Characterization of biotechnological processes and products using high-performance liquid chromatography (HPLC) IV. SEC,HIC, and IEC separations of proteins

Proteins are separated by means of size-exclusion (SEC), hydrophobic-interaction (HIC) and ion-exchange chromatography (IEC). Analytical and semipreparative HPLC glass columns are the basis of the chromatographic analyses. Using a short (100 × 3.8 mm i.d.) column packed with Si 200 Polyol standard of different molecular weights (ovalbumin, MW = 43 000; chymotrypsinogen A, MW = 25 000; ribonuclease, MW = 13 700 and contrycal, MW = 6512) could be distinguished. Basic proteins (e.g., chymotrypsinogen A, cytochrome C) are separated on aluminium oxide (Li-Chrosorb Alox T) by cation-exchange chromatography. Correlations between the retention times of proteins and their isoelectric points or the buffer concentration of the mobile phase are investigated. Furthermore, two examples of liquid chromatographic purification procedures for enzymes of biotechnological interest are demonstrated. One enzyme extract (thermostable protease) is separated by hydrophobic-interaction chromatography, another one (β-galactosidase from a thermophilic microorganism) is purified on a weakly basic anion-exchange resin (pore size: 130 nm) based on a styrene-divinylbenzene copolymer.     

Synthesis and Perkow Reaction of Uridine Derivatives

Abstract

Several uridine derivatives with 0-p-toluene sulfonyl groups in 2′- or 3′-position were prepared and their oxidation to the corresponding uloses studied. By Perkow reaction these α-tosyl ketones led to enol-phosphates which were subjected to hydrogenation and hydrolysis. This procedure represents a facile access to uridine derived deoxy uloses.     

Artificial N-functionalized UDP-glucosamine analogues as modified substrates for N-acetylglucosaminyl transferases

Analogues of UDP-GlcNAc modified at the 2-acetamido group of the GlcNAc moiety were prepared in order to study their role in the mechanism of N-acetylglucosaminyl transferase mediated glycosylation reactions. The structural analogues with N-formyl-, N-propionyl-, N-butyryl- and N-isobutyryl-groups were synthesized, utilizing the morpholidate coupling method starting from d-glucosaminyl-1-phosphate after selective N-acylation of its amino group with the appropriate N-acyloxysuccinimide esters as well as a chlorinated formylformiate.     

The status of fishways in Canada: trends identified using the national CanFishPass database

The disruption of river connectivity through the construction of barriers used for hydropower development and water control purposes can severely damage river ecosystems, reduce the quality of fish habitat, and prevent the upstream migration of fishes. Fishways function as a means of passage around barriers for fish migrating both upstream and downstream. In 2009, the CanFishPass project was initiated in a partnership with Fisheries and Oceans Canada and Carleton University to create a searchable database containing specific information on fishways in Canada built to enable upstream passage. In this paper we evaluate the information gathered in the CanFishPass database to identify trends concerning fishways and fish passage in Canada, yielding, we believe, the first national-scale trend analysis related to fishways anywhere in the world. Although CanFishPass may not include all fishways in Canada, our analysis identified 211 which are primarily located along the coasts and along major rivers and water bodies such as the Great Lakes. British Columbia has the largest number of fishways in Canada (62) and Prince Edward Island has the fewest (2). The most popular type of fishway is the pool and weir fishway (85), followed by vertical slot (37) and Denil type fishways (23). Fishway construction has proceeded at a steady rate since the 1970’s, although there has been an increase in the number of nature-like fishways since the year 2000. The majority of fishways are installed to pass salmonids in Canada, although some fishways on warmwater systems pass large components of the fish community. Only 9 % of the fishways in Canada have been studied using methods that enable proper evaluation of biological effectiveness. We recommend that evaluations be carried out at new and existing fishways and that these evaluations enable the determination of attraction and passage efficiency. Additionally, we recommend that future fishway projects and evaluations in Canada be advised to submit details of their work to CanFishPass so that knowledge of these fishways is centralized. Similar efforts on a global scale could lead to opportunities to identify patterns in fishway design and biological effectiveness that would ultimately inform decision making and improve connectivity where deemed necessary.     

A Multi-country Study of the Household Willingness-to-Pay for Dengue Vaccines: Household Surveys in Vietnam,Thailand, and Colombia

BackgroundThe rise in dengue fever cases and the absence of dengue vaccines will likely cause governments to consider various types of effective means for controlling the disease. Given strong public interests in potential dengue vaccines, it is essential to understand the private economic benefits of dengue vaccines for accelerated introduction of vaccines into the public sector program and private markets of high-risk countries.Conclusions/SignificanceKnowing that dengue vaccines are not yet available, our study provides critical information to both public and private sectors. The study results can be used to ensure broad coverage with an affordable price and incorporated into cost benefit analyses, which can inform prioritization of alternative health interventions at the national level.     

Syntheses of imido-substituted glycosans and their photocyclisation towards highly functionalised heterotricycles.

Reaction of O-protected amino-1,6-anhydro-beta-D-hexopyranoses with succinic or glutaric anhydride and subsequent intramolecular acylation afforded the succinimido- and glutarimido-substituted glycosans. Irradiation with UV light of 254 nm wavelength led to gamma-hydrogen abstraction at the pyranose ring by the excited carbonyl function. The stereoselective recombination of the resulting 1,4-diradicals gave annelated azetidinols, which fragmented by a retrotransannular ring opening reaction to give the glycosan-annelated azepanedione and azocanedione systems, respectively.     

Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection.     

Hydrolase-catalyzed β-mannosylations

Transmannosylations catalysed by -mannosidase from snail viscera or -galactosidase from Aspergillus oryzae were accomplished with 4-nitrophenyl D -mannopyranoside as donor substrate. With suitable hydrophobic acceptor molecules preferentially 1-4-linked disaccharides were obtained. The activities of both glycosidases in buffer cosolvent mixtures were determined, and conditions for their immobilization were elaborated and optimized. A model of the enzymic transfer mechanism is suggested.