Atsushi Fujita

Engineering of Yeast Put4 Permease and Its Application to Lager Yeast for Efficient Proline Assimilation

The Saccharomyces cerevisiae Put4 permease is significant for the transport of proline, alanine, and glycine. Put4p downregulation is counteracted by npi1 mutation that affects the cellular ubiquitination function. Here we describe mutant Put4 permeases, in which up to nine lysine residues in the cytoplasmic N-terminal domain have been replaced by arginine. The steady-state protein level of the mutant permease Put4-20p (Lys9, Lys34, Lys35, Lys60, Lys68, Lys71, Lys93, Lys105, Lys107 → Arg) was largely higher compared to that of the wild-type Put4p, indicating that the N-terminal lysines can undergo ubiquitination and the subsequent degradation steps. Proline is the only amino acid that yeast assimilates with difficulty under standard brewing conditions. A lager yeast strain provided with Put4-20p was able to assimilate proline efficiently during beer fermentations. These results suggest possible industrial applications of the mutant Put4 permeases in improved fermentation systems for beer and other alcoholic beverages based on proline-rich fermentable sources.

In vivo activation of the constitutive androstane receptor β (CARβ) by treatment with dehydroepiandrosterone (DHEA) or DHEA sulfate (DHEA-S)

We investigated whether dehydroepiandrosterone (DHEA) or DHEA‐sulfate (S) affected the activities of nuclear receptors, with special reference to constitutive androstane receptor β (CARβ). Administration of DHEA or DHEA‐S enhanced the DNA binding of hepatic nuclear extracts to responsive elements for the retinoic acid receptor, the retinoic acid receptor β 2 and the peroxisome proliferator activated receptor. The bound complexes were shown to be the CARβ‐RXR heterodimer by antibody‐supershift assays. The expression of a target gene of CARβ, Cyp2b10, was increased in liver by DHEA or DHEA‐S treatment, suggesting that DHEA or DHEA‐S actually activated CARβ in vivo. It was suggested that the metabolic conversion of DHEA, DHEA‐S to CARβ ligands could occur in vivo and the metabolites could regulate the expression of CARβ target gene expression. Our results provide new insights into the in vivo relationship between DHEA/DHEA‐S and CARβ activation.

Focal hepatic steatosis surrounding a metastatic insulinoma

Reported herein is a case of focal hepatic steatosis surrounding a metastatic insulinoma in the liver of a 69‐year‐old woman. The patient complained of losing consciousness after meals, and hypoglycemia and hyperinsulinemia were confirmed. On CT and abdominal angiography a mass, 1u2003cm in diameter, was seen in the tail of the pancreas. In the early phase of dynamic CT a mass, 5u2003mm in diameter, was seen in the liver. In the late phase this mass appeared to be 3u2003cm in diameter. An arterial calcium stimulation/venous sampling test showed insulin levels after calcium injections in the hepatic artery to be extremely high. Thus, the liver tumor was diagnosed as a metastatic insulinoma, and distal pancreatectomy and partial resection of the liver were performed. The pancreatic tumor cells were immunohistochemically positive for insulin. The liver tumor was pale yellow. A white area surrounded the tumor. Histologically, the liver tumor was an insulinoma and the white area was focal fatty change of the liver. High insulin levels are said to inhibit oxidation of free fatty acids into triglycerides, causing free fatty acids to accumulate in hepatocytes. Focal hepatic steatosis caused by the local effects of insulin can present as a focal rim surrounding a metastatic insulinoma.

Single point mutations in Met4p impair the transcriptional repression of MET genes in Saccharomyces cerevisiae.

Transcription of MET genes in Saccharomyces cerevisiae depends on a transcriptional activator, the MET4 gene product (Met4p). Using in vitro mutagenesis, we isolated two mutant MET4 alleles encoding [Pro215]Met4p and [Ser156]‐Met4p. These mutations impeded Met4ps responsiveness to methionine in the media, and yeast cells carrying mutant alleles exhibited enhanced transcription of MET genes under repressing conditions. The enhanced transcription was dependent on the CBF1 gene, but did not compete with an excess of wild‐type Met4p, suggesting that some changes in the affinity of Met4p to other factors might be involved in S‐adenosylmethionine‐mediated transcriptional regulation