Quantitative analysis of a synthetic peptide, NR58-3.14.3, in serum by LC-MS with inclusion of a diastereomer as internal standard.

A method for quantifying an intramolecularly linked all-d-amino acid peptide, NR58-3.14.3, in rat serum by LC-MS using selected ion monitoring with inclusion of a diastereomer as internal standard was developed. The reproducible quantitation of multiply charged compounds by LC-MS using single ion or selective reaction monitoring is often a challenge as the intensity ratio of the ions in a series of different charge states can vary. Good precision was obtained in the selected ion monitoring mode by integrating the summed ion currents of the singly, doubly, and triply charged molecular ions. Since stable isotope analogs are costly and integration of residual unlabeled material can be of concern, a diastereomer of NR58-3.14.3, NR58-3.14.5, was used as internal standard. The diastereomers were indistinguishable by electrospray MS, but fully separated by reversed-phase LC. Consequently, interference due to isotopic impurities or coelution was not encountered. The calibration plot was linear throughout a concentration range of 0.2 to 200.0 microg/ml (r(2) = 0.9996). Intraday precision of the standards analyzed was less than 12% RSD over the calibration range and the accuracy within +/-11% RE. Serum pharmacokinetics were in good agreement with the pharmacokinetic profiles of small, ionic, and polar molecules.

Quantification of jasmonic acid, methyl jasmonate, and salicylic acid in plants by capillary liquid chromatography electrospray tandem mass spectrometry.

Jasmonic acid, methyl jasmonate, and salicylic acid have been reported to occur in plants and are thought to be essential for the regulation of systemic defense responses. This work describes a method for the quantitation in plant tissue of these regulators by reverse-phase capillary liquid chromatography interfaced to an electrospray tandem mass spectrometer. Inclusion during sample preparation of hydrogenated and/or deuterated internal standards corresponding to analogs of the regulators compensated for sample loss and permitted quantitation using the multiple reaction monitoring mode of the mass spectrometer. The free acids were analyzed in a negative-ion mode, whereas methyl jasmonate was analyzed in a positive-ion mode. Using these procedures an extract of fresh hybrid poplar leaves was found to contain per gram of leaf tissue 2.6 micrograms of jasmonic acid, 1.3 micrograms of methyl jasmonate, and 31.0 micrograms of salicylic acid. The techniques used should be applicable to other plant materials.

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco.

Wounding hybrid poplar (Populus trichocarpa x P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of the win6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases. Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induces win6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that a win6 promoter-beta-glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplar win6 promoter has regulatory elements that are responsive to 'wound signals' in the heterologous host. In addition, win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur for win6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.

Endonuclease activity associated with purified simian virus 40 virions.

Purified simian virus 40 has associated with it an endonuclease activity which converts form I (double-stranded, circular) simian virus 40 deoxyribonucleic acid to a nicked form that sediments as a homogeneous peak in alkaline sucrose gradients. The enzyme is dependent on magnesium ions for activity and is completely inhibited by ethylenediaminetetraacetic acid (0.02 m) or heat (80 C for 10 min). In tris(hydroxymethyl)aminomethane-hydrochloride buffer it exhibits optimal activity between pH 6.7 and 7.1 at 37 C. Gel electrophoretic analysis of purified, disrupted virus indicates the absence of detectable host cell protein contamination.

Analysis of simian virus 40-induced transformation of hamster kidney tissue in vitro. 8. Induction of infectious simian virus 40 from virogenie transformed hamster cells by amino acid deprivation or cycloheximide treatment.

Clones of virogenic simian virus 40 (SV40)-transformed hamster kidney cells were exposed to medium deficient in the essential amino acids leucine, arginine, or methionine. Infectious virus was induced after deprivation periods of from 24 to 32 hr. The highest yields of infectious SV40 were obtained from cultures deprived for 3 to 4 days. Infectious virus was also induced in cells that were treated with the metabolic inhibitor cycloheximide. Pulse labeling experiments revealed that both protein synthesis and deoxyribonucleic acid (DNA) synthesis were inhibited by concentrations of cycloheximide which were effective for virus induction. It is suggested that inhibition of protein synthesis by either amino acid deprivation or by cycloheximide was responsible for the induction of infectious virus from virogenic cells. We postulate that the inhibition of protein synthesis caused a temporary inhibition of DNA synthesis which resulted in the induction of infectious virus.