a gaseous adsorbate on a granular adsorbent by ultra-rapid-scanning Fourier transform infrared spectrometry

A novel method for determining the rates of adsorption of gaseous adsorbates on granular adsorbents in the millisecond time regime is described. Mixtures of the adsorbate and nitrogen are passed through a solenoid valve and 100 mg of the adsorbent for periods of (typically) 100 ms. The concentration of the adsorbate passing into a low-volume, long path length gas cell is measured with an ultra-rapid-scanning Fourier transform infrared spectrometer capable of gathering 200 mid-infrared spectra per second with 6-cm(-1) resolution. The pressure of the gas entering the cell is measured simultaneously with a capacitance manometer. A dynamic mathematical model was developed to analyze and describe the results in terms of a Langmuir isotherm. The success of this approach is demonstrated by the estimation of the rate of adsorption of vapor-phase acetaldehyde on aminopropylsilylated granular silica gel.     

Three-dimensional nonlinear optical chromophores based on metal-to-ligand charge-transfer from ruthenium(II) or iron(II) centers

In this article, we describe a series of new complex salts in which electron-rich transition-metal centers are coordinated to three electron-accepting N-methyl/aryl-2,2':4,4' ':4',4' '-quaterpyridinium ligands. These complexes contain either Ru(II) or Fe(II) ions and have been characterized by using various techniques, including electronic absorption spectroscopy and cyclic voltammetry. Molecular quadratic nonlinear optical (NLO) responses beta have been determined by using hyper-Rayleigh scattering at 800 nm and also via Stark (electroabsorption) spectroscopic studies on the intense, visible d --> pi* metal-to-ligand charge-transfer bands. The latter experiments reveal that these putatively octupolar D(3) chromophores exhibit two substantial components of the beta tensor which are associated with transitions to dipolar excited states. Computations involving time-dependent density-functional theory and the finite field method serve to further illuminate the electronic structures and associated linear and NLO properties of the new chromophoric salts.     

A fluorescent molecular imaging probe with selectivity for soluble tau aggregated protein

Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and Aβ proteins were assessed. Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation constants (K_d) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for tau over Aβ aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau protein ex vivo was also supported with studies on human brain tissue containing tau and Aβ pathology. To the best of our knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggests that pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzheimer''s disease and other tauopathies.

pTP-TFE imaging probe can distinguish soluble tau aggregated proteins from other aggregated proteins enabling earlier detection of neurodegenerative diseases.     

Electron-density-dependent fused-sphere surfaces derived from pseudopotential calculations

Fused-sphere surfaces can be used to mimic a molecular boundary associated with a constant value of the electron density. The simplest of such fused-sphere models are constructed by using the atomic radii for the spherical isodensity surfaces of individual atoms. In this work, we discuss the extension of this model to molecules containing atoms beyond the second row. In these many- electron systems, the computation of electron densities is usually simplified by adopting a pseudopotential (or effective-core potential) approach. Here, we discuss the performance of large- and small-core pseudo-potential calculations as a tool to derive atomic radii. Our results provide an optimum set of variable radii that can be used to build fused-sphere surfaces. This continuum of surfaces provides a simple approximation to the low-electron-density regions around molecules with heavy atoms.