A novel mechanism regulates H(2) S and SO(2) production in Saccharomyces cerevisiae.

Sulfite (SO(2) ) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H(2) S) has an undesirable aroma. To discover the cellular processes that control SO(2) and H(2) S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H(2) S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H(2) S and SO(2) , because the adenosylphophosulfate kinase Met14p is a substrate of SCF(Skp2) and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S-adenosylmethionine, S-adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H(2) S/SO(2) production.

Development of bottom-fermenting saccharomyces strains that produce high SO2 levels, using integrated metabolome and transcriptome analysis.

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO(2) is desirable, it is complicated by the fact that undesirable H(2)S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO(2)-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO(2) and H(2)S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO(2), resulting in high SO(2) and low H(2)S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO(2) than the parent but produced parental levels of H(2)S.

Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein.

Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

Needle-free jet injection of a mixture of Japanese encephalitis DNA and protein vaccines: a strategy to effectively enhance immunogenicity of the DNA vaccine in a murine model.

Combined immunization with gene-based and protein-based vaccines can increase vaccine effectiveness. We previously demonstrated, using a murine model for Japanese encephalitis (JE), that simultaneous immunization with a DNA vaccine (pcJEME) by the intramuscular route and a protein vaccine consisting of subviral extracellular particles (EPs) by the subcutaneous route provided a synergistic increase in immunogenicities of these vaccines. Here, we investigated a novel immunization protocol consisting of a single inoculation with a mixture of DNA and protein vaccines using a needle-free jet injector. Immunization of ddY mice with 1 microg of pcJEME mixed with 1 microg of EPs or a 1/100 dose of commercial inactivated JE vaccine (JEVAX) induced neutralizing antibody titers of 1:40 to 1:80 (90% plaque reduction) 6 weeks after immunization, whereas immunization with DNA or protein alone only induced low titers (< or =1:10). Co-immunization with pcDNA3, a CpGcontaining vector of the vaccine plasmid, increased immunogenicity of JEVAX to some extent. IgG1/IgG2a isotype profiles supported increased production of EPs in pcJEME-inoculated mice by needle-free injection and an adjuvant effect of the vector on immunogenicity of JEVAX.

Needle-free jet injection of small doses of Japanese encephalitis DNA and inactivated vaccine mixture induces neutralizing antibodies in miniature pigs and protects against fetal death and mummification in pregnant sows.

Japanese encephalitis (JE) virus causes abortion and stillbirth in swine, and encephalitis in humans and horses. We have previously reported that immunogenicity of a DNA vaccine against JE was synergistically enhanced in mice by co-immunization with a commercial inactivated JE vaccine (JEVAX) under a needle-free injection system. Here, we found that this immunization strategy was also effective in miniature pigs. Because of the synergism, miniature pigs immunized twice with a mixture of 10 µg of DNA and a 1/100 dose of JEVAX developed a high neutralizing antibody titer (1:190 at 90% plaque reduction assay). Even using 1 µg of DNA, 3 of 4 pigs developed neutralizing antibodies. Following challenge, all miniature pigs with detectable neutralizing antibodies were protected against viremia. Pregnant sows inoculated with 10 or 1 µg of DNA mixed with JEVAX (1/100 dose) developed antibody titers of 1:40-1:320. Following challenge, fetal death and mummification were protected against in DNA/JEVAX-immunized sows.

Dengue tetravalent DNA vaccine increases its immunogenicity in mice when mixed with a dengue type 2 subunit vaccine or an inactivated Japanese encephalitis vaccine.

We previously developed a dengue tetravalent DNA vaccine that can induce neutralizing antibodies against four dengue viruses in mice. Here, we demonstrated that immunogenicity of our tetravalent vaccine is synergistically increased in mice by co-immunization with dengue type 2 virus (DENV2) subviral extracellular particles (D2EPs) or inactivated Japanese encephalitis vaccine (JEVAX). A single immunization with a mixture of 100 microg of the tetravalent vaccine and 150 ng of D2EPs or a 1/10 dose of JEVAX induced moderate levels of neutralizing antibodies in a 90% plaque reduction assay. Immunized mice were protected from "artificial" viremia created by intravenous injection with DENV2.

Dengue tetravalent DNA vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice.

We developed a dengue tetravalent DNA vaccine consisting of plasmids expressing premembrane and envelope genes of each of four serotypes of dengue viruses. BALB/c mice immunized twice with the tetravalent vaccine at a dose of 100 microg (25 microg for each serotype) using a needle-free jet injector developed neutralizing antibodies against all serotypes. There was no interference among the four components included in this combination vaccine. Tetravalent vaccine-immunized mice showed anamnestic neutralizing antibody responses following challenge with each dengue serotype: responses to challenges from serotypes different to those used for neutralization tests were also induced.

Simultaneous immunization with DNA and protein vaccines against Japanese encephalitis or dengue synergistically increases their own abilities to induce neutralizing antibody in mice.

Gene-based and protein-based vaccines are two distinct types of vaccines. In this report, we examined if combined use of DNA and protein vaccines would increase their own abilities to induce neutralizing antibody in murine models for Japanese encephalitis (JE) or dengue type 2 (DEN2). DNA vaccines for JE (pcJEME) or DEN2 (pcD2ME) were inoculated intramuscularly, and protein vaccines consisting of subviral extracellular particles (EPs) containing JE (JEEP) or DEN2 (D2EP) virus antigens were inoculated subcutaneously with Freund's adjuvant. Two immunizations of ICR mice with pcJEME and/or JEEP in the prime-boost protocol indicated that levels of neutralizing antibody induced by the pcJEME prime-JEEP boost vaccination were two to eight-fold higher than those induced by pcJEME alone, but were equivalent to those induced by JEEP alone and slightly higher than those induced by the JEEP prime-pcJEME boost regimen. On the other hand, simultaneous immunization of ICR mice with pcJEME and JEEP provided synergistically higher neutralizing antibody titers than those provided by immunization with either immunogen. Immunization with graded doses of pcJEME and JEEP confirmed the synergism. The synergistic increase in neutralizing antibody titer by simultaneous immunization with DNA and protein vaccines was also shown by immunization with pcD2ME and D2EP in ICR and ddY mice. Both IgG1 and IgG2a antibodies were induced by combined immunization with pcJEME and JEEP.