DNA shuffling of subgenomic sequences of subtilisin.

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.     

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.     

Unified display of Arabidopsis thaliana physical maps from AtDB, the A.thaliana database.

In the past several years, there has been a tremendous effort to construct physical maps and to sequence the genome of Arabidopsis thaliana. As a result, four of the five chromosomes are completely covered by overlapping clones except at the centromeric and nucleolus organizer regions (NOR). In addition, over 30% of the genome has been sequenced and completion is anticipated by the end of the year 2000. Despite these accomplishments, the physical maps are provided in many formats on laboratories' Web sites. These data are thus difficult to obtain in a coherent manner for researchers. To alleviate this problem, AtDB (Arabidopsis thaliana DataBase, URL: http://genome-www.stanford.edu/Arabidopsis/) has constructed a unified display of the physical maps where all publicly available physical-map data for all chromosomes are presented through the Web in a clickable, 'on-the-fly' graphic, created by CGI programs that directly consult our relational database.     

Efficacy of biorational insecticides against chilli thrips,Scirtothrips dorsalis (Thysanoptera: Thripidae), infesting roses under nursery conditions

We evaluated biopesticides based on entomopathogenic fungi, azadirachtin and horticultural oils for the management of the chilli thrips, Scirtothrips dorsalis Hood, an economically significant invasive pest in the United States. Insecticides were applied four times at 7‐ to 10‐day intervals against established S. dorsalis infestations on shrub roses KnockOut^®, Rosa x ‘Radrazz’ under simulated nursery conditions. When applied as stand‐alone treatments, Beauveria bassiana GHA (BotaniGard^® ES), Metarhizium brunneum F52 (Met‐52 EC), a horticultural oil (SuffOil‐X^®) and azadirachtin (Molt‐X^®) at label rates provided significant control, reducing populations of S. dorsalis by 48–71% compared with control over 4–6 weeks. Similar results were observed when the biopesticides were applied in rotation with each other. A conventional standard, spinosad (Conserve^® SC), was consistently the most effective treatment in these studies, reducing thrips populations by >95% overall. In another study, more effective control (87%–92%) was achieved in biopesticide rotation programmes that included spinosad, when compared with those that did not. Results also showed that these biopesticides can be tank‐mixed. However, there was no evidence that B. bassiana or M. brunneum combined with azadirachtin resulted in additive or synergistic control, as neither tank‐mix treatment improved control compared with azadirachtin alone. These findings highlight the potential use of biopesticides in rotation programmes with conventional insecticides to manage S. dorsalis on roses. Biopesticides evaluated in this study can be incorporated into an IPM programme for roses.     

Simultaneous PET-MRI: a new approach for functional and morphological imaging

Noninvasive imaging at the molecular level is an emerging field in biomedical research. This paper introduces a new technology synergizing two leading imaging methodologies: positron emission tomography (PET) and magnetic resonance imaging (MRI). Although the value of PET lies in its high-sensitivity tracking of biomarkers in vivo, it lacks resolving morphology. MRI has lower sensitivity, but produces high soft-tissue contrast and provides spectroscopic information and functional MRI (fMRI). We have developed a three-dimensional animal PET scanner that is built into a 7-T MRI. Our evaluations show that both modalities preserve their functionality, even when operated isochronously. With this combined imaging system, we simultaneously acquired functional and morphological PET-MRI data from living mice. PET-MRI provides a powerful tool for studying biology and pathology in preclinical research and has great potential for clinical applications. Combining fMRI and spectroscopy with PET paves the way for a new perspective in molecular imaging.     

Radiolabeling rhesus monkey CD34+ hematopoietic and mesenchymal stem cells with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for microPET imaging

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34+ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34+ hematopoietic cells were radiolabeled with 0 to 40 microCi/mL 64Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34+ cells radiolabeled with 20 microCi/mL and rhMSCs radiolabeled with 10 microCi/mL 64Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34+ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 microCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50x10(4) CD34+ cells (1.1 pCi/cell) and 6.25x10(3) rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34+ hematopoietic cells and rhMSCs can be safely radiolabeled with 64Cu-PTSM without adverse cellular effects.     

Trade-offs in plant responses to herbivory influence trophic routes of production in a freshwater wetland

Responses of aquatic macrophytes to leaf herbivory may differ from those documented for terrestrial plants, in part, because the potential to maximize growth following herbivory may be limited by the stress of being rooted in flooded, anaerobic sediments. Herbivory on aquatic macrophytes may have ecosystem consequences by altering the allocation of nutrients and production of biomass within individual plants and changing the quality and quantity of aboveground biomass available to consumers or decomposers. To test the effects of leaf herbivory on plant growth and production, herbivory of a dominant macrophyte, Nymphaea odorata, by chrysomelid beetles and crambid moths was controlled during a 2-year field experiment. Plants exposed to herbivory maintained, or tended to increase, biomass and aboveground net primary production relative to controls, which resulted in 1.5 times more aboveground primary production entering the detrital pathway of the wetland. In a complementary greenhouse experiment, the effects of simulated leaf herbivory on total plant responses, including biomass and nutrient allocation, were investigated. Plants in the greenhouse responded to moderate herbivory by maintaining aboveground biomass relative to controls, but this response occurred at the expense of belowground growth. Results of these studies suggest that N. odorata may tolerate moderate levels of herbivory by reallocating biomass and resources aboveground, which in turn influences the quantity, quality and fate of organic matter available to herbivores and decomposers.