N-cyanoimidazole and diimidazole imine: water-soluble condensing agents for the formation of the phosphodiester bond

The reaction of BrCN with imidazole results in the formation of N-cyanoimidazole and diimidazole imine. These compounds were shown to be useful condensing agents for the formation of the phosphodiester bound in aqueous solution.     

Differential cleavage and developmental rates and their correlation with cell numbers and sex ratios in buffalo embryos generated in vitro

In vitro matured and fertilized buffalo oocytes were co-cultured with buffalo oviductal epithelial cells (BOEC) in CRlaa medium. Cleaved embryos were separated according to the time of completion of first cleavage (i.e., before 30 h and after 30 h post insemination) and cultured for 5 to 10 d and allowed to develop to the blastocyst stage. Zygotes cleaving before 30 h were termed fast-cleaving while those cleaving after 30 h were termed slow-cleaving. The results indicated that fast-cleaving embryos are more likely to develop into blastocysts (25%) than slow-cleaving embryos (7.8%). The quality and viability of fast-cleaving and fast-developing blastocysts was found to be better than that of slow-cleaving, slow-developing blastocysts as judged by cell numbers (67.7 +/- 3.7 vs 35.2 +/- 2.1). However, the mitotic index was not different between the 2 groups. The sex of fast-developing and slow-developing blastocysts was determined via the polymerase chain reaction (PCR) to correlate the rate of embryonic development with the sex ratio of the embryos. Embryos produced by Bull 293 and Bull M-82, irrespective of their being fast or slow-developing, gave rise to more females and males, respectively. From these results, we suggest that there may be a sire effect on sex ratio of in vitro produced buffalo embryos.     

Low multiplicity infection of insect cells with a recombinant baculovirus: The cell yield concept

In vitro infection of insect cells with baculoviruses is increasingly considered a viable means for the production of biopesticides, recombinant veterinary vaccines, and other recombinant products. Batch fermentation processes traditionally employ intermediate to high multiplicities of infection necessitating two parallel scale-up processes-one for cells and one for virus. In this study, we consider the use of multiplicities of infection as low as 0.0001 plaque-forming units per cell, a virus level low enough to enable infection of even large reactors (e.g., 10 m(3)) directly from a frozen stock. Using low multiplicities in the Sf9/beta-gal-AcNPV system, recombinant protein titers comparable with the maximum titer observed in high multiplicity infections were achieved. Cultures yielding the maximum titer were characterized by reaching a maximum cell density between 3 and 4 x 10(9) cell L(-1). This optimal cell yield did not depend on the multiplicity of infection, supporting the existing view that batch cultures are limited by availability of substrate. Up to a certain cell density, product titer will increase almost linearly with availability of biocatalyst, that is, cells. Beyond this point any further cell formation comes at the expense of final product titer. Low multiplicity infections were found not to cause any significant dispersion of the protein production process. Hence, product stability is not a major issue of concern using low multiplicities of infection. The sensitivity to initial conditions and disturbances, however, remains an issue of concern for the commercial use of low multiplicity infections. (c) 1996 John Wiley & Sons, Inc.     

Cell Expansion and Tracheary Element Differentiation Are Regulated by Extracellular pH in Mesophyll Cultures of Zinnia elegans L

The effects of medium pH on cell expansion and tracheary element (TE) differentiation were investigated in differentiating mesophyll suspension cultures of Zinnia elegans L. In unbuffered cultures initially adjusted to pH 5.5, the medium pH fluctuated reproducibly, decreasing about 1 unit prior to the onset of TE differentiation and then increasing when the initiation of new Tes was complete. Elimination of large pH fluctuations by buffering the culture medium with 20 mM 2-(N-morpholino)ethanesulfonic acid altered both cell expansion and TE differentiation, whereas altering the starting pH of unbuffered culture medium had no effect on either process. Cell expansion in buffered cultures was pH dependent with an optimum of 5.5 to 6.0. The direction of cell expansion was also pH dependent in buffered cultures. Cells elongated at pH 5.5 to 6.0, whereas isodiametric cell expansion was predominant at pH 6.5 to 7.0. The onset of TE differentiation was delayed when the pH was buffered higher or lower than 5.0. However, TEs eventually appeared in cultures buffered at pH 6.5 to 7.0, indicating that a decrease in pH to 5.0 is not necessary for differentiation. Very large TEs with secondary cell wall thickenings resembling metaxylem differentiated in cultures buffered at pH 5.5 to 6.0, which also showed the greatest cell expansion. The correlation between cell expansion and delayed differentiation of large, metaxylem-like TEs may indicate a link between the regulatory mechanisms controlling cell expansion and TE differentiation.     

'Concealed' antigens: expanding the range of immunological targets

For many parasites, the interaction between the immunogenicity of the parasite and the immunological response of the host is a dynamic equilibrium that allows both to survive, albeit often with severe consequences for the host. Vaccines, if intended as a means of parasite control, are unlikely to be generally successful if they do no more than mimic an immunological equilibrium that would be reached after natural exposure to the parasites. The situation must be tipped in favour of the host. It has been difficult to find ways around this impasse. One approach has been receiving practical attention over recent years, an approach that Peter Willadsen, Craig Eisemann and Ross Tellam have called vaccination against 'concealed' antigens.