James K. Waters

Microwave digestion and ultrasonic nebulization for determination of boron in animal tissues by inductively coupled plasma atomic emission spectrometry with internal standardization and addition of mannitol

Determinations of boron in animal tissues were performed by inductively coupled plasma atomic emission spectrometry (ICP-AES) with an ultrasonic nebulizer. The method is based on the microwave digestion of tissues with HNO 3 –H 2 O 2 and the use of mannitol as a modifier. It was found that mannitol can significantly enhance the analytical sensitivity, improve the precision and minimize the memory effect for the determination of boron. Acid effect and matrix effects were controlled by using the Be I 234.861 nm line as the internal standard. Six animal tissue samples, including two NIST standard reference materials (Oyster Tissue and Bovine Liver), were analyzed to test the reliability of the method. A limit of detection (3 σ ) of 0.7 µg l -1 and recoveries of added boron from selected matrices between 95.0 and 100.2% were obtained.

Proteomic analysis of soybean nodule cytosol

An isolation procedure for soybean (Glycine max L. cv Williams 82) nodule cytosol proteins was developed which greatly improved protein resolution by two-dimensional polyacrylamide gel electrophoresis. The most abundant proteins were selected and analyzed by mass spectrometry. The identified proteins were categorized by function (% of total proteins analyzed): carbon metabolism (28%), nitrogen metabolism (12%), reactive oxygen metabolism (12%) and vesicular trafficking (11%). The first three categories were expected based on the known physiological functions of the symbiotic nitrogen fixation process. The number of proteins involved in vesicular trafficking suggests a very active exchange of macromolecules and membrane components. Among the 69 identified proteins were the enzymes of the three carbon portion of glycolysis, which were further characterized to support their roles in the sucrose synthase pathway to provide malate for the bacteroids. Proteomic analysis provides a functional tool by which to understand and further investigate nodule function.

piggyBac Transposon plus Insulators Overcome Epigenetic Silencing to Provide for Stable Signaling Pathway Reporter Cell Lines

Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular.

Further evidence for the uniformity of the microsymbiont population from soybean nodules.

Summary The microsymbiont population in soybean root nodules (Glycine max L.cv.Williams 82 inoculated with Bradyrhizobium japonicum 2143) was characterized during symbiotic development.The microsymbiont population obtained from nodules of plants at different ages was subjected to sucrose density centrifugation.The microsymbionts were fractionated according to their buoyant densities and characterized with regard to several enzyme activities and O2 uptake.No differences were observed in the specific activities of these enzymes in the microsymbionts obtained at various buoyant densities.This would suggest the absence of discrete subpopulations.These results are consistent with the concept of a single microsymbiont population.

Quality of Intrathecal Baclofen From Different Sources

To compare the quality of intrathecal baclofen obtained from a national compounding pharmacy (AnazaoHealth) with the manufactured product (Lioresal) with regard to accuracy and precision of baclofen concentration, and the content of the baclofen degradation product, 4‐(4‐chlorophenyl)‐2‐pyrrolidinone (PYR).

Determination of total boron in soils by inductively coupled plasma atomic emission spectrometry using microwave‐assisted digestion

Abstract A method is described in which total soil boron (B) was determined by inductively coupled plasma atomic emission spectrometry (ICP‐AES). The method is based on microwave‐assisted digestion of soil samples with nitric acid (HNO3), hydrofluoric acid (HF) and hydrogen peroxide (H2O2). Excess HF was eliminated by adding silicon (IV) oxide (SiO2). The B 208.959 nm line was chosen as the analytical line to avoid the spectral interferences of iron (Fe). A detection limit of 0.0045 mg L‐1 was obtained with the selected analytical line under the optimized operating conditions. Four National Institute of Standards and Technology (NIST) standard reference materials (three soils and one river sediment) and four different type of practical soils were analyzed to test the reliability of the method. The total B concentration in selected samples ranged from 19 to 76 mg kg‐1. The excellent recoveries of the spike (98.5–101%) indicate that the proposed procedure is effective and feasible for the determination of t...

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24–30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.

Fluorescence studies with malate dehydrogenase from Bradyrhizobium japonicum 3I1B-143 bacteroids: A two-tryptophan containing protein

Abstract A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3I1B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or d -malate.

The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

Poster 342: Quality of Intrathecal Baclofen From Different Sources

codes. Preoperative VAS ranged from 1.5-10 (mean 5.94, SD 2.00). Of the patients, 70.7% had more than 6 months of symptoms before evaluation. The population was 41% men, 9.8% older than 65 years old, 2.4% with diabetes, 7.3% actively smoking, and 36.6% on opiates. Of the patients, 39.0% had medial dye spread with TFESI. Medial dye spread is not associated with differential response to the procedure (P .64 by Fisher test, P .59 by Barnard test). Age, gender, diabetes, smoking, and opiates are not associated with differential response to the treatment. However, obese patients (BMI 25) are more likely to respond positively to the injection (OR 9.34 [CI, 1.77-68.3]). Conclusions: TFESI is an effective treatment for radicular back pain. Medial dye spread is not associated with differential response to the procedure. We discovered that BMI is associated with greater pain relief with TFESI. Further studies across institutions will be needed to validate these findings.