Synthesis of safflomide and its HPLC measurement in mouse plasma after oral administration

Safflomide (N-caffeoyltryptamine) is a compound belonging to a group of phenylpropanoid amides found in plants. In this study, safflomide was chemically synthesized and confirmed by LC-MS, LC-MS/MS and NMR spectroscopic methods, and a high-performance liquid chromatography (HPLC) method was developed for quantifying safflomide in biological samples. The synthesis was simple, and the yield of safflomide was greater than 50%. Using the synthesized safflomide as a standard, HPLC separation was performed on a Nova-Pak C18 column using an isocratic buffer, and the separation was detected using a coulometric electrochemical detector. The detection of safflomide yielded an excellent peak resolution at the retention time of 21 min, and the lower limit of the detection was as little as 100 fmol. Using this HPLC method, the plasma concentrations of safflomide were determined in mouse blood, collected at 5, 10, 15, 20, 25, 30, and 35 min following its oral administrations (1 and 3 mg/30 g body weight). This HPLC method standardized with safflomide is the first reported method able to quantify the compound in standard and plasma samples with good detection limit and consistent reproducibility.     

Determination of beraprost in human plasma by a high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of beraprost, a stable, orally active prostacyclin analogue with vasodilatory, antiplatelet and cytoprotective effects. The analyte and internal standard, indomethacin, were extracted by solid-phase extraction using OASIS HLB cartridge. The chromatographic separation was performed on a C18 column with a mobile of 0.1% formic acid-methanol (30:70, v/v). The highest daughter ion of deprotonated analyte was quantitated in negative ionization by multiple reactions monitoring with a mass spectrometer. The mass transitions m/z 397>269 and m/z 356>312 were used to measure beraprost and internal standard, respectively. The assay exhibited a linear range from 0.02 to 2 ng/mL for beraprost in human plasma. The lower limit of quantitation was 20 pg/mL with a relative standard deviation of less than 20%. The method was validated with respect to linearity, sensitivity, specificity, recovery, accuracy and precision. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic study.     

Identification and Biochemical Studies on Novel Non-Nucleoside Inhibitors of the Enzyme Adenosine Kinase

The enzyme adenosine kinase (AK) plays a key role in the regulation of intracellular and extracellular concentration of adenosine (Ado), which exhibits potent hormonal activity in cardiovascular, nervous and immune systems. In view of the pharmacological effects of Ado, there is much interest in identifying inhibitors of AK, which can augment its tissue-protective effects. In this study, we have screened 1040 compounds from a chemical library of putative kinase inhibitors for their effect on purified human recombinant AK. These studies have identified 8 novel, non-nucleoside AK inhibitors. Four of these compounds (viz. 2-tert-butyl-4H-benzo[1,2,4]thiadiazine-3-thione (2759–0749); N-(5,6-diphenyl-furo[2,3-d]pyrimidin-4-yl)-propionamide (3998–0118); 3-[5,6-Bis-(4-methoxy-phenyl)-furo[2,3-d]pyrimidin-4-ylamino]-propan-1-ol (4072–2732); and 2-[2-(3,4-dihydroxy-phenyl)-5-phenyl-1H-imidazol-4-yl]-fluoren-9-one (8008–6198)), which inhibited human AK in a concentration-dependent manner in a low micromolar range (IC₅₀ = 0.38 ∼ 1.98 μM) were further studied. Kinetic and structural studies on these compounds provide evidence that inhibition of AK by these compounds was competitive with respect to Ado and non-competitive for ATP. All of these compounds also inhibited uptake of Ado and its metabolism in cultured mammalian cells at comparable concentrations indicating their efficient cellular penetrability. These AK inhibitors, whose chemical structures differ significantly from all previously known inhibitors, provide useful lead compounds for identification of more potent but less toxic AK inhibitors that may prove useful for therapeutic purposes.     

High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Bioaugmented sulfur-oxidizing denitrification system with <Emphasis Type="Italic">Alcaligenes defragrans</Emphasis> B21 for high nitrate containing wastewater treatment

The performance of enriched sludge augmented with the B21 strain of Alcaligenes defragrans was compared with that of enriched sludge, as well as with pure Alcaligenes defragrans B21, in the context of a sulfur-oxidizing denitrification (SOD) process. In synthetic wastewater treatment containing 100–1,000 mg NO₃⁻-N/L, the single strain-seeded system exhibited superior performance, featuring higher efficiency and a shorter startup period, provided nitrate loading rate was less than 0.2 kg NO₃⁻-N/m³ per day. At nitrate loading rate of more than 0.5 kg NO₃⁻-N/m³ per day, the bioaugmented sludge system showed higher resistance to shock loading than two other systems. However, no advantage of the bioaugmented system over the enriched sludge system without B21 strain was observed in overall efficiency of denitrification. Both the bioaugmented sludge and enriched sludge systems obtained stable denitrification performance of more than 80% at nitrate loading rate of up to 2 kg NO₃⁻-N/m³ per day.     

Membrane proteomic analysis of human mesenchymal stromal cells during adipogenesis

Mesenchymal stromal cells (MSCs) have proven useful for cell and immune therapy, but the molecular constituents responsible for their functionalities, in particular, those on the plasma membrane, remain largely unknown. Here we employed both gel and nongel based MS to analyze human MSCs' membrane proteome before and after adipogenesis. 2-DE of cells that were pretreated with membrane impermeable fluorescent dyes revealed that both the whole cell proteome and the cell surface subproteome were independent of donors. LC coupled with tandem MS analysis of the plasma membrane-containing fraction allowed us to identify 707 proteins, approximately half of which could be annotated as membrane-related proteins. Of particular interest was a subset of ectodomain-containing membrane-bound proteins that encompass most known surface markers for MSCs, but also contain a multitude of solute carriers and ATPases. Upon adipogenic differentiation, this proteomic profile was amended to include several proteins involved in lipid metabolism and trafficking, at the expense of, most noticeably, ectoenzymes. Our results here provide not only a basis for future studies of MSC-specific molecular mechanisms, but also a molecular inventory for the development of antibody-based cell isolation and identification procedures.     

Proteomic analysis of glutamate-induced toxicity in HT22 cells

In the present study, we have investigated the proteome changes associated with glutamate-induced HT22 cell death, a model system to study oxidative stress-mediated toxicity. Among a number of HT22 proteins exhibiting altered expression, several molecular chaperones demonstrated substantial changes. For example, the levels of Hsp90 and Hsp70 decreased as cell death progressed whereas that of Hsp60 increased dramatically. Interestingly, cytosolic Hsp60 increased more prominently than mitochondrial Hsp60. Concomitantly, the accumulation of poly-ubiquitylated proteins and differential regulation of the peptidase activities and the subunits of 26S proteasomes were observed in glutamate-treated HT22 cells. Our findings that the molecular chaperones and the ubiquitin-proteasome system undergo changes during glutamate-induced HT22 cell death may suggest the importance of a protein quality control system in oxidative damage-mediated toxicity.     

Differential analysis of Bacillus anthracis after pX01 plasmid curing and comprehensive data on Bacillus anthracis infection in macrophages and glial cells

Bacillus anthracis is a gram-positive bacterial organism responsible for anthrax. This organism has two pathogenic plasmids: pX01 and pX02. The genetic function of pX01, which comprises about 198 kb, is not known, except for a region called the pathogenic island, which contains three genes-pag, lef, and cya-that code for three toxic proteins. A 2-D difference gel electrophoresis (2-D DIGE) system was used to verify the existence of proteins controlled by the pX01 plasmid, and protein regulation data were obtained using DeCyder software. A total of 1728 proteins were identified in the wild-type strain of this organism and 1684 in the pX01 plasmid. Twenty-seven of these proteins disappeared and eight appeared when the pX01 plasmid was removed. An additional 52 proteins were downregulated and 15 were upregulated when this plasmid was removed. A total of 102 proteins have been identified using the MALDI-TOF method of analysis, including 49 whose functions are unknown. Among these, 31 participate in metabolic processes, two in cellular processes, 15 in the processing of genetic information, and five in the processing of extracellular information. Another seven proteins participate in bacterial virulence and pathogenesis. We investigated the functions of these proteins in other bacteria, particularly the B. anthracis derivative H9041. Bacterial growth differed between pX01+/pX02+ B. anthracis and its pX01-/pX02+ derivative as did the cytotoxicity of macrophages infected by pX01+/pX02+ B. anthracis and the pX01-pX02+ derivative. We also found that S100B protein levels increased in the host infected with pX01+/pX02+ B. anthracis or its pX01-/pX02+ derivative. These data suggest that the pX01 plasmid plays a key role in the regulation of protein functions in B. anthracis.