James J. Greene

Purified dengue 2 virus envelope glycoprotein aggregates produced by baculovirus are immunogenic in mice

The full-length dengue 2 virus envelope glycoprotein (Egp) was expressed in insect cells by recombinant (r) baculovirus and found to form multimeric aggregates that were recovered in the void volume of gel filtration columns and by ultracentrifugation. An immunoblot confirmed that rEgp aggregrates disrupted with SDS sample buffer released a monomeric form that migrated with a molecular weight similar to native dengue 2 virus Egp on polyacrylamide gels. The rEgp aggregates reacted strongly with a panel of monoclonal antibodies specific for the native Egp and which identify critical structural and functional epitopes. The rEgp aggregates were purified by ultracentrifugation through 30% sucrose, and were shown to be the major protein band on a polyacrylamide gel and corresponding immunoblot. Purified rEgp aggregates in combination with aluminum hydroxide induced high titer neutralizing antibodies in adult mice. The generation of full-length dengue 2 rEgp aggregates in insect cells facilitated development of a simple, effective procedure for purification of the recombinant protein, and represents a good approach for producing highly immunogenic dengue 2 rEgp as a component of a subunit vaccine.

Influence of the cellular redox state on NF‐κB‐regulated gene expression

The redox state has been shown to regulate a variety of biochemical functions including cellular proliferation. Previous studies from our laboratory and others have shown that the binding of many transcription factors to their cognate DNA sequences is sensitive to the redox environment. Therefore, it is likely that redox status serves as an additional regulatory control for the activity of transcription factors and that this may mediate the redox regulation of proliferation. To assess this possibility, the influence of altering the redox state on NF‐κB‐regulated gene expression was studied. A more‐reducing environment favored higher levels of expression of gro, an endogenous gene associated with proliferation, when the redox levels were changed either naturally by altering culture density or chemically by treatment with modulators of glutathione synthesis. Furthermore, nuclear runoff studies showed that a more‐reducing redox increased transcription of gro. In order to ascertain the singular effect of the redox state on the activity of NF‐κB, expression of a secreted alkaline phosphatase (SEAP) reporter gene solely under the control of an NF‐κB response element was measured under varying redox conditions. Changes in the redox state modulated the expression of this reporter system. Taken together, these results suggest the involvement of a redox mechanism regulating signaling events operating through the control of gene expression by transcription factors. J. Cell. Physiol. 183:45–52, 2000.

Host Cell Compatibility in Protein Expression

The expression of cloned genes in prokaryotic or eukaryotic host cells provides the means not only for the study of gene function but also for the production of substantial amounts of protein and nonprotein molecules for commercial and investigational use. In the case of proteins, strategies for determining the most appropriate vector-host combination for the expression of an exogenous gene depend on a diverse range of factors that relate ultimately to the properties of the gene and its product. The approach used in the downstream purification of the product is another factor that impinges on this selection. However, among the most important considerations in the choice of vector and host in ensuring the maximal amount of expression is the compatibility of the host cells to translate the RNA transcript, to ensure the proper folding of the product, and to sustain the protein in the intact and functional state.

Preferential modulation of embryonic cell proliferation and differentiation by embryonic interferon

Embryonic-interferon (E-IFN), a novel species of interferon (IFN) produced solely by embryo cells, inhibited the proliferation of embryo cells in early gestation preferentially vis-à-vis fetal cells in late gestation but had little effect on the in vitro differentiation of embryonic pre-adipocytes to adipocytes. In contrast, mature-interferon (M-INF) did not exhibit this preferential inhibition of cell proliferation and did inhibit pre-adipocyte differentiation. These results suggest that the function of E-INF is different from that of M-IFN and that this function may involve modulation of the developmental process.

Redox-mediated bypass of restriction point via skipping of G1pm

BackgroundIt is well known that cancer cells bypass the restriction point, R, and undergo uncontrolled cell proliferation.Hypothesis and evidenceWe suggest here that fibrosarcoma cells enter G1ps directly from M, skipping G1pm, hence bypassing R, in response to redox modulation. Evidence is presented from the published literature that demonstrate a shortening of the cycle period of transformed fibroblasts (SV-3T3) compared to the nontransformed 3T3 fibroblasts, corresponding to the duration of G1pm in the 3T3 fibroblasts. Evidence is also presented that demonstrate that redox modulation can induce the CUA-4 fibroblasts to bypass R, resulting in a cycle period closely corresponding to the cycle period of fibrosarcoma cells (HT1080).ConclusionThe evidence supports our hypothesis that a low internal redox potential can cause fibrosarcoma cells to skip the G1pm phase of the cell cycle.

Measurement of gene-sepcific transcription by nuclease protection of pulse-labeled nuclear RNA

A gene-specific transcription assay was developed that is based on pulse-labeled incorporation of [3H]uridine into nuclear RNA. Transcription is quantified by scintillation counting of [3H]uridine incorporated into nuclear RNA that is protected from S1 nuclease digestion by hybridization with cold gene probes. This assay was dependent upon partial degradation of nuclear RNA and optimization of hybridization and nuclease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell lines using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in serum-stimulated fibrosarcoma HT-1080 cells determined by [3H]uridine incorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in transcription rate within two hours of stimulation that declined to basal levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greater at 6-9-fold. The relative transcriptional activities of the beta-actin gene to that of the c-myc gene were virtually identical using the two assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were measured in log-phase HL-60 leukemia cells.