Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Echinococcus multilocularis: responses to infection in Mongolian gerbils

Mongolian gerbils (Meriones unguiculatus) inoculated intraperitoneally with three acephalic cysts of Echinococcus multilocularis were very susceptible to infection. Aspects of the responses of gerbils to this infection were examined to determine if they could be related to the progress of the infection. Hematologic changes observed during the infection included anemia, reticulocytosis, lymphocytopenia, neutrophilia, monocytosis, and eosinopenia; these changes were related to the size of the infection. Infected gerbils also produced specific protein-A binding antibodies to E. multilocularis. At 14 weeks after inoculation, infected gerbils showed splenomegaly and somewhat elevated serum transaminase levels, although serum 5'-nucleotidase levels were normal.     

Synthesis and possible role of carbohydrate moieties of yeast glycoproteins

The pathways for protein N- and O-glycosylation in yeast cells are summarized. Evidence is presented that the terminal glucosyl residues of the dolichyl-PP-oligosaccharide intermediate are responsible for decreasing the Km for the peptide to be N-glycosylated. A liposomal model system is introduced that allows the study of a dolichyl phosphate (Dol-P) dependent transmembrane transport of mannosyl residues. The results obtained so far suggest that the mannosylation of Dol-P and the transmembrane translocation of Dol-P-Man are catalysed by the enzyme more or less simultaneously. However, only about 8-10% of the enzyme molecules incorporated into the liposomes seem to carry out the 'coupled' reaction. The glycosylation of carboxypeptidase Y is not required for this protein to reach the vacuole, its target organelle. In the presence of low concentrations of tunicamycin, however, yeast cells do stop growth. This does not seem to be due to the inhibition of secretion of glycoproteins like external invertase. It is postulated that protein glycosylation is crucial for a cell cycle event during the G1 phase.     

An error estimation of Michaelis-Menten (Monod)-type kinetics

Due to research on biochemistry and genetic engineering, mathematical models of microbial growth have become more complicated but Michaelis-Menten or Monod type expressions have still been used for conversion rates, uptake rates, etc. It is worth examining the error that can be caused by these quasi-steady-state-hypotheses. This paper presents a simple but very effective rationale function that describes the error of the quasi-steady-state hypothesis in enzyme kinetics. A simplified fermentation kinetic model was used for comparison of microbial growth but no analytical error function has been found for batch cultivation. In the case of continuous fermentation the error can be given in an analytical form. Many simulations, based on real SCP experiments, show a significant effect of the quasi-steady-state hypothesis. Since the rate constants of intracellular events are not really known, we have to be very careful when taking into account Michaelis-Menten type expressions in the building of complicated models. Correspondence to: L. Szigeti     

Epstein-Barr virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping, and endocytosis

The type 2 complement receptor, CR2, a B lymphocyte surface glycoprotein, is known to be a component of the EBV receptor. We now demonstrate that the major EBV outer membrane glycoprotein, gp350/220, is a highly specific ligand for CR2. EBV or beads coated with purified recombinant gp350/220 adsorb to normal B lymphocytes, cap with CR2, become endocytosed into vesicles, and are released into the cytoplasm. This is the first demonstration of herpesvirus glycoprotein-cell glycoprotein receptor interaction in viral adsorption and penetration. The capping of CR2 in response to virus, gp350/220-coated beads, or anti-CR2 monoclonal antibodies is associated with cocapping of surface immunoglobulin. Interaction between CR2 and surface immunoglobulin may be important in modulating the B cell activation that normally follows EBV infection or exposure to antigen.     

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.