Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.     

Factors associated with a rust infection (Sphenosphora saphena) in an epiphytic orchid (Tolumnia variegata)

The interaction of a rust infection (Sphenosphora saphena) with the orchid Tolumnia (Oncidium) variegata was studied in northern Puerto Rico to determine which intrinsic and extrinsic factors are associated with the development of the infection. The relationship between anatomical and morphological characters, life-history stage, density, herbivory, horizontal and vertical location, and frequencies of allo- and autoinfection were investigated. Stomatal densities and plant age were not associated with the incidence of disease. However, larger plants were more frequently infected than small ones. Censuses showed that seedlings were affected less frequently than adults, but inoculations demonstrated that both stages were equally susceptible to infection. Infection frequencies varied considerably over time and space. Population density was positively correlated with incidence of infection in two out of five censuses within a 3-year period. Vertical distribution of the orchid was not related to the incidence of infection. Infection frequencies were related to site location, but this varied with time. Infections were highly localized, and new shoots were more likely to become infected if the previous shoot was diseased. Herbivory was more frequent among noninfected plants and may impart some degree of resistance. Our results suggest that the infection frequencies are associated more strongly with infection histories, plant size, and ecological conditions rather than with age, life histories, or anatomical traits of the orchid.     

Gynogenesis in the vine cacti <Emphasis Type="Italic">Hylocereus</Emphasis> and <Emphasis Type="Italic">Selenicereus</Emphasis> (Cactaceae)

Gynogenesis was investigated on the allotetraploid Selenicereus megalanthus and the diploid Hylocereus polyrhizus and Hylocereus undatus vine cactus species. Unpollinated ovules from developing flower buds containing microspores at middle uninucleate developmental stage were cultured on MS basal medium containing 2,4-D/TDZ with different sucrose concentrations. Ovule size increased under dark culture conditions in all the three species and the level of response was species and sucrose concentration dependent. The best responses were achieved in the two S. megalanthus accessions, E-123 and J-80, at 0.18 and 0.26 M sucrose. Only ovule enlargement was obtained in H. undatus and both ovule enlargement and callus were obtained in H. polyrhizus. Development in both species ceased and embryoids were not formed. Plant regeneration was directly and indirectly obtained in both S. megalanthus accessions. Ploidy level was determined for a total of 29 S. megalanthus gynogenic plants using flow cytometry: 15 were found to be dihaploid (plants with the gametophytic chromosome number) and the other 14 were found to have higher ploidy levels. This is the first report of successful gynogenesis in Cactaceae. The dihaploids of S. megalanthus successfully produced by ovule culture techniques opens new perspectives in vine cacti breeding.     

Age class influence on the yield of edible fungi in a managed Mediterranean forest

Lack of information and difficulty in predicting wild edible sporocarp yields is blocking its integration in forest management. In the Mediterranean area, this nontimber forest product has increased its market value, consumption demand, and interest over the last decade. In this work, sampling year and stand age effects are analyzed in order to advance knowledge of edible fungi community structure, dynamics, and production. Weekly autumnal sporocarp monitoring was performed from 1997 to 2011 in a Pinus pinaster managed forest in central Spain. After applying a random stand age-stratified survey, 21 plots of 150 m² have been set with three per stand age class. The forest age classes have been defined as follows: 0–10 years, mixture of parent and regenerated trees, 11–20, 21–40, 41–60, 61–90, and over 90 years. A total of 153 species belonging to 56 genera were recorded, 55 of which are edible. The production of edible sporocarps was 19.8 kg ha^?1, representing 31 % of total production. Sporocarp production presents a sharp interannual variability with autumns 62 times more productive than others. The most abundant edible species in terms of fresh weight per hectare has been Lactarius deliciosus with 7.0 kg ha^?1. Edible fungi yields registered a significant decline in 10 years following regenerative cutting. The presence of parent trees significantly increases production with regard to the first class. The highest production of edible species occurs in the middle age, 41–60 years, and in the following classes, a decrease is produced. L. deliciosus production registered differences with age, manifesting in a high yield in young stands (11–20 years) and significant recovery in woodlands near to the cutting.     

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.     

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.