Role of electrostatics in the binding of charged metallophthalocyanines to neutral and charged phospholipid membranes

Binding of the cationic tetra(tributylammoniomethyl)-substituted hydroxoaluminum phthalocyanine (AlPcN₄) to bilayer lipid membranes was studied by fluorescence correlation spectroscopy (FCS) and intramembrane field compensation (IFC) methods. With neutral phosphatidylcholine membranes, AlPcN₄ appeared to bind more effectively than the negatively charged tetrasulfonated aluminum phthalocyanine (AlPcS₄), which was attributed to the enhancement of the coordination interaction of aluminum with the phosphate moiety of phosphatidylcholine by the electric field created by positively charged groups of AlPcN₄. The inhibitory effect of fluoride ions on the membrane binding of both AlPcN₄ and AlPcS₄ supported the essential role of aluminum-phosphate coordination in the interaction of these phthalocyanines with phospholipids. The presence of negative or positive charges on the surface of lipid membranes modulated the binding of AlPcN₄ and AlPcS₄ in accord with the character (attraction or repulsion) of the electrostatic interaction, thus showing the significant contribution of the latter to the phthalocyanine adsorption on lipid bilayers. The data on the photodynamic activity of AlPcN₄ and AlPcS₄ as measured by sensitized photoinactivation of gramicidin channels in bilayer lipid membranes correlated well with the binding data obtained by FCS and IFC techniques. The reduced photodynamic activity of AlPcN₄ with neutral membranes violating this correlation was attributed to the concentration quenching of singlet excited states as proved by the data on the AlPcN₄ fluorescence quenching.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

On the rotational brownian motion of a bacterial idle motor. II. Theory of fluorescence correlation spectroscopy

The photon flux autocorrelation function of a fluorescent label attached to a bacterial motor shaft is calculated for the case in which the bacterial motor is considered to be actively but idly rotating. It is shown that even when the fluorescent label has a very short lifetime, fluorescence correlation spectroscopy should provide a useful tool for determining the rate of revolution of the bacterial motor under various solution conditions.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Determination of trace levels of fatty acid metal salts by high-performance liquid chromatography with fluorescence prelabeling

A simple method is described for picomole determinations of fatty acid metal salts. Fatty acid salts are directly labeled with 4-bromomethyl-7-methoxycoumarin in the presence of excess ethylenediaminetetraacetic acid tripotassium salt without any solvent extractions. The fluorescence derivatives of fatty acids are separated by reverse-phase high-performance liquid chromatography followed by fluorometric detection. The response of each fatty acid (C8-C18) calcium salt is linear from 1 to 50 micrograms/ml of samples. The detection limit is about 7 pmol. Good recoveries are obtained for the calcium salts of myrystic acid and soap (C8-C18, C18:1,2). The new method is successfully applied to the study on biodegradation of fatty acids in river water.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

Focus: A Multifaceted Battle Against Cancer: Extracting Knowledge from Chemical Imaging Data Using Computational Algorithms for Digital Cancer Diagnosis

Fourier transform infrared (FTIR) spectroscopic imaging is an emerging microscopy modality for clinical histopathologic diagnoses as well as for biomedical research. Spectral data recorded in this modality are indicative of the underlying, spatially resolved biochemical composition but need computerized algorithms to digitally recognize and transform this information to a diagnostic tool to identify cancer or other physiologic conditions. Statistical pattern recognition forms the backbone of these recognition protocols and can be used for highly accurate results. Aided by biochemical correlations with normal and diseased states and the power of modern computer-aided pattern recognition, this approach is capable of combating many standing questions of traditional histology-based diagnosis models. For example, a simple diagnostic test can be developed to determine cell types in tissue. As a more advanced application, IR spectral data can be integrated with patient information to predict risk of cancer, providing a potential road to precision medicine and personalized care in cancer treatment. The IR imaging approach can be implemented to complement conventional diagnoses, as the samples remain unperturbed and are not destroyed. Despite high potential and utility of this approach, clinical implementation has not yet been achieved due to practical hurdles like speed of data acquisition and lack of optimized computational procedures for extracting clinically actionable information rapidly. The latter problem has been addressed by developing highly efficient ways to process IR imaging data but remains one that has considerable scope for progress. Here, we summarize the major issues and provide practical considerations in implementing a modified Bayesian classification protocol for digital molecular pathology. We hope to familiarize readers with analysis methods in IR imaging data and enable researchers to develop methods that can lead to the use of this promising technique for digital diagnosis of cancer.     

X-ray absorption spectroscopy of selenium-containing amino acids

Received: 2 April 1999 / Accepted: 17 September 1999     

Increase in lipid microviscosity of unilamellar vesicles upon the creation of transmembrane potential

Summary Diffusion potential of potassium ions was formed in unilamellar vesicles of phosphatidyl choline. The vesicles, which included potassium sulfate buffered with potassium phosphate, were diluted into an analogous salt solution made of sodium sulfate and sodium phosphate. The diffusion potential was created by the addition of the potassium-ionophore, valinomycin. The change in lipid microviscosity, ensuing the formation of membrane potential, was measured by the conventional method of fluorescence depolarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid microviscosity was found to increase with membrane potential in a nonlinear manner, irrespective of the potential direction. Two tentative interpretations are proposed for this observation. The first assumes that the membrane potential imposes an energy barrier on the lipid flow which can be treated in terms of Boltzmann-distribution. The other interpretation assumes a decrease in lipid-free volume due to the pressure induced by the electrical potential. Since increase in lipid viscosity can reduce lateral and rotational motions, as well as increase exposure of functional membrane proteins, physiological effects induced by transmembrane potential could be associated with such dynamic changes.