Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica.

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3 µg/mL (CI95: 25.9-28.9 µg/mL) corresponding to 9.6 µM (CI95: 9.1-10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR.The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4-6″) and single linked B-type units.This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

Photoacoustic monitoring of trace gases by use of a diode-based difference frequency laser source.

We present a compact mid-infrared laser spectrometer for trace-gas monitoring. Difference frequency generation in periodically poled LiNbO(3) is used as laser source, yielding a tuning range 3.2-3.7mum at a linewidth of 154 MHz. The relatively high average power of 3 to 5 mW favors detection with a small resonant photoacoustic gas cell. Measurements of methane yield a detection limit in the low parts in 10(6) by volume concentration range.

Compact gas sensor using a pulsed difference-frequency laser spectrometer.

We present a novel compact pulsed laser spectrometer based on difference-frequency mixing of a cw tunable external-cavity diode laser (795-825 nm) and a pulsed Nd:YAG laser (1064 nm) in bulk LiNbO(3) . The pulsed mid-IR source is continuously tunable from 3.16 to 3.67microm and exhibits a linewidth of only 154 MHz, a peak power of approximately 50microW , and a pulse duration of 6 ns at a 6.5-kHz repetition rate. Spectra of methane in room air and formaldehyde have been recorded at room-temperature operation in a multipass cell with deduced detection limits of 10 and 40 parts in 10(9) , respectively.

On-line multicomponent trace-gas analysis with a broadly tunable pulsed difference-frequency laser spectrometer.

The design and application of a novel automated room-temperature laser spectrometer are reported. The compact instrument is based on difference-frequency generation in bulk LiNbO(3). The instrument employs a tunable cw external-cavity diode laser (795-825 nm) and a pulsed diode-pumped Nd:YAG laser (1064 nm). The generated mid-IR nanosecond pulses of 50-microW peak power and 6.5-kHz repetition rate, continuously tunable from 3.16 to 3.67 microm, are coupled into a 36-m multipass cell for spectroscopic studies. On-line measurements of methane are performed at concentrations between 200 ppb (parts in 10(9) by mole fraction) and approximately 1%, demonstrating a large dynamic range of 7 orders of magnitude. Furthermore computer-controlled multicomponent analysis of a mixture containing five trace gases and water vapor with an overall response time of 90 s at an averaging time of only approximately 30 s is reported. A minimum detectable absorption coefficient of 1.1 x 10(-7) cm(-1) has been achieved in an averaging time of 60 s, enabling detection limits in the ppb range for many important trace gases, such as CH(4), C(2)H(6), H(2)CO, NO(2), N(2)O, HCl, HBr, CO, and OCS.

Electron-electron spin-spin interaction in spin-labeled low-spin methemoglobin.

Nitroxyl free radical electron spin relaxation times for spin-labeled low-spin methemoglobins were measured between 6 and 120 K by two-pulse electron spin echo spectroscopy and by saturation recovery electron paramagnetic resonance (EPR). Spin-lattice relaxation times for cyano-methemoglobin and imidazole-methemoglobin were measured between 8 and 25 K by saturation recovery and between 4.2 and 20 K by electron spin echo. At low temperature the iron electron spin relaxation rates are slow relative to the iron-nitroxyl electron-electron spin-spin splitting. As temperature is increased, the relaxation rates for the Fe(III) become comparable to and then greater than the spin-spin splitting, which collapses the splitting in the continuous wave EPR spectra and causes an increase and then a decrease in the nitroxyl electron spin echo decay rate. Throughout the temperature range examined, interaction with the Fe(III) increases the spin lattice relaxation rate (1/T1) for the nitroxyl. The measured relaxation times for the Fe(III) were used to analyze the temperature-dependent changes in the spin echo decays and in the saturation recovery (T1) data for the interacting nitroxyl and to determine the interspin distance, r. The values of r for three spin-labeled methemoglobins were between 15 and 15.5 A, with good agreement between values obtained by electron spin echo and saturation recovery. Analysis of the nitroxyl spin echo and saturation recovery data also provides values of the iron relaxation rates at temperatures where the iron relaxation rates are too fast to measure directly by saturation recovery or electron spin echo spectroscopy. These results demonstrate the power of using time-domain EPR measurements to probe the distance between a slowly relaxing spin and a relatively rapidly relaxing metal in a protein.

Labeling of the mitochondrial membrane D-3-hydroxybutyrate dehydrogenase (BDH) with new bifunctional phospholipid analogues.

D-3-Hydroxybutyrate dehydrogenase (BDH), an inner mitochondrial protein, is a well-known phospholipid dependent enzyme. It is a primary dehydrogenase of the oxidative phosphorylation system and is involved in the redox balance of the NAD+/NADH pool. The preparation of fluorescent phospholipids and newly synthesized bifunctional phospholipid analogues (fluorescent and photoactivatable) allowed us to study the structural requirement for lipid activation of the purified enzyme. This paper reports the chemical synthesis protocols to prepare these new phospholipids and their characterization. Illumination experiments of complexes between bifunctional phospholipids and BDH which lead to a cross-linked polypeptide indicate that both the polar head and the hydrophobic moiety of phospholipids interact with BDH. The bifunctional phospholipids were also tested on other lipid-binding proteins, i.e., horse cytochrome c and bovine serum albumin, and demonstrated the promising potential of this new type of photoactivatable molecules which can be followed merely by fluorescence without radioactive labeling.

Influence of selected reinforcers on the cardiorespiratory exercise behavior of profoundly mentally retarded youth.

6 profoundly mentally retarded youth were provided a 5-wk. stair-climbing program to improve cardiorespiratory fitness behavior. Three subjects were provided verbal plus food reinforcement and the other three received verbal reinforcement during the intervention phase. Based on the visual inspection of the data, both types of reinforcement increased the number of steps taken and exercise time.