Characterization of short-term temperature, exposure, and solubilization effects on library compound quality

Many compound collections are stored under the same temperature conditions, which can limit flexibility by increasing the processing time required for high-demand compounds. In this study, the authors wanted to evaluate the impact of a hybrid-storage approach where high-demand compounds are stored for a shortened time period at room temperature to expedite processing operations. The use of a Covaris adaptive-focused acoustics platform was also characterized as a potential enhancement or alternative to storage at elevated temperatures. This study evaluated the impact of temperature, exposure, and solubilization on overall compound quality for short-term storage. A small library of 25 representative compounds was evaluated over an 18-week period to monitor the change in purity and concentration by high-performance liquid chromatography with ultraviolet detection. The authors concluded that temperature had a significant impact on compound concentration, and the effects due to exposure cycles were minimal. A storage time of 12 weeks at room temperature resulted in minimal compound loss, but storage times beyond this would be unacceptable because of a >20% decrease in concentration. Finally, the acoustic solubilization protocol also increased the number of compounds at the target concentration with no impact on overall purity, leading to a potential for increased storage times at frozen temperatures.     

Radioimmunoassay for quantitative determination of morphine in capsules of Papaver somniferum

A radioimmunoassay (RIA) procedure for the determination of pmol quantitites of morphine in capsule samples of Papaver somniferum was developed. An antiserum developed against a conjugate of morphine-3-hemisuccinate-BSA was relatively specific for morphine and possessed moderated cross-reactivity with codeine and mild cross-reactivity with thebaine, but none with narceine, papaverine, or noscapine. The standard curve was linear over a range of 0.01–0.20 ng. This assay allows for the rapid, sensitive and precise determination of morphine in unpurified aqueous extracts of capsule samples. The amounts of morphine in the aqueous extracts determined by radioimmunoassay were validated by high performance liquid chromatography (HPLC). The two methods show a high correlation coefficient (r = 0.98) with no significant difference in determinations of morphine content by RIA and HPLC.     

THE DEVELOPMENT OF THE SEED OF ABUTILON THEOPHRASTI I. OVULE AND EMBRYO

Winter , Dorothy M. (Iowa State U., Ames.) The development of the seed of Abutilon theophrasti. I. Ovule and embryo. Amer. Jour. Bot. 47(1): 8–14. Illus. 1960.—Abutilon theophrasti Medic, is a widespread annual weed which produces an abundance of seed in capsules which mature within 20 days after pollination. Ovule differentiation may be observed at least 8 days before anthesis when a sporogenous cell becomes evident and 2 integuments are initiated. An 8-nucleate embryo sac is produced from the chalazal megaspore approximately 2 days before anthesis. The outer integument of the mature campylotropous ovule consists of 2 cell layers, the inner integument has 6 to 15 cell layers. The initially free-nucleate endosperm becomes cellular betwen 3 and 7 days after pollination. At maturity a thin layer of gelatinous endosperm encases the embryo. The Asterad-type proembryo of Abutilon has a stout suspensor and develops rapidly. Four days after pollination cotyledons are initiated; 4 days later a leaf primordium is evident. Fifteen days after pollination the embryo, which has essentially completed its growth, consists of a large hypocotyl with root promeristem and root cap at its basal end, and 2 flat, folded, leaflike cotyledons enclosing a small epicotyl at its upper end. The epicotyl consists of an embryonic leaf and a stem apex.     

Potential for control of seedling blight of wheat caused by Fusarium graminearum and related species using the bacterial endophyte Bacillus mojavensis

Fusarium-infected wheat seed decreases germination, seedling emergence, and causes post emergence seedling death, and can contribute to wheat scab and ear rot of maize, with consequent production of mycotoxins such as deoxynivalenol and zearalenone. Current seed treatments have proved ineffective in controlling seedling blight and scab. A patented endophytic bacterial strain, Bacillus mojavensis RRC 101, and several other strains of this species were studied to determine in vitro antagonism to some Fusarium species and to assess the potential of this bacterium to serve as an endophytic biocontrol for seedling blight of wheat produced by species within the F. graminearum complex, as well as other species of Fusarium. Seedling emergence and seed germination were two tests used as indicators of seedling blight. These tests were conducted in growth rooms with two wheat cultivars highly susceptible to scab, Norm and Pioneer 2552, and other cultivars with varying resistance to scab. The results indicated that all strains of this bacterium were antagonistic in vitro to the strains of F. graminearum and its seven related species, as well as four strains of F. pseudograminearum and the two strains of F. verticillioides. Germination of the highly scab susceptible cultivar 2552 was increased from 77 to 97% when planted in soil containing a mixed inoculum of F. graminearum and related species. Seedling emergence in the very susceptible wheat cultivar Norm increased from 20 to 82% when treated with the bacterium. The data indicated that inoculating wheat kernels with B. mojavensis reduced seedling blight of wheat produced by F. graminearum and related Fusarium species indicating the potential for this bacterium as a biocontrol under field condition.     

Characterization of fall armyworm mitochondrial DNA (Lepidoptera: Noctuidae)

Mitochondrial DNA from the fall armyworm, Spodoptera frugiperda (J.E. Smith), was cloned and characterized using restriction enzyme mapping. Genome size is approximately 16.3 kilobase (Kb), consistent with that of most animals. Three fragments, 8.1 Kb, 6.2 Kb, and 2.0 Kb, were produced by digestion with restriction enzyme Xbal and cloned into a pUC19 vector. Twenty-nine restriction enzymes were used to generate a detailed physical restriction enzyme map of the three cloned fragments. These data represent the first detailed characterization of a lepidopteran mitochondrial genome. © 1992 Wiley-Liss, Inc.     

Some Properties of Heat-Resistant and Heat-Sensitive Strains of Clostridium perfringens I. Heat Resistance and Toxigenicity

Heat resistance at 100 C (D-values), sporulating ratios, toxigenicity for mice, and lecithinase activity (as micrograms per milliliter of enzyme, ascertained by the lecithovitellin reaction) were determined for four strains of Clostridium perfringens. A definite inverse relationship between thermal resistance and toxigenicity was found. The D-values ranged from 17.6 for the most heat-resistant strain to 0.3 for the strain possessing the least heat resistance, with corresponding lecithinase activities from 25 to 133 mug/ml of enzyme. The sporulating ratios did not differ greatly between the strains. The heat stability of the toxin was greater at 100 C than at 75 C. There was a noticeable difference between the heat stabilities of the toxin in the culture fluids of the heat-sensitive and heat-resistant strains at pH 7.0 when the toxic filtrates were held at 100 C. At a holding temperature of 75 C, a similar but lesser difference was observed at pH 5.5. Heat resistance and lecithinase activity did not change when a substrain of the least heat-resistant parent strain was obtained through heat selection by a single transfer, or when the most heat-resistant strain was transferred serially 12 times.     

Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial

Background

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF.

Methodology/Principal Findings

Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-α and IFN-γ. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation.

Conclusions/Significance

These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells'' vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials.