Novel method for the synthesis of dinucleoside-(N3′ → P5′)-phosphoramidothioates

A new approach for the synthesis of dinucleoside-(N3′ → P5′)-phosphoramidothioates based on the Atherton–Todd reaction has been developed using nucleoside H-thiophosphonates and 3′-amino-2′,3′-dideoxynucleosides with an unprotected 5′-hydroxyl group. A mixture of P-diastereomers of dinucleosides was separated by column chromatography into fast migrating and slow migrating isomers. Based on single crystal X-ray diffraction analysis, the absolute configuration at the phosphorus atom in the slow eluting diastereomer of the dinucleoside-(N3′ → P5′)-phosphoramidothioate G_NPSG_NHTr was assigned as Rp.     

Some Generalized Problems for Polyharmonic Functions

In the first section of this paper, some non-local boundary value problem for the polyharmonic equation in the plane is considered. This problem consists in determining solution of the polyharmonic equation satisfying some special non-local-type boundary condition on two curves. The existence theorem is proved. In the second section, an example for the case of the biharmonic equation is considered. In the third section, some non-local, non-linear problem of Riquier type is examined. The Riquier-type problem consists in determining the polyharmonic function in the plane whose value together with its successive Laplacians are prescribed on the boundary. The existence theorem is proved and an example for the case of the biharmonic equation is considered.     

The effect of the dipalmitoylphosphatidylcholine lipid bilayer state on the adsorption of phenyltins

The nonspecific adsorption of amphiphilic molecules onto the membrane depends both on the properties of the adsorbate and the state of the lipid bilayer. Electrostatic interactions drive the adsorption of charged molecules and hydrophobicity determines partition of the adsorbate into the membrane, whereas the steric compatibility of the lipid bilayer and the amphiphilic molecule is an additional factor to be accounted for when considering interaction between the adsorbate and the membrane. The adsorption of phenyltins was evaluated from changes in Fluorescein‐PE fluorescence intensity. The pH sensitivity of fluorophore, located at the membrane surface, was utilized to detect charges introduced onto the membrane by adsorbing compounds. It has been shown that the state of the membrane affects phenyltin adsorption in accordance with the number of phenyl rings on the molecule. Furthermore, the membrane surface topology determines interfacially located triphenyltin adsorption, with a much weaker effect on deeply embedded diphenyltin. When the dipalmitoylphosphatidylcholine (DPPC) model membrane is in the ripple phase, with complex surface morphology, phenyltin adsorption is greatly enhanced. Results presented in this paper show that steric constraints imposed on rigid and bulky amphiphilic compounds by ordered alkyl chains and membrane surface topology affect nonspecific molecule adsorption onto the membrane. Copyright © 2000 John Wiley & Sons, Ltd.     

Adsorption of phenyltin compounds onto phosphatidylcholine / cholesterol bilayers

Phenyltin compounds are known to be biologically active and, whan widely spread, are potentially hazardous. As their chemical structure suggests, they interact with the lipid fraction of the cell membrane. Their effect on the model phosphatidylcholine/cholesterol bilayer has been studied using fluorescence and ¹H NMR techniques. The change in the fluorescein‐PE fluorescence intensity indicates the amount of charge added by phenyltin compounds to the membrane surface. Although the presence of cholesterol alone does not alter membrane interface properties measured with fluorescein‐PE, ¹H NMR measurements show that lipid mobility is altered throughout the hydrophobic core of the membrane. Cholesterol in the phosphatidylcholine bilayer does not alter tetraphenyltin interaction with the membrane, though the effect of diphenyltin dichloride, penetrating deeply into the hydrophobic core of the membrane, is reduced when the amount of cholesterol in the membrane is increased, suggesting decreased compound adsorption. Triphenyltin chloride has a qualitatively different effect on the lipid bilayer, when observed using this fluorescence technique. The adsorption of triphenyltin onto the phosphatidylcholine/cholesterol membrane induces a lateral phase separation of membrane components. Since triphenyltin chloride is known to be adsorbed onto the interface of the lipid bilayer, this separation mechanism must originate in this region and does not seem to be electrostatic in origin. ¹H NMR measurements have confirmed the observation that these two active phenyltin compounds interact with the phosphatidylcholine/cholesterol membrane differently, disrupting different regions of the bilayer to a different degree. Copyright © 2000 John Wiley & Sons, Ltd.     

Nanomaterials in complex biological systems: insights from Raman spectroscopy

The interaction of nanomaterials with biomolecules, cells, and organisms plays an important role in cell biology, toxicology, and nanotechnology. Spontaneous Raman scattering can be used to probe biomolecules, cells, whole animals, and nanomaterials alike, opening interesting avenues to study the interaction of nanoparticles with complex biological systems. In this review we discuss work in biomedical Raman spectroscopy that has either been concerned directly with nanostructures and biosystems, or that indicates important directions for successful future studies on processes associated with nano-bio-interactions.     

Development of a fluorescence-based microtiter plate method for the measurement of glutathione in yeast

The present work was aimed to the development of a fluorescence assay using the universal 96-well microplate format, for the measurement of reduced glutathione (GSH) in yeast cells. The method relies upon the reaction between GSH and a highly selective fluorogenic probe, i.e. naphthalene-2,3-dicarboxaldehyde (NDA). The optimization of the method included the extraction step of GSH from cultured yeast cells in a cold perchloric acid solution, derivatization conditions (10-min reaction at pH 8.6 and at 20 ± 2 °C in darkness) and stability studies of the resulting fluorescent adduct. Full selectivity was observed versus other endogenous thiols (except for γ-glutamylcysteine), glutathione disulfide (GSSG) and enzymatic reducing reagents of GSSG. Linearity was verified in the range 0.3-6.5 μM (R² > 0.98) and limits of quantification and detection were 0.3 and 0.05 μM, respectively. Relative standard deviation corresponding to repeatability (n = 3) and inter-day precision (n = 5) were 2.8 and 6.1%, respectively. Mean GSH recovery from cell extracts was 95%. The method appeared highly correlated (R² = 0.96) with a previously reported HPLC method.The method was then applied to the monitoring of GSH in the yeast strain Kluyveromyces lactis during its growth period and in the presence of an inhibitor of GSH biosynthesis. The method presents the main advantage of a high throughput for the measurement of biological samples. The extent of the method to the study of the redox couple GSSG/GSH by including an enzymatic reduction step and the enhancement of the fluorescence signal using cyclodextrins were discussed.