Chimeric yeast G-protein α subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae

Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-Gα interactions. Here, using the yeast endogenous Gα protein (Gpa1p) as template, we constructed various chimeric Gα proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric Gα protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.     

Apoptosis supercedes necrosis in mitochondrial DNA-depleted Jurkat cells by cleavage of receptor-interacting protein and inhibition of lysosomal cathepsin

In the present study, we used mitochondrial DNA-depleted Jurkat subclones (rho0 cells) to demonstrate that Fas agonistic Ab (CH-11), at the concentrations that evoke apoptotic death of the parental Jurkat cells, induced necrosis mainly through generation of excess reactive oxygen species, lysosomal rupture, and sequential activation of cathepsins B and D, and in minor part through activation of receptor-interacting protein (RIP). In the rho0 cells treated with CH-11, ATP supplementation converted necrosis into apoptosis by the formation of the apoptosome and subsequent activation of procaspase-3. In these ATP-supplemented rho0 cells (ATP-rho0), generation of excess ROS and lysosomal rupture were still seen, yet cathepsins B and D were inactivated and RIP was degraded. The conversion of necrosis to apoptosis, RIP degradation, and cathepsin inactivation in ATP- rho0 cells were blocked by caspase-3 inhibitors. Activities of cathepsins B and D in the lysate of necrotic rho0 cells were inhibited by the addition of apoptotic parental Jurkat cell lysate. Thus, apoptosis may supercede necrosis.     

Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA

In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen.     

Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries

A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a “yeast cell chip.” A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface.     

Metabolic consequence of long-term exposure of pancreatic beta cells to free fatty acid with special reference to glucose insensitivity.

Long-term exposure of the pancreatic beta cells to free fatty acid (FFA) reportedly inhibits glucose-stimulated insulin secretion. We here studied the impact of FFA on glucose and lipid metabolism in pancreatic beta cells with special reference to insulin secretion. Pancreatic beta-cell line MIN6 was exposed to various concentrations of palmitate for 3 days. Glucose-stimulated insulin secretion and insulin content were decreased corresponding to the concentration of the palmitate exposed. Glycolytic flux and ATP synthesis was unchanged, but pyruvate-stimulated change in NAD(P)H concentration was decreased. Pyruvate carboxylase was decreased at the protein level, which was restored by the removal of palmitate or the inhibition of beta-oxidation. Intracellular content of triglyceride and FFA were elevated, beta-oxidation was increased, and de novo lipogenesis from glucose was decreased. NADPH content and citrate output into the medium, which reflected pyruvate malate shuttle flux, were decreased, but malic enzyme activity was unaffected. The malic enzyme inhibitor alone inhibited insulin response to glucose. In conclusion, long-term exposure of FFA to beta cells inhibits glucose-stimulated insulin secretion via the decreased NADPH contents due to the inhibition of pyruvate carboxylase and malate pyruvate shuttle flux.     

A Comparison Between Machine Flexibility and Routing Flexibility

In this paper, we evaluate two types of flexibility, machine flexibility and routing flexibility, in terms of manufacturing performance in various shop environments. A simulation-based investigation was conducted to analyze the impact of these types of flexibility on the average flow time of parts under various job flow pattern conditions, which characterize the shop nature from a random job shop to a flow shop, operation time variance, setup time, and shop load. The experimental results show how these types of flexibility affect the average flow time of parts and which type is superior under what conditions. Management can obtain better insight and guidelines for determining priorities or the scale, or scope, of various decision items relating to design standardization, process and operations improvement, investment in new equipment and tools, and the like.     

Simultaneous quantification by HPLC of purines in umami soup stock and evaluation of their effects on extracellular and intracellular purine metabolism

Ribonucleotide flavor enhancers such as inosine monophosphate (IMP) and guanosine monophosphate (GMP) provide umami taste, similarly to glutamine. Japanese cuisine frequently uses soup stocks containing these nucleotides to enhance umami. We quantified 18 types of purines (nucleotides, nucleosides, and purine bases) in three soup stocks (chicken, consommé, and dried bonito soup). IMP was the most abundant purine in all umami soup stocks, followed by hypoxanthine, inosine, and GMP. The IMP content of dried bonito soup was the highest of the three soup stocks. We also evaluated the effects of these purines on extracellular and intracellular purine metabolism in HepG2 cells after adding each umami soup stock to the cells. An increase in inosine and hypoxanthine was evident 1 h and 4 h after soup stock addition, and a low amount of xanthine and guanosine was observed in the extracellular medium. The addition of chicken soup stock resulted in increased intracellular and extracellular levels of uric acid and guanosine. Purine metabolism may be affected by ingredients present in soups.     

Protection of Coral Larvae from Thermally Induced Oxidative Stress by Redox Nanoparticles

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNP^O) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNP^O treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNP^O treatment, whereas these proteins were absent in RNP^O-treated larvae, which suggested that RNP^O effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNP^O treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.     

Expression of the SNF1 gene from Candida tropicalis is required for growth on various carbon sources, including glucose

SNF1 of Saccharomyces cerevisiae is an essential gene for the derepression of glucose repression. A homolog of SNF1 (CtSNF1) was isolated from an n-alkane-assimilating diploid yeast, Candida tropicalis. CtSNF1 could complement the snf1 mutant of S. cerevisiae. The previously published method for introducing the exogenous DNA into C. tropicalis was employed to construct SNF1/ snf1 heterozygote and snf1/snf1 homozygote strains. The successfully constructed SNF1/snf1 heterozygote was named KO-1. Disruption of the second CtSNF1 allele was unsuccessful, suggesting that CtSNF1 might be essential for cell viability. Therefore, in order to control the expression of CtSNF1, a strain (named KO-1G) in which the promoter region of CtSNF1 was replaced with the GAL10 promoter of C. tropicalis was constructed, and the growth of strains KO-1 and KO-1G was compared with that of the parental strain. The growth of strain KO-1 on glucose, sucrose, or acetate did not differ from the growth of the parental strain, but strain KO-1 showed a slight growth retardation on n-alkane. The growth of strain KO-1G on galactose was normal, but the cells stopped growing when transferred to glucose-, acetate-, or n-alkane-containing medium. Northern blot analysis against mRNA from the n-alkane-grown KO-1G strain demonstrated a close relationship between the presence of CtSNF1 mRNA and the growth of the cells, indicating that CtSNF1 is essential for cell viability. Moreover, mRNA levels of isocitrate lyase, which is localized in peroxisomes of C. tropicalis, were significantly affected by the level of CtSNF1 mRNA. Received: 3 May 1999 / Accepted: 14 July 1999