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.     

Anatomical correlates of the functional organization in the human occipitotemporal cortex

The connectivity between functionally distinct areas in the human brain is unknown because of the limitations posed by current postmortem anatomical labeling techniques. Diffusion tensor imaging (DTI) has previously been used to define large white matter tracts based on well-known anatomical landmarks in the living human brain. In the present study, we used DTI coupled with functional magnetic resonance imaging (fMRI) to assess neuronal connections between human striate and functionally defined extrastriate ventral cortical areas. Functional areas were identified with conventional fMRI mapping procedures and then used as seeding points in a DTI analysis to ascertain connectivity patterns between cortical areas, thus yielding the pattern of connections between human occipitoventral visual areas in vivo.     

Coexistence of gapless excitations and commensurate charge-density wave in the 2H transition metal dichalcogenides

An unexpected feature common to 2H transition metal dichalcogenides ( 2H TMDs) is revealed with a first-principles Wannier function analysis of the electronic structure of the prototype 2H TaSe2: The low-energy Ta "5d(z2)" bands governing the physics of a charge-density wave (CDW) is dominated by hopping between next-nearest neighbors. With this motivation we develop a minimal effective model for the CDW formation, in which the unusual form of the hopping leads to an approximate decoupling of the three sublattices. In the CDW phase one sublattice remains undistorted, leaving the bands associated with it ungapped everywhere in the Fermi surface, resolving the long-standing puzzle of the coexistence of gapless excitations and commensurate CDW in the 2H TMDs.     

Picosecond pump-probe measurement of bandgap changes in SiO2/TiO2 one-dimensional photonic bandgap structures

A picosecond pump-probe nonlinear optical measurement is performed in SiO2/TiO2 one-dimensional photonic bandgap structures fabricated by a solgel method. Both high and low band edges were examined by varying the probe wavelengths and angle tuning was also employed to further clarify the mechanism of a nonlinear optical response. The third-order nonlinear optical response in one-dimensional photonic bandgap structures that comprise TiO2 films is responsible for the nonlinear optical transmissions at both bandgap edges, with an 8% decrease at the low-energy edge and a 4.5% increase at the high-energy edge for a 355 nm pump intensity of 430 MW/cm2.     

Atomic arrangements inside Ru and Os nanoislands spontaneously deposited on Pt(111)

The atomic arrangements inside Ru and Os nanoislands spontaneously deposited on Pt(111) electrode surface were observed with electrochemical scanning tunneling microscopy. The surperlattice of the pristine Ru nanodeposits is (square root of 3 x square root of 3)R30 degrees -RuO+. Upon reduction, the Ru nanodeposits are compressed to a uniaxially incommensurate (square root of 3 x square root of 2)R30 degrees -Ru structure, which does not change during the following reoxidation. The atomic arrangement inside the pristine Os nanodeposits is an incommensurate (square root of 2 x square root of 2)-OsO+ structure, which does not transform during the subsequent reduction-oxidation cycles. The structures of the Ru and Os nanodeposits are discussed in terms of removal and insertion of oxygen ions depending on electrode potential.     

Interaction of amphotericin B and its selected derivatives with membranes: molecular modeling studies

Amphotericin B (AmB) is a well-known antifungal antibiotic that has been used in the clinic for about five decades. Despite its chemotherapeutic importance, AmB is quite toxic and many efforts have been made to improve its pharmacological properties, e.g., by chemical modifications. The lipid membrane is a molecular target for AmB, however, due to heterogeneity of its components, the molecular mechanism of AmB action is still unclear. The lack of this knowledge hinders rational designing of new and less toxic AmB derivatives. Our review is a critical presentation of the current understanding of AmB molecular mechanism of action at the membrane level. Except the experimental approach, the extensive overview of molecular modeling studies, performed mostly in our lab, is presented. The results of interactions between AmB or some of its derivatives and lipid model membranes are discussed. In our studies, different biomembrane models and different associate states of the antibiotic were included. Presented molecular modeling approach is especially valuable with regard to a new paradigm of the structure of lipid membrane containing liquid-ordered domains. Hopefully, all these complementary experimental/computational approaches are going to reach the point at which a new hypothesis about molecular mechanism of AmB activity and selectivity will be put forward.     

Analysis of urine for cysteine, cysteinylglycine, and homocysteine by high-performance liquid chromatography

A new analytical method is proposed for simultaneous determination, by liquid chromatography, of the three main urinary thiols–cysteine, cysteinylglycine, and homocysteine. To measure the total amount of these thiols urine is reduced with sodium borohydride, to convert disulfides to thiols which are then derivatized with 2-chloro-1-methylquinolinium tetrafluoroborate. Separation and quantitation of the 2-S-quinolinium thiol derivatives formed were achieved by high-performance liquid chromatography with detection at 355 nm. Validation showed the method enabled reliable simultaneous determination of these aminothiols in urine. The calibration graphs for each analyte, obtained by use of normal urine spiked with increasing amounts of cysteine, cysteinylglycine, and homocysteine, were linear (R ²≥0.997) over the range covering most practical situations. The recovery of the assay was 98–100% and sensitivity was 0.12–0.25 μmol L⁻¹. The method was applied to 91 different samples of normal urine to establish reference values for the aminothiols, normalized on creatinine.     

Applications of carbon in lead-acid batteries: a review

Journal of Solid State Electrochemistry - A review presents applications of different forms of elemental carbon in lead-acid batteries. Carbon materials are widely used as an additive to the...