The advent of thermostable enzymes has led to great advances in molecular biology, such as the development of PCR and ligase chain reaction. However, isolation of naturally thermostable enzymes has been restricted to those existing in thermophylic bacteria. Here, we show that the disaccharide trehalose enables enzymes to maintain their normal activity (thermostabilization) or even to increase activity at high temperatures (thermoactivation) at which they are normally inactive. We also demonstrate how enzyme thermoactivation can improve the reverse transcriptase reaction. In fact, thermoactivated reverse transcriptase, which displays full activity even at 60°C, was powerful enough to synthesize full length cDNA without the early termination usually induced by stable secondary structures of mRNA.The usefulness of thermostable enzymes is indisputable; they allowed the development of outstanding techniques such as PCR and ligase chain reaction (1, 2). However, the isolation of thermostable enzymes has been restricted to those existing in thermophylic organisms. To expand the availability of thermostable enzymes, we explored a completely new way-the thermal stabilization of those enzymes that are normally thermolabile by the addition of structure stabilizing molecules. In particular, we explored the properties of molecules that are normally involved in heat shock response, and we anticipated being able to confer thermal stability to enzymes. Among them, there is the disaccharide trehalose. Trehalose synthesis is induced by heat shock in the Saccharomyces cerevisiae yeast (3), suggesting its possible role in this response and in desiccation tolerance (4). In fact, yeast mutants defective in trehalose synthesis show a significant reduction in thermotolerance (5). It has been reported that enzymes could be protected from irreversible heat aggregation-heat denaturation in vitro by trehalose, suggesting its chaperonin-like function (6). Trehalose also has been used to confer stability to dried enzymes (7).In the present study, we discovered that trehalose can be used as a reaction additive to stabilize or stimulate enzymatic activity at unusually high temperatures, enabling the use of thermosensitive enzymes as though they would be thermostable. This property should be useful for converting a wide range of thermosensitive enzymes to thermostable and thermoactive ones for wide applications in biological, medical, and industrial fields.To show the power of trehalose-mediated thermal activation, we subsequently applied this new method to the synthesis of full length cDNA. The major obstacle to preparing high quality cDNA libraries has been the low efficiency of reverse transcriptase (RT) to synthesize full length cDNA, which is due to the strong secondary structure of mRNA, which cause the RT to stop the synthesis and subsequently to be released from the hybrid mRNA͞incomplete cDNA. To overcome problems associated with the secondary structure of mRNA, both denaturing of sample before the reaction and increased temper...

Lipopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotela intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h exposure, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various SalmoneUla species with S and R chemotypes and from bacterial and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-la induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-la did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-y) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with SalmoneUla LPS, compared with nontreated cells. Natural human EFN-j exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-I added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-I enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.

Vascular endothelial growth factor (VEGF) is a potent mitogen in endothelial cells, but little is known about its activity in other cell types. To clarify the role of VEGF in human dental pulp cells and pulp tissue, we investigated the effects of VEGF on the chemotaxis, proliferation, and differentiation of human dental pulp cells. VEGF induced a strong chemotactic response in human dental pulp cells in a dose-dependent manner. VEGF also marginally enhanced the proliferation of human dental pulp cells and induced an increase in alkaline phosphatase in human dental pulp cells. However, these effects of VEGF were not observed in reference to human skin fibroblasts. Analyses by the reverse-transcription/polymerase-chain-reaction method and flow cytometry showed that the mRNAs of two VEGF receptors, fins-like tyrosine kinase and kinase insert domain-containing receptor, were expressed in human dental pulp cells, whereas only fms-like tyrosine kinase mRNA was expressed in human skin fibroblasts. VEGF induced the activation of activator protein 1 (AP-1) and c-fos mRNA expression in human dental pulp cells. The AP-1 inhibitor curcumin strongly inhibited VEGF-induced alkaline phosphatase production in human dental pulp cells. In addition, VEGF antisense oligonucleotide suppressed the production of VEGF and alkaline phosphatase in human dental pulp cells. These results suggest that VEGF produced by human dental pulp cells acts directly upon human dental pulp cells in an autocrine manner, and may promote the chemotaxis, proliferation, and/or differentiation of human dental pulp cells via the utilization of kinase insert domain-containing receptor and in part through AP-1 by increasing c-fos.

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We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.