Effects of Cosolvents on the Folding and Catalytic Activities of the Hammerhead Ribozyme

The RNA cleavage activity of the hammerhead ribozyme has been compared in various mixed aqueous solutions containing cosolvents. Kinetic analysis revealed that the tested cosolvents enhanced the ribozyme activity, particularly at low MgCl₂ concentrations. These enhancements, in some cases of more than tenfold, resulted from a reduction in the Mg²⁺ concentration required for substrate cleavage. An inverse correlation was found between the MgCl₂ concentration essential for efficient catalysis and the dielectric constant values. In contrast, FRET measurements showed no substantial influence of cosolvents on the Mg²⁺‐induced structural transitions. The results suggest that the solution environment has various effects on the Mg²⁺ interactions involved in the catalysis and global folding of the ribozyme.     

Gel—sol transition in gellan gum solutions. II. DSC studies on the effects of salts

The thermal properties of sodium form gellan gum solutions with and without sodium chloride, potassium chloride, calcium chloride and magnesium chloride were studied by differential scanning calorimetry (DSC). The DSC cooling or heating curves for 1% gellan gum solutions without salt showed a single exothermic or endothermic peak at ~30°C. DSC cooling curves showed a single exothermic peak, with the setting temperature (Ts) shifting to progressively higher temperatures with increasing concentration of the added NaCl or KCl. At low concentrations of NaCl or KCl, DSC heating curves showed a single endothermic peak; however with more addition of salt the endothermic peak gradually developed a bimodal character and eventually split into more than two distinct peaks. The onset of detectable splitting occurred at a high salt concentration which coincided with that at which elastic gels are formed at even a higher temperature as was observed by viscoelastic measurements. With a sufficient addition of monovalent cations the endothermic curve became again a single peak shifting to higher temperatures. In the presence of divalent cations, although Ts shifted to higher temperatures with increasing concentration of added CaCl₂ or MgCl₂, the melting temperature (Tm) in heating DSC curves shifted to higher temperatures (up to a certain temperature) and then shifted to lower temperatures with increasing concentration of salt. With increasing concentration of CaCl₂ or MgCl₂, the exothermic and endothermic enthalpies estimated for a main peak increased up to a certain salt concentration and then decreased; however many other peaks were observed at higher temperatures. The endothermic peaks for gels with excessive divalent cations were too broad to be resolved from the baseline; in contrast the exothermic peaks were much sharper and readily recognized. In comparing thermal properties with rheological properties, gellan gum solutions with excessive divalent cations form firm gels on cooling to below the setting temperature, and then it was difficult to remelt them. This was quite different from the behaviour of thermoreversible gels formed in the presence of monovalent cations. It seems that the mechanism of gel formation in gellan gum with divalent cations is markedly different from that with monovalent cations.     

Cell type-specific modulation of cell growth by transforming growth factor beta 1 does not correlate with mitogen-activated protein kinase activation

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-beta 1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-beta 1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-beta 1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-beta 1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-beta 1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-beta 1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.     

Toward Engineered Biosynthesis of Drugs in Human Cells

Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.