Development of combinatorial bioengineering using yeast cell surface display--order-made design of cell and protein for bio-monitoring

A genetic system to display proteins as their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae, has been exploited. Surface-engineered (arming) cells displaying amylase or cellulase could assimilate starch or cellulose as the sole carbon source, although S. cerevisiae can not intrinsically assimilate them. Arming cells with a green fluorescent protein (GFP) from Aequorea victoria can emit green fluorescence from the cell surface in response to the environmental conditions. From these results, we attempted to construct a system to monitor the foreign protein production in yeast by simultaneous displaying the enhanced GFP (EGFP). The expression in yeast of the Escherichia coli beta-galactosidase-encoding gene was examined as an example of intracellular production and that of the human interferon-alpha (omega, IFN-omega)-encoding gene as an example of extracellular production. Their productions and the simultaneous surface-display of EGFP as a reporter were controlled by the same promoter, GAL1. The relationship among fluorescence signals and their productions was evaluated. The surface-display system, unlike one using tag-proteins, would be able to facilitate the monitoring of native protein productions in bioprocesses using living cells in real time by the combination of promoters and GFP variants.     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.     

A PPAR agonist improves TNF-alpha-induced insulin resistance of adipose tissue in mice

Thiazolidinediones (TZDs), agonists for PPARs, have been shown to block the inhibitory effects of TNF-alpha on insulin action using cultured cells. In order to clarify the in vivo effects of TZDs on the inhibition of insulin sensitivity by TNF-alpha, insulin action in muscles and adipose tissues was assessed in the TNF-alpha-overexpression mice model using transplantation of cells secreting the TNF-alpha protein. After the pioglitazone treatment for 4 weeks, glucose uptake, insulin-induced IRS-1 phosphorylation, and lipoprotein lipase mRNA levels were analyzed. Pioglitazone did not ameliorate TNF-alpha-induced hyperinsulinemia in this model, as assessed by the OGTT. Glucose uptake and lipoprotein lipase mRNA levels were decreased by TNF-alpha in adipose tissues from the TNF-alpha-overexpressing mice, and pioglitazone blocked these inhibitions by TNF-alpha. On the other hand, in muscles, pioglitazone did not reverse the effects of TNF-alpha on insulin-induced phosphorylation of IRS-1, glucose uptake, and lipoprotein lipase mRNA levels. Present study revealed the different sensitivities of pioglitazone for the recovery of decreased insulin action in a TNF-alpha-overexpressing model using cell transplantation. These results suggest that the effect of TZDs is dependent on the fat distribution and accumulation in humans.     

Production of alkyl glucoside from cellooligosaccharides using yeast strains displaying Aspergillus aculeatus β-glucosidase 1

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.     

Kinetics of microbial death by combined treatment with heat and antimicrobial agents

Studies were made of the death kinetics of Escherichia coli cells heated at 46 to 56°C in 0.05M phosphate buffer (pH 7.0) containing either an amphoteric surfactant (Tego 15DL, 1–10 μg/ml) or sorbic acid (0.5 to 3%). A linear relationship was obtained on the Arrhenius plot for the death of cells heated with each antimicrobial agent. The kinetics of the action of the surfactant, however, differed from that of sorbic acid. With the amphoteric surfactant, the activation enthalpy of the death reaction decreased from 108 to 51 kcal/mol as the concentration of surfactant was increased in the range tested although the death rate remained high; whereas with sorbic acid the activation enthalpy remained fairly constant (104 ± 9 kcal/mol) independent of its concentration and the death rate was similarly high. Further, in the action of the amphoteric surfactant, a thermodynamic compensation effect was observed, the compensation temperature being 334°K (61°C), i.e., relatively close to the observed temperatures. For sorbic acid, however, this temperature seemed to be too high to observe when determined from the Arrhenius plot. The data of the dependency of the death-rate constant upon the concentration of antimicrobial agent indicated a similar difference in the action of the two agents. Based on our results and on data obtained by other workers, we propose that antimicrobial agents that enhance cellular death induced by heating can be characterized into two types.     

Diurnal change in bioelectric potential of Phaseolus plant in relation to the leaf movement and light condition

A regular pattern of diurnal change in electric potentials developedrepeatedly from the pulvinus in the Phaseolus plant. Four kindsof bioelectric waves were discerned in the pattern; p-waves,a photoelectric response; l-waves, long and slow waves of negativepotential; e- and f-waves, rhythmic change in electric potentialswith high frequencies. ³ Present address: Tanagura High School, Tanagura 961, Japan. (Received May 8, 1975; )     

Continuous measurement of tympanic temperature with a new infrared method using an optical fiber

The purpose ofthis study was to investigate the utility of an infrared tympanicthermometry by using an optical fiber for measuring tympanictemperature (T_ty). In the headcooling and facial fanning tests during normothermia, rightT_ty measured by this method(infrared-T_ty) and esophagealtemperature (T_es) were notaffected by decreased temple and forehead skin temperatures, suggestingthat the infrared sensor in this system measured the infrared radiationfrom the tympanic membrane selectively. Eight male subjects took partin passive-heat-stress and progressive-exercise tests. No significantdifferences among infrared-T_ty,the left T_ty measured bythermistor (contact-T_ty), andT_es were observed at rest or atthe end of each experiment, and there was no significant difference inthe increase in these core temperatures from rest to theend. Furthermore, there were no significant differences inthe core temperature threshold at the onset of sweating and slope (therelationship of sweating rate vs.infrared-T_ty and vs.contact-T_ty). Theseresults suggest that this method makes it possible to measureT_ty accurately, continuously, andmore safely.

     

A radioimmunoassay for γ1-melanotropin and evidence that the smallest pituitary γ-melanotropin is amidated at the COOH-terminus

Specific radioimmunoassays for γ₁-melanotropin (γ₁-MSH) and γ₂-melanotropin (γ₂-MSH) have been developed. The γ₁-MSH antibody recognizes the portion between His⁵ and Phe¹¹-NH₂ of γ₁-MSH and shows no significant cross-reactivities with other related peptides. The γ₂-MSH antibody cross-reacts with γ₁-MSH and γ₃-MSH to the extent of 0.004% and 0.04%, respectively, on a weight basis. Using these two different antisera on bovine pituitary extracts, two γ₁-MSH-like peptides were detected only in the intermediate lobe, whereas γ₂-MSH-like peptides were not detectable. Furthermore, it is likely that the smallest γ-MSH produced in the bovine intermediate pituitary is a γ₁-MSH-like peptide with the COOH-terminus amidated.     

Phylogeographic and demographic modeling analyses of the multiple origins of the rheophytic goldenrod Solidago yokusaiana Makino

Understanding adaptation mechanisms is important in evolutionary biology. Parallel adaptation provides good opportunities to investigate adaptive evolution. To confirm parallel adaptation, it is effective to examine whether the phenotypic similarity has one or multiple origins and to use demographic modeling to consider the gene flow between ecotypes. Solidago yokusaiana is a rheophyte endemic to the Japanese Archipelago that diverged from Solidago virgaurea. This study examined the parallel origins of S. yokusaiana by distinguishing between multiple and single origins and subsequent gene flow. The haplotypes of noncoding chloroplast DNA and genotypes at 14 nuclear simple sequence repeat (nSSR) loci and single-nucleotide polymorphisms (SNPs) revealed by double-digest restriction-associated DNA sequencing (ddRADseq) were used for phylogeographic analysis; the SNPs were also used to model population demographics. Some chloroplast haplotypes were common to S. yokusaiana and its ancestor S. virgaurea. Also, the population genetic structures revealed by nSSR and SNPs did not correspond to the taxonomic species. The demographic modeling supported the multiple origins of S. yokusaiana in at least four districts and rejected a single origin with ongoing gene flow between the two species, implying that S. yokusaiana independently and repeatedly adapted to frequently flooding riversides.Subject terms: Structural variation, Plant evolution