Action spectra of photosystems II and I and quantum yield of photosynthesis in leaves in State 1

The spectral global quantum yield (Y_II, electrons/photons absorbed) of photosystem II (PSII) was measured in sunflower leaves in State 1 using monochromatic light. The global quantum yield of PSI (Y_I) was measured using low-intensity monochromatic light flashes and the associated transmittance change at 810 nm. The 810-nm signal change was calibrated based on the number of electrons generated by PSII during the flash (4 · O₂ evolution) which arrived at the PSI donor side after a delay of 2 ms. The intrinsic quantum yield of PSI (y_I, electrons per photon absorbed by PSI) was measured at 712 nm, where photon absorption by PSII was small. The results were used to resolve the individual spectra of the excitation partitioning coefficients between PSI (a_I) and PSII (a_II) in leaves. For comparison, pigment–protein complexes for PSII and PSI were isolated, separated by sucrose density ultracentrifugation, and their optical density was measured. A good correlation was obtained for the spectral excitation partitioning coefficients measured by these different methods. The intrinsic yield of PSI was high (y_I = 0.88), but it absorbed only about 1/3 of quanta; consequently, about 2/3 of quanta were absorbed by PSII, but processed with the low intrinsic yield y_II = 0.63. In PSII, the quantum yield of charge separation was 0.89 as detected by variable fluorescence F_v/F_m, but 29% of separated charges recombined (Laisk A, Eichelmann H and Oja V, Photosynth. Res. 113, 145–155). At wavelengths less than 580 nm about 30% of excitation is absorbed by pigments poorly connected to either photosystem, most likely carotenoids bound in pigment–protein complexes.     

Photobiomodulation of human adipose-derived stem cells using 810 nm and 980 nm lasers operates via different mechanisms of action

Photobiomodulation (PBM) using red or near-infrared (NIR) light has been used to stimulate the proliferation and differentiation of adipose-derived stem cells. The use of NIR wavelengths such as 810 nm is reasonably well accepted to stimulate mitochondrial activity and ATP production via absorption of photons by cytochrome c oxidase. However, the mechanism of action of 980 nm is less well understood. Here we study the effects of both wavelengths (810 nm and 980 nm) on adipose-derived stem cells in vitro. Both wavelengths showed a biphasic dose response, but 810 nm had a peak dose response at 3 J/cm² for stimulation of proliferation at 24 h, while the peak dose for 980 nm was 10–100 times lower at 0.03 or 0.3 J/cm². Moreover, 980 nm (but not 810 nm) increased cytosolic calcium while decreasing mitochondrial calcium. The effects of 980 nm could be blocked by calcium channel blockers (capsazepine for TRPV1 and SKF96365 for TRPC channels), which had no effect on 810 nm. To test the hypothesis that the chromophore for 980 nm was intracellular water, which could possibly form a microscopic temperature gradient upon laser irradiation, we added cold medium (4 °C) during the light exposure, or pre-incubated the cells at 42 °C, both of which abrogated the effect of 980 nm but not 810 nm. We conclude that 980 nm affects temperature-gated calcium ion channels, while 810 nm largely affects mitochondrial cytochrome c oxidase.     

Synthesis of α-tocohexaenol (α-T6) a fluorescent,oxidatively sensitive polyene analogue of α-tocopherol

A polyunsaturated analogue of α-tocopherol was synthesized that is both fluorescent and sensitive to peroxidative chemistry that occurs in phospholipid membranes. α-Tocohexaenol 1, [(S)-2,5,7,8-tetramethyl-2-((1E/Z,3E,5E,7E,9E)-4,8,12-trimethyltrideca-1,3,5,7,9,11-hexaenyl)chroman-6-ol, α-T6] was prepared by condensing a known triene fragment triphenyl-(2,6-dimethyl-octa-2,4,6-trienoic acid methyl ester)-phosphonium bromide with a protected chromanol aldehyde, (2S)-6-{[tert-butyl(dimethyl)silyl]oxy}-2,5,7,8-tetra-methyl-3,4-dihydro-2H-chromene-2-carbaldehyde. The full side chain was then completed with isopentyl(tri-n-butyl)phosphonium bromide to give 1. The geometry of the C1′?C2′ alkene appears to be Z (cis) although the coupling constants of the olefinic protons are intermediate between values normally assigned to E and Z-isomers. In ethanol, α-T6 has a maximum absorption at 368 nm with an absorption coefficient of 45,000 M^?1 cm^?1, and displays a maximum fluorescence emission at 523 nm. The susceptibility of α-T6 to peroxidative chemistry was dependent on the concentration of azo-initiators of lipid oxidation in acetonitrile solution as well as in phospholipid vesicles. A loss of fluorescence at 520 nm was observed when α-T6 (vesicles or α-T6-lipid mixtures) was exposed to peroxidative conditions, and this loss mirrored the production of conjugated dienes and trienes during the peroxidation of bulk phospholipids. Addition of natural α-tocopherol during the AMVN induced oxidation of 4 μM α-T6 and 0.5 mg/ml soybean PC induced a characteristic lag phase, after which the fluorescence of α-T6 began to lessen. Thus, α-T6 may be a useful reporter not only of tocopherol location in cells, but also of the extent of peroxidative events.     

Chemical access to the mononuclear Mn(III) [(mL)Mn(OMe)]+ complex (mLH = N,N′-bis-(2-pyridylmethyl)-N-(2-hydroxybenzyl)-N′-methyl-ethane-1,2-diamine) and electrochemical oxidation to the Mn(IV) [(mL)Mn(OMe)]2+ species

Chemical oxidation in acetonitrile of the previously reported phenolato-bridged binuclear Mn(II) complex [(mL)MnMn(mL)]²⁺ (1), where mLH is pentadentate N,N′-bis-(2-pyridylmethyl)-N-(2-hydroxybenzyl)-N′-methyl-ethane-1,2-diamine ligand [C. Hureau, et al., Chem. Eur. J. 2004, 10, 1998–2010] using iodosylbenzene PhIO (dissolved in methanol) is described. The addition of one to four equivalents of PhIO per Mn ion leads to the transient formation of the mono-μ-oxo binuclear Mn₂(III,III) complex [(mL)Mn(μ-O)Mn(mL)]²⁺ (2), previously studied. After addition of five equivalents of PhIO per Mn ion, the mononuclear Mn(III) species [(mL)Mn(OMe)]⁺ (3) is quantitatively generated. The UV–Vis spectrum of 3 displays a broad band at 456 nm (ε = 1000 L mol⁻¹ cm⁻¹) attributed to phenolato to Mn(III) charge transfer transition. Complex 3 exhibits a reversible oxidation wave at E^1/2 = 0.68 V versus SCE, and the mononuclear Mn(IV) complex [(mL)Mn(OMe)]²⁺ (3^ox) can thus be generated by exhaustive electrolysis at 1.0 V versus SCE. The 9.4 GHz EPR spectrum of complex 3^ox shows a strong transition near g = 4 consistent with a rhombically distorted S = 3/2 system with a zero-field splitting dominating the Zeeman effect. UV–Vis spectrum displays a large phenolato to Mn(IV) charge transfer transition at 670 nm (ε = 2450 L mol⁻¹ cm⁻¹).     

Determination of malondialdehyde in biological fluids by high-performance liquid chromatography using rhodamine B hydrazide as the derivatization reagent

Malondialdehyde (MDA) is a biomarker for lipid peroxidation, and studies of sensitive and selective analytical methods for it are very important for pathological research. The aim of this work was to develop and validate a novel HPLC method for the quantification of MDA in biological fluids using rhodamine B hydrazide (RBH) as the derivatization reagent. After pretreatment and derivatization in acid medium at 50 °C for 40 min, the RBH-derivatized MDA was separated on a Kromasil C₁₈ column at 25 °C and detected by a fluorescence detector at excitation wavelength of 560 nm and emission wavelength of 580 nm. The results showed linearity in the range of 0.8–1500.0 nM with a detection limit of 0.25 nM (S/N = 3). The recovery of MDA from plasma and urine was 91.50 to 99.20%, with a relative standard deviation range of 1.45 to 3.26%. In comparison to other methods reported for the determination of MDA, the proposed method showed superiority in simplicity, more sensitivity, shorter derivatization time, and less interference. The developed method was applied to quantification of MDA in human biological fluids collected from five volunteers with a concentration range of 24.62–245.00 nM.     

Optimization of Zn2+-containing mobile phase for simultaneous determination of kynurenine,kynurenic acid and tryptophan in human plasma by high performance liquid chromatography

In the present work we have developed a standard-addition HPLC method using a mobile phase containing low concentration of ZnAc₂ to determine physiological level of kynurenine (KYN), kynurenic acid (KYNA) and tryptophan (TRP) in human plasma simultaneously. The method greatly improved the sensitivity of KYNA, the resolution of KYNA and TRP, and avoided clotting risk caused by high concentration of ZnAc₂ in mobile phase. Samples were deproteinized by addition of equal volume of 0.6 mol/L HClO₄. Analytes in supernatants were separated by an Agilent HC-C18 (2) analytical column; an aqueous mobile phase containing 20 mmol/L NaAc, 3 mmol/L ZnAc₂ and 7% acetonitrile at flow rate of 1.0 mL/min. Detections were performed by a variable wavelength detector at wavelength 365 nm for KYN and a fluorescence detector at wavelengths excitation 344 nm and emission 398 nm for KYNA and TRP. Good linear responses were found with r² > 0.999 for all analytes within the concentration range of physiological levels. The limit of detection of the developed method was 0.03 μmol/L, 0.9 nmol/L and 0.4 μmol/L for KYN, KYNA and TRP respectively. Recoveries from spiked human plasma were 95.4–99.7% for KYN, 98.9–104% for KYNA and 96.5–100.2% for TRP. All CVs for the repeatability and intermediate precision were less than 5%. We conclude that the developed method is helpful for the research investigations in KYN pathway of TRP metabolism.     

Excited state properties of dinaphthyl ditelluride

Di-1-naphthyl ditelluride (Te₂naphthyl₂) is characterized by two low-energy excited states. The corresponding electronic transitions nTe → σ¹ Te–Te and nTe → π¹ naphthyl CT give rise to absorptions at λ_max = 403 and 311 nm, respectively. In solution nTe → σ¹ excitation leads to the cleavage of the Te–Te bond. In contrast to Te₂naphthyl₂ in the dissolved state the solid compound shows a luminescence (λ_max = 576 nm) which originates from nTe → π¹ naphthyl CT triplet.     

Single-walled carbon nanotubes as an effective adsorbent in solid-phase microextraction of low level methyl tert-butyl ether,ethyl tert-butyl ether and methyl tert-amyl ether from human urine

Carbon nanotubes (CNTs) are a kind of new carbon-based nano-materials which have drawn great attention in many application fields. The potential single-walled carbon nanotubes (SWCNTs) as solid-phase microextraction (SPME) adsorbents for the preconcentration of environmental pollutants have been investigated in recent years. The goal of this work was to investigate the feasibility of SWCNTs used as adsorbents for solid-phase microextraction of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) in human urine. SWCNTs were attached onto a stainless steel wire through organic binder. Potential factors affecting the extraction efficiency were optimized, including extraction time, extraction temperature, desorption time, desorption temperature, and salinity. The developed method showed good performance according to the ICH performance criteria for bioanalytical methods. The calibration curves of the ethers were linear (r² ≥ 0.992) in the range from 10 to 5000 ng L^?1. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng L^?1 for all the analytes. In addition, compared with the commercial carboxen/polydimethylsiloxane (CAR/PDMS) fiber, the SWCNT fiber showed better thermal stability (over 350 °C) and longer life span (over 150 times). The developed method was applied successfully to determine trace level of the ethers in urine of 10 healthy male volunteers.     

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT^−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT^−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT^−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT^−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.     

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 °C for 15 min in borate buffer (20 mmol l^?1, pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 °C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l^?1, pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7–4.6% and 0.4–5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6–125 nmol (mg creatinine)^?1 (mean ± SD: 21.6 ± 26.6 nmol (mg creatinine)^?1, n = 20).     

Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography–mass spectrometry

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography–mass spectrometry. The limits of detection are 0.2 ng ml⁻¹ for midazolam and 0.1 ng ml⁻¹ for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml⁻¹ for midazolam, 6.7% at 2 ng ml⁻¹ for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml⁻¹ for 4-hydroxymidazolam.     

Structure and water permeability of fully hydrated diphytanoylPC

Diphytanoylphosphatidylcholine (DPhyPC) is a branched chain lipid often used for model membrane studies, including peptide/lipid interactions, ion channels and lipid rafts. This work reports results of volume measurements, water permeability measurements P_f, X-ray scattering from oriented samples, and X-ray and neutron scattering from unilamellar vesicles at T = 30 °C. We measured the volume/lipid V_L = 1426 ± 1 Å³. The area/lipid was found to be 80.5 ± 1.5 Å² when both X-ray and neutron data were combined with the SDP model analysis (Ku?erka, N., Nagle, J.F., Sachs, J.N., Feller, S.E., Pencer, J., Jackson, A., Katsaras, J., 2008. Lipid bilayer structure determined by the simultaneous analysis of neutron and X-ray scattering data. Biophys. J. 95, 2356–2367); this is substantially larger than the area of DOPC which has the largest area of the common linear chain lipids. P_f was measured to be (7.0 ± 1.0) × 10^?3 cm/s; this is considerably smaller than predicted by the recently proposed 3-slab model (Nagle, J.F., Mathai, J.C., Zeidel, M.L., Tristram-Nagle, S., 2008. Theory of passive permeability through lipid bilayers. J. Gen. Physiol. 131, 77–85). This disagreement can be understood if there is a diminished diffusion coefficient in the hydrocarbon core of DPhyPC and that is supported by previous molecular dynamics simulations (Shinoda, W., Mikami, M., Baba, T., Hato, M., 2004. Molecular dynamics study on the effects of chain branching on the physical properties of lipid bilayers. 2. Permeability. J. Phys. Chem. B 108, 9346–9356). While the DPhyPC head–head thickness (D_HH = 36.4 Å), and Hamaker parameter (H = 4.5 × 10^?21 J) were similar to the linear chain lipid DOPC, the bending modulus (K_C = 5.2 ± 0.5 × 10^?21 J) was 30% smaller. Our results suggest that, from the biophysical perspective, DPhyPC belongs to a different family of lipids than phosphatidylcholines that have linear chain hydrocarbon chains.     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

Dual purpose system that treats anaerobic effluents from pig waste and produce Neochloris oleoabundans as lipid rich biomass

Dual purpose systems that treat wastewater and produce lipid rich microalgae biomass have been indicated as an option with great potential for production of biodiesel at a competitive cost. The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing Neochloris oleoabundans and to evaluate its growth, lipid content and lipid profile of the harvested biomass and the removal of nutrients from the media. Cultures of N. oleoabundans were established in 4 L flat plate photobioreactors using diluted effluents from two different types of anaerobic filters, one packed with ceramic material (D1) and another one packed with volcanic gravel (D2). Maximum biomass concentration in D1 was 0.63 g L⁻¹ which was significantly higher than the one found in D2 (0.55 g L⁻¹). Cultures were very efficient at nutrient removal: 98% for NNH₄⁺ and 98% for PO₄³⁻. Regarding total lipid content, diluted eflluents from D2 promoted a biomass containing 27.4% (dry weight) and D1 a biomass containing 22.4% (dry weight). Maximum lipid productivity was also higher in D2 compared to D1 (6.27 ± 0.62 mg L⁻¹ d⁻¹ vs. 5.12 ± 0.12 mg L⁻¹ d⁻¹). Concerning the FAMEs profile in diluted effluents, the most abundant one was C18:1, followed by C18:2 and C16:0. The profile in D2 contained less C18:3 (linolenic acid) than the one in D1 (4.37% vs. 5.55%). In conclusion, this is the first report demonstrating that cultures of N. oleoabundans treating anaerobic effluents from pig waste are very efficient at nutrient removal and a biomass rich in lipids can be recovered. The maximum total lipid content and the most convenient FAMEs profile were obtained using effluents from a digester packed with volcanic gravel.     

Efficient light absorbers based on thiophene-fused boron dipyrromethene (BODIPY) dyes

We present the development of the thiophene-fused boron dipyrromethene derivatives as efficient light absorbers. The two strategies for the evolution of the optical properties such as the peak positions of absorption wavelengths and molar extinct coefficients were established by the substituent effects: by introducing iodine groups, the bathochromic shifts of the peak positions (+15 nm) and the enhancement of molar extinct coefficients were simultaneously received owing to the heavy atom effect. Next, it was found that the modification with the trifluoromethyl group contributed to the large bathochromic shift (+60 nm) because of the lowering effect on the lowest unoccupied molecular orbital of the dye by the substituent. Finally, we obtained the dyes with large molar extinct coefficients (184,140 M^?1 cm^?1 at 592 nm, 72,180 M^?1 cm^?1 at 623 nm), sharp absorption bands, and low emissions.     

Lower dipeptidyl peptidase-4 following exercise training plus weight loss is related to increased insulin sensitivity in adults with metabolic syndrome

Dipeptidyl peptidase-4 (DPP-4) is a circulating glycoprotein that impairs insulin-stimulated glucose uptake and is linked to obesity and metabolic syndrome. However, the effect of exercise on plasma DPP-4 in adults with metabolic syndrome is unknown. Therefore, we determined the effect of exercise on DPP-4 and its role in explaining exercise-induced improvements in insulin sensitivity. Fourteen obese adults (67.9 ± 1.2 years, BMI: 34.2 ± 1.1 kg/m²) with metabolic syndrome (ATP III criteria) underwent a 12-week supervised exercise intervention (60 min/day for 5 days/week at ∼85% HR_max). Plasma DPP-4 was analyzed using an enzyme-linked immunosorbent assay. Insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp (40 mU/m²/min) and estimated by HOMA-IR. Visceral fat (computerized tomography), 2-h glucose levels (75 g oral glucose tolerance), and basal fat oxidation as well as aerobic fitness (indirect calorimetry) were also determined before and after exercise. The intervention reduced visceral fat, lowered blood pressure, glucose and lipids, and increased aerobic fitness (P < 0.05). Exercise improved clamp-derived insulin sensitivity by 75% (P < 0.001) and decreased HOMA-IR by 15% (P < 0.05). Training decreased plasma DPP-4 by 10% (421.8 ± 30.1 vs. 378.3 ± 32.5 ng/ml; P < 0.04), and the decrease in DPP-4 was associated with clamp-derived insulin sensitivity (r = −0.59; P < 0.04), HOMA-IR (r = 0.59; P < 0.04) and fat oxidation (r = −0.54; P < 0.05). Increased fat oxidation also correlated with lower 2-h glucose levels (r = −0.64; P < 0.02). Exercise training reduces plasma DPP-4, which may be linked to elevated insulin sensitivity and fat oxidation. Maintaining low plasma DPP-4 concentrations is a potential mechanism whereby exercise plus weight loss prevents/delays the onset of type 2 diabetes in adults with metabolic syndrome.     

Crystal structures and coordination-site exchange reactions of palladium(II) and platinum(II) complexes containing tris[2-(tert-butylthio)ethyl]amine

Palladium(II) and platinum(II) complexes, [PdX(NS₃ ¹Bu)]BPh₄ (X=Cl, Br, I; NS₃ ¹Bu=tris[2-(tert-butylthio)ethyl]amine) and [PtCl(NS₃ ¹Bu)]BPh₄, were prepared, and their structures were determined by X-ray analyses. The geometry around the palladium and platinum atoms is square planar. The NS₃ ¹Bu ligand functions as a tridentate ligand and one sulfur atom is not coordinated to the metal. The ¹H NMR spectrum of [PdCl(NS₃ ¹Bu)]BPh₄ in acetone-d₆ exhibited a dynamic behavior. At 20°C the spectrum showed a singlet signal at 1.60 ppm that can be assigned to tert-butyl protons, whereas at −70°C three singlet signals were observed at 1.36, 1.61, and 1.70 ppm with an intensity ratio of 1: 0.25: 2. The signals at 1.36 and 1.70 ppm are assigned to the tert-butyl protons in the square-planar structure, and these signals are consistent with the X-ray structure. The signal at 1.61 ppm can be assigned to the tert-butyl protons in a trigonal-bipyramidal structure where the three tert-butyl groups are magnetically equivalent. Thus, we concluded that the coordination-site exchange occurred via a trigonal-bipyramidal intermediate. The square-planar and trigonal-bipyramidal species of [PdCl(NS₃ ¹Bu)]BPh₄ are in equilibrium in acetone-d₆. The equilibrium was shifted toward the square-planar species on decreasing the temperature. The ¹H NMR spectra for [PdX(NS₃ ¹Bu)]BPh₄ (X=Cl, Br, and I) were similar to one another at the same temperature, suggesting that the site-exchange process is insensitive to the kind of coexisting halogen ligand. The site exchange reaction of [PtCl(NS₃ ¹Bu)]BPh₄ seems to occur more slowly than that of the palladium(II) analogue.     

Complete separation of urinary metabolites of xylene in HPLC/DAD using β-cyclodextrin: Application for biological monitoring

To determine the biomarkers of exposure to xylene, urinary 2-, 3- and 4-methyl-hippuric acids, a new HPLC/DAD analytical method has been developed, which uses β-cyclodextrin as an additive for elution; its complexing abilities are exploited to achieve complete chromatographic separation of the three isomers. The mobile phase was a 3% aqueous solution of β-cyclodextrin, pH 3, and methanol, 80:20, in isocratic conditions, with a flow rate of 1 mL/min. To optimize quantitative analysis three wavelengths were employed for detection: λ = 198 nm, λ = 200 nm, and λ = 202 nm. SPE was applied for the extraction from urine samples of analytes. Validation parameters show recoveries always above 82%; LOD was set at 1 μg/mL with an LOQ of 3 μg/mL. The linear dynamic range (from 4 to 100 μg/mL) showed excellent correspondence. This method is rapid and inexpensive and can be applied to several samples simultaneously using a manifold for SPE extraction. The analytes were separated completely and could be fully quantified. The method was used for the analysis of urine samples from 54 workers exposed to xylene in hospital laboratories and showed a good applicability while allowing quantification even at low doses.     

Characterization of methylated azopyridine as a potential electron transfer mediator for electroenzymatic systems

N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron transfer mediator for oxidation reactions catalyzed by NAD⁺- and pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity with MAZP was defined as (2.2 ± 0.1) × 10⁵ M⁻¹ s⁻¹, whereas the bimolecular rate constant of reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was determined to be (4.7 ± 0.1) × 10⁴ M⁻¹ s⁻¹. The use of MAZP for the regeneration of the cofactors was investigated by applying the electrochemical oxidation of the mediator. The total turnover numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD⁺-dependent alcohol dehydrogenase depended on the concentration of the substrate and the duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable, and the turnover number of the enzyme reached a value of 2.3 × 10³. In addition, oxidation of 1,2-propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded enantioselectively to yield l-lactic acid.     

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy

closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 10¹² neutrons/cm²).