Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a lambda gt11 expression library using 125I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 and CMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62,999 Da) and CMP2 protein (68,496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 2B (calcineurin). The products of the CMP1 and CMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62,000 and 64,000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wildtype neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His 96 by alanine leads to complete loss of heme, indicating that His 96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the lowspin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His 64 is the endogenous ligand and that Lys 67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the (Fe-His) stretching frequency appears at 221 cm ؊1 , which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the (Fe-CO) stretching frequency was detected at 521 and 494 cm ؊1 in WT, M69L, and K67T, while only the 494 cm ؊1 component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm ؊1 component is attributed to the presence of polar His 64 . The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His 64 and Lys 67 comprise a unique distal heme pocket in neuroglobin.

The present results show that the mGPS is an independent prognostic factor in patients with PC, especially for advanced-stage disease.

The increase in k suggests the acceleration of gastric emptying in the later phase. For the first time, this study has revealed that acute smoking speeds the gastric emptying of solids in Japanese habitual smokers.

BackgroundKojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications. Thus, an efficient and cost-effective kojic acid production process would be valuable. However, little is known about the complete set of genes for kojic acid production. Currently, kojic acid is produced from glucose. The efficient production of kojic acid using cellulose as an inexpensive substrate would help establish cost-effective kojic acid production.ResultsA kojic acid transcription factor gene over-expressing the A. oryzae strain was constructed. Three genes related to kojic acid production in this strain were transcribed in higher amounts than those found in the wild-type strain. This strain produced 26.4 g/L kojic acid from 80 g/L glucose. Furthermore, this strain was transformed with plasmid harboring 3 cellulase genes. The resultant A. oryzae strain successfully produced 0.18 g/L of kojic acid in 6 days of fermentation from the phosphoric acid swollen cellulose.ConclusionsKojic acid was produced directly from cellulose material using genetically engineered A. oryzae. Because A. oryzae has efficient protein secretion ability and secondary metabolite productivity, an A. oryzae-based cell factory could be a platform for the production of various kinds of bio-based chemicals.