Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).     

Crystallization and Reconstitution of Yeast Aconitase

The combination of flow injection analysis and the electrochemical method was investigated for measuring the organic acid content of citrus fruits. The controlled-potential four-electrode method was used for conductance measurements. The instrumentation of the measuring apparatus used in this study is defined. The optimum conditions of the flow system were as follows: injection volume 35 μ1, mixing coil length 100 cm, flow rate 20ml/min and dilution ratio of samples 1:130. A linear correlation between the peak conductivity and the organic acid content was obtained with high correlation coefficient (r=0.992). It is possible to analyze about 50 samples within an hour using the method, and the coefficient of variation with any given juice for 20 assays was less than 0.5%.     

Effect of Ala replacement with Aib in amphipathic cell-penetrating peptide on oligonucleotide delivery into cells

A number of cell-penetrating peptides (CPPs) have been characterized and their usefulness as delivery tools has been clarified. As one of the CPPs, model amphipathic peptide (MAP) was developed by integrating both hydrophobic and hydrophilic amino acids in its sequence. In our previous work, we designed MAP(Aib) by replacing five alanine (Ala) residues on the hydrophobic face of the helix in the MAP sequence with α-aminoisobutyric acid (Aib) residues, and the replacement resulted in higher helix propensity, stronger resistance to protease, and higher cell membrane permeability than MAP. As a next step, we examined the efficiency of oligonucleotide (ODN) delivery into cells by MAP(Aib) in comparison with that by MAP. The electrostatically formed MAP(Aib)/ODN complex was more easily taken up by cells than the MAP/ODN complex, and the ODN delivery by MAP(Aib) was via an endocytic pathway. We demonstrated that the incorporation of Aib residues into CPPs enhances the delivery of hydrophilic molecules, such as ODN, into cells.     

Increased bone mass in mice after single injection of anti-receptor activator of nuclear factor-kappaB ligand-neutralizing antibody: evidence for bone anabolic effect of parathyroid hormone in mice with few osteoclasts

Receptor activator of nuclear factor-κB ligand (RANKL) is a pivotal osteoclast differentiation factor. To investigate the effect of RANKL inhibition in normal mice, we prepared an anti-mouse RANKL-neutralizing monoclonal antibody (Mab, clone OYC1) and established a new mouse model with high bone mass induced by administration of OYC1. A single subcutaneous injection of 5 mg/kg OYC1 in normal mice significantly augmented the bone mineral density in the distal femoral metaphysis from day 2 to day 28. The OYC1 treatment markedly reduced the serum level of tartrate-resistant acid phosphatase-5b (TRAP-5b, a marker for osteoclasts) on day 1, and this level was undetectable from day 3 to day 28. The serum level of alkaline phosphatase (a marker for osteoblasts) declined significantly following the reduction of TRAP-5b. Histological analysis revealed few osteoclasts in femurs of the treated mice on day 4, and both osteoclasts and osteoblasts were markedly diminished on day 14. Daily injection of parathyroid hormone for 2 weeks increased the bone mineral density in trabecular and cortical bone by stimulating bone formation in the OYC1-treated mice. These results suggest that parathyroid hormone exerted its bone anabolic activity in mice with few osteoclasts. The mouse anti-RANKL neutralizing antibody OYC1 may be a useful tool to investigate unknown functions of RANKL in vivo.     

Discovery of novel 7-membered cyclic amide derivatives that inhibit 11beta-hydroxysteroid dehydrogenase type 1

A series of novel 5-trans-hydroxyadamantan-2-yl-5,6,7,8-tetrahydropyrazolo[4,3-c]azepin-4(1H)-ones that inhibit 11beta-hydroxysteroid dehydrogenase type 1 are described. We discovered these 7-membered cyclic amide derivatives by introducing a distinctive linker through pharmacophore analysis of known ligands included in X-ray co-crystal structures. Further optimization using docking studies led to highly potent inhibitors 15b and 27, which furthermore showed the potent efficacy in in vivo studies.     

Theonellamide F, a Bicyclic Peptide Marine Toxin, Induces Formation of Vacuoles in 3Y1 Rat Embryonic Fibroblast

The effect of a marine bicyclic peptide, theonellamide F, on vacuole formation in exponentially growing 3Y1 rat embryonic fibroblasts was studied in comparison with the effect of monensin. Many abnormally large vacuoles appeared around the nucleus in the cells treated with 6 μM theonellamide F within 24 hours. Following prolonged treatment with this agent, the number of enlarged vacuoles increased. Such vacuoles accumulated many granules that showed Brownian movement. The effect of theonellamide F on the cells was more drastic in an amino-acid-deficient medium, in which all cells died within 1 hour at a 3-μM concentration of the agent. Theonellamide F probably affects cellular autophagy, inhibiting the degradation of the organelles and turnover of proteins. Monensin, a well-known Na⁺ ionophore that disintegrates the Golgi apparatus, induced similar types of vacuole formation, although the vacuoles were localized in a region slightly distant from the nucleus. Monensin readily affected cell morphology, resulting in cell death. We propose that theonellamide F, like monensin, is a useful agent for investigating membrane structures in cells. Received November 25, 1998; accepted February 10, 1999     

Change in nutritional status modulates the abundance of critical pre-initiation intermediate complexes during translation initiation in vivo

In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).     

Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses

Tk-subtilisin [the mature domain of Pro-Tk-subtilisin in active form (Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis is matured from Pro-Tk-subtilisin [a subtilisin homologue from T. kodakaraensis in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide. Pro-Tk-subtilisin is characterized by extremely slow maturation at mild temperatures, but this maturation rate is greatly increased by a single Gly56 → Ser mutation in the propeptide region. To analyze the role of Gly56, which assumes a left-handed conformation, Pro-Tk-subtilisin variants with complete amino acid substitutions at Gly56 were constructed. A comparison of their halo-forming activities suggests that all variants, except for Pro-G56W [Pro-G56X, Pro-Tk-subtilisin with Gly56 → X mutation (X = any amino acid)], mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S, were overproduced, purified, and characterized. SDS-PAGE analyses and Tk-subtilisin activity assay indicated that their maturation rates increased in the order WT ≤ Pro-G56W < Pro-G56S < Pro-G56E. The propeptides of these variants were also overproduced, purified, and characterized. The stability and inhibitory potency of these propeptides decreased in the order Tk-propeptide [propeptide of Tk-subtilisin (Gly1-Leu69)] ≥ G56W-propeptide > G56S-propeptide > G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-subtilisin and its derivatives. The crystal structures of these propeptides determined in complex with S324A-subtilisin indicate that the conformation of the propeptide is altered by the mutation, such that nonglycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease. These results indicate that Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of Tk-propeptide to Tk-subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and slow maturation of Pro-Tk-subtilisin.