Virtual resource development in the glycosciences

The development of Internet-based virtual resources is a relatively new area of scientific and technical activity that is currently undergoing rapid expansion. Major factors fuelling recent growth include the emergence of multimedia capabilities through the rapid evolution of the World Wide Web, the reduction in cost of high quality personal computers and graphics workstations and the provision of mass-marketed provider services. Prior to 1995 the presence of Internet resources in the glycosciences was virtually non-existent. Existing scientific knowledge was primarily made available on the Net through the provision of databases from gopher and ftp sites. A particular example in the glycosciences is the Carbbank database of biological carbohydrate sequences. We will describe here our efforts in 1994–95 in establishing The Glycoscience Network (TGN, http://bellatrix.pcl.ox.ac.uk/TGN/). These activities included the establishment of a newsgroup, mailing lists, Web resources and the running of the First Electronic Glycoscience Conference (EGC-1, http://bellatrix.pcl.ox.ac.uk/egc/). EGC-1 included many novel initiatives in the glycosciences including electronic posters and papers, a Virtual Conference Centre, a Web-based hyperglossary, Virtual Trade and Employment Centres, refereed electronic publishing, and the creation of a Virtual Reality Gallery. We would like to look towards the near future and discuss several initiatives in virtual resource creation that we believe will have significant scientific impact on the glycosciences including the development of bioinformatics-based servers, sophisticated interactive databases, and videoconferencing. Furthermore, we cherish the belief that these resources will foster international scientific collaboration and progress of an extent never previously possible. Finally, we indulge in speculation and make some suggestions on the form and long-term impact of Glycoscience Virtual Resources. We predict that their development may completely reconstruct the scientific environment that we work in as scientists and we reflect on the probable benefits and pitfalls to be encountered.This paper was presented at the First Electronic Glycoscience Conference (EGCI) on the World Wide Web, September 1995.     

Temperature and host effects on key morphological characters of Hemisarcoptes cooremani and Hemisarcoptes malus (Acari: Hemisarcoptidae)

We investigated the influence of temperature and resources on six morphological characters thought to distinguish two North American species of Hemisarcoptes (H. malus and H. cooremani). We raised mites at three temperatures (15, 24 or 30°C) and on two different scale insect prey (Aspidiotus nerii or Aonidiella aurantii) which were cultured on two different substrates (potato tubers and lemon fruit). In general, the temperature had more of an influence on the character variation than did the host and the highest temperature resulted in the smallest mean body size. The two species did not respond to changes in the temperature or host in a symmetrical fashion. The temperature significantly influenced the lengths of the external scapular setae (sce) of H. malus and the sce and first coxal setae (1a) of H. cooremani. The relative lengths of the setae sce and 1a of H. cooremani were significantly influenced by the temperature, while the host type significantly influenced the paraproctal setae (ps ₂). Major-axis regressions indicated that H. cooremani had an absolutely longer mean setal length for 1a and for ps ₂, than H. malus, but a relatively shorter sce. An ANOVA of the size-adjusted shield characters of H. malus resulted in non-significant effects of the temperature or host on either the prodorsal shield area or and area in H. cooremani. Regressions of the shield area (size) on body length, resulted in two clear groupings by species. Hemisarcoptes cooremani had an absolutely larger shield area and increased circumference (complexity), as compared to H. malus. A plot of the shield circumference in relation to the shield area, however, resulted in a single trajectory, indicating that shield complexity is an allometric consequence of an increase in body size in both species. Though characters can be influenced significantly by environmental parameters, the species-specific patterns of some characters of North American Hemisarcoptes are distinctive enough to allow diagnosis and identification.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

A general assay for restriction endonucleases and other DNA-modifying enzymes with plasmid substrates

A procedure for measuring the activities of enzymes that alter the covalent structure of DNA is described. The assay utilizes covalently closed circles of DNA as the substrate and yields quantitative data on the fraction of this DNA converted to both open-circle and linear forms.     

Differential surface characteristics of M cells from mouse intestinal Peyer''s and caecal patches

Summary The mouse caecal patch is located near the blind end of the caecum, and consists of a group of lymphoid follicles. In common with the Peyer's patches, the follicle-associated epithelium overlying these follicles is largely composed of enterocytes, goblet cells and membranous epithelial (M) cells. Each of these types of cell was readily identified by electron microscopy, although caecal patch enterocytes and M cells were morphologically distinct from those of the Peyer's patches. Staining for alkaline phosphatase activity demonstrated that the majority of caecal follicle-associated epithelial cells were alkaline phosphatase-negative, positive cells consisting of a mixture of enterocytes and M cells. In contrast, it has previously been found that Peyer's patch enterocytes are positive for alkaline phosphatase while the M cells are relatively lacking in alkaline phosphatase activity. Lectin histochemistry revealed that surface glycoconjugate expression differs between the caecal and Peyer's patch follicle-associated epithelial cells; in particular, the characteristic staining of Peyer's patch M cells by Ulex europaeus agglutinin 1 was absent on the caecal patch follicle-associated epithelium. These altered surface characteristics indicate that the development of the caecal patch follicle-associated epithelial cells is influenced by the local environment, and these altered properties may be indicative of modified functional roles for the cells at this site.