Papers by Jules Beekwilder
American Journal of Roentgenology, Dec 1, 1991
ton, vA 22091. Address reprint requests to J. Sunshine. Because of concern about pressures from h... more ton, vA 22091. Address reprint requests to J. Sunshine. Because of concern about pressures from hospitals for changes in their contracts with radiology practices, the American College of Radiology undertook a stratified sample survey of radiology practices and their contracts with hospitals in late 1989 and early 1990. After three remailings, the survey obtained 904 valid responses for a 64% response rate. It found (weighting data to represent all radiology practices in the nation) that 91% of radiology practices provide services in hospitals. There are contracts at 57% of these hospital sites, and hospitals use tax-exempt bond financing in 54% of the sites with contracts. One year is the most common duration of contracts, and 90 days is the most common cancellation period (89% of contracts can be canceled during the contract's term). Eighty-six percent of contracts are exclusive; 73% are automatically renewable ("evergreen"). Separate billing exists in well over 90% of hospftals with contracts. Two percent of contracts require radiologists to provide equipment 4% require them to provide support staff. There are fixed-fee arrangements in 9% of contracts and percent compensation arrangements in 3%. HospItals share in radiologists' fees as a charge for contractual privileges in 5% of contracts. Twenty-seven percent of contracts require hospital approval for radiologists' fee increases; 19% require radiologists to participate with Blue Shield or Medicare. If a contract characteristic is predominant nationally, it is predominant in every region, for solo, small, medium, and large practices, and whether or not hospitals use tax-exempt bond financing. There is, however, some modest variation in frequency of contract provisions. Multivariate analysis shows that most such variation reflects true effects of region and other variables, not statistical artifacts. Region was a statistically significant determinant of 1 1 of 17 contract characteristics studied. Practice size was a statistically significant determinant of only about half as many characteristics. The survey found some interference in the independence of radiology practices (e.g., required hospftal approval of fee increases), questionable clauses (e.g., mandated provision of equipment), and even illegal ones (hospftals charging a fee for contract privileges). Knowledge of the survey findings can make individual radiology practices more effective in negotiations with hospftals and assist the profession in forming a strategy to oppose disadvantageous and illegal provisions.
Please cite this article in press as: Beekwilder, J., et al., Polycistronic expression of a -car... more Please cite this article in press as: Beekwilder, J., et al., Polycistronic expression of a -carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to -ionone production. J. Biotechnol. (2014), http://dx.
Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide... more Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules. Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 μM). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 μM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 μM were reached. The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.
Carboxypeptidase activity participates in the protein digestion process in the gut of lepidoptera... more Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent. r
For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies... more For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at −20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°C.
Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootk... more Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential.
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Papers by Jules Beekwilder