Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Determination of paroxetine levels in human plasma using gas chromatography with electron-capture detection

A simple, rapid and sensitive procedure using gas chromatography with electron-capture detection to measure paroxetine levels in human plasma has been developed. The analyte was extracted from plasma with ethyl acetate after basification of the plasma and then derivatized with heptafluorobutyric anhydride before gas chromatographic separation. The calibration curves were linear, with typical r² values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.     

Use of short high-performance liquid chromatography columns and tandem-mass spectrometry for the rapid analysis of a prostaglandin analog, fluprostenol, in rat plasma

A short reversed-phase HPLC column and a tandem mass spectrometer were used to develop a stable-isotope-dilution assay for the rapid and sensitive analysis of fluprostenol, a prostaglandin analog, in rat plasma. A Waters Symmetry ODS column (2.1×10 mm) afforded rapid isocratic elution of fluprostenol (t_R=40 s) but still provided a relatively large k′ value of 4. The use of tandem mass spectrometry allowed the interference-free detection of fluprostenol under the rapid elution conditions, with a limit of quantitation of 25 pg ml⁻¹ fluprostenol, using 0.2 ml plasma sample volumes. The method was linear over three orders of magnitude, yielded accurate and precise results and allowed the pharmacokinetic profile of fluprostenol to be defined following intravenous administration in rats.     

Raindrop Momentum Triggers Growth of Leaf-Associated Populations of Pseudomonas syringae on Field-Grown Snap Bean Plants

Observational and microclimate modification experiments were conducted under field conditions to determine the role of the physical environment in effecting large increases in phyllosphere population sizes of Pseudomonas syringae pv. syringae, the causal agent of bacterial brown spot disease of snap bean (Phaseolus vulgaris L.). Comparisons of daily changes in population sizes of P. syringae on three plantings of snap bean cultivar Cascade and one of cultivar Eagle with weather conditions indicated a strong association of rainfalls with periods of 1 to 3 days in duration during which increases in bacterial population sizes were greater than 10-fold and up to 1,000-fold. The effects of rain on populations of P. syringae were explored further by modifying the microclimate of bean plants in the field with polyethylene shelters to shield plants from rain and fine-mesh inert screens to modify the momentum of raindrops. After each of three separate intense rains, the greater-than-10-fold increases in population sizes of P. syringae observed on plants exposed to the rains did not occur on plants in the shelters or under the screens. The screens decreased the velocity and, thus, the momentum of raindrops but not the volume or quality of rainwater that fell on plants under the screens. Thus, the absence of increases in population sizes of P. syringae on plants under the screens suggests that raindrop momentum plays a role in the growth-triggering effect of intense rains on populations of P. syringae on bean plants under field conditions.     

A binding-lipoprotein-dependent oligopeptide transport system in Streptococcus gordonii essential for uptake of hexa- and heptapeptides.

Cells of the oral bacterium Streptococcus gordonii express three cytoplasmic membrane-bound lipoproteins with apparent molecular masses of 76 to 78 kDa that are the products of three genes (designated hppA, hppG, and hppH). The lipoproteins are immunologically cross-reactive, contain 60% or more identical amino acid residues, and are highly similar to the AmiA, AliA (PlpA), and AliB substrate-binding protein components of an oligopeptide permease in Streptococcus pneumoniae. Insertional inactivation of the hppA or hppH gene resulted in loss of the ability of S. gordonii cells to utilize specific peptides of five to seven amino acid residues for growth. An insertion within the COOH-terminal coding region of hppG that caused apparent truncation of the HppG polypeptide had a similar effect; however, S. gordonii mutants in which HppG polypeptide production was abolished were still able to grow on all oligopeptides tested. Inactivation of hppA gene (but not inactivation of the hppG or hppH gene) caused reduced growth rate of cells in complex medium, slowed the rate of development of competence for transformation, reduced the efficiency of transformation, and increased the resistance of cells to aminopterin. These results suggest that the formation of a solute-binding-protein complex consisting of at least the HppA and the HppH lipopolypeptides is necessary for binding and subsequent uptake of primarily hexa- or heptapeptides by a Hpp (Hexa-heptapeptide permease) system in S. gordonii. In addition, Hpp may play a role in the control of metabolic functions associated with the growth of streptococcal cells on complex nitrogen sources and with the development of competence.     

Changes in gravity inhibit lymphocyte locomotion through type I collagen

Summary Immunity relies on the circulation of lymphocytes through many different tissues including blood vessels, lymphatic channels, and lymphoid organs. The ability of lymphocytes to traverse the interstitium in both nonlymphoid and lymphoid tissues can be determined in vitro by assaying their capacity to locomote through Type I collagen. In an attempt to characterize potential causes of microgravity-induced immunosuppression, we investigated the effects of simulated microgravity on human lymphocyte function in vitro using a specialized rotating-wall vessel culture system developed at the Johnson Space Center. This very low shear culture system randomizes gravitational vectors and provides an in vitro approximation of microgravity. In the randomized gravity of the rotating-wall vessel culture system, peripheral blood lymphocytes did not locomote through Type I collagen, whereas static cultures supported normal movement. Although cells remained viable during the entire culture period, peripheral blood lymphocytes transferred to unit gravity (static culture) after 6 h in the rotating-wall vessel culture system were slow to recover and locomote into collagen matrix. After 72 h in the rotating-wall vessel culture system and an additional 72 h in static culture, peripheral blood lymphocytes did not recover their ability to locomote. Loss of locomotory activity in rotating-wall vessel cultures appears to be related to changes in the activation state of the lymphocytes and the expression of adhesion molecules. Culture in the rotating-wall vessel system blunted the ability of peripheral blood lymphocytes to respond to polyclonal activation with phytohemagglutinin. Locomotory response remained intact when peripheral blood lymphocytes were activated by anti-CD3 antibody and interleukin-2 prior to introduction into the rotating-wall vessel culture system. Thus, in addition to the systemic stress factors that may affect immunity, isolated lymphocytes respond to gravitational changes by ceasing locomotion through model interstitium. These in vitro investigations suggest that microgravity induces non-stress-related changes in cell function that may be critical to immunity. Preliminary analysis of locomotion in true microgravity revealed a substantial inhibition of cellular movement in Type I collagen. Thus, the rotating-wall vessel culture system provides a model for analyzing the microgravity-induced inhibition of lymphocyte locomotion and the investigation of the mechanisms related to lymphocyte movement.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.