Studies on Oxygen Transfer in Submerged Fermentations

This paper refers to the application of gas analyzers for the determination of oxygen transfer rate, showing examples in the studies and the performances of submerged fermentations. Oxygen and carbon dioxide analyzers were set to monitor the gas streams to and from the fermentor. Continuous data on the concentrations of oxygen and carbon dioxide in the air streams were thus provided throughout the fermentation. Distinctive characters of this method were applicability to fermentors in practice and ability of obtaining data directly relating to the fermentations.

The modification of sulfite oxidation method for the determination of oxygen transfer rate from air into liquid or of a measure of aeration effectiveness was made. The proposed method was the application of gas analyzers in the studies on submerged fermentation. Some comparative discussions were made between this and the conventional titrimetric method. This modified method could be applied to biological systems with no alteration, therefore, it was made possible to compare the sulfite solution with the biological systems in relation to the problems on oxygen transfer.     

Studies on the Behaviors of Impurities on the Crystallization of L-Glutamie Acid

The influences of some factors at the both sides of the solid and liquid phases on the solubilities of L-glutamic acid in α and β-forms in water were examined.

As differences in the conditions of the liquid phase, the effects of co-existing inorganic neutral salts and amino acids on the solubilities were investigated; and as for the inorganic neutral salts applied here, salting-in effects were recognized, the degrees of which were in the following order, i.e., NH₄Cl, (NH₄)₂SO₄>KCl>NaCl. The effect in the β-form was larger than that of in the α-form. The influence of co-existing amino acids was very little if the amount was not so much, but the tendency to increase the solubility could be recognized.

As a difference in the condition of the solid phase, the purity of the crystals was picked up, and it was clarified that the crude crystal of L-glutamic acid in α-form gave a larger value of the solubility than the purified one.     

Induction of histone acetylation on the CRBPII gene in perinatal rat small intestine

The expression of genes associated with lipid and vitamin A metabolism is elevated when the small intestinal mucosa is maturing rapidly during the perinatal period. We have previously reported that cellular retinol-binding protein type II (CRBPII) mRNA levels rise abruptly in the rat small intestine during this period. In this study, we examined whether the acetylation of histones H3 and H4 is involved in the intestinal expression of CRBPII during the perinatal stage. The expression of cyclin D1 and cyclin B1 genes, which are markers of cell proliferation, decreased markedly during the perinatal period, whereas expression of CRBPII as well as villin, a marker of intestinal maturation, increased rapidly. Using a ChIP assay, we showed rapid induction of acetylation of the histones H3 and H4 which interacted with the promoter/enhancer region of the CRBPII gene at this time. The binding of CBP and p300, which have histone acetyltransferase activity, as well as binding of retinoid X receptor alpha (RXRalpha) increased on the CRBPII promoter/enhancer region during the perinatal period. These results suggest that CRBPII gene expression during the perinatal period is associated with abrupt acetylation of histones H3 and H4 followed by the binding of CBP/p300 and RXRalpha.     

Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains

Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.     

Detection of substances with alcoholic or phenolic hydroxyl groups by generation of hydrogen peroxide with imidazole and peroxyoxalate chemiluminescence

On-line detection of substances with an alcoholic or phenolic hydroxyl group using imidazole and peroxyoxalate chemiluminescence was investigated qualitatively using a flow-injection method. The substances tested included six polyphenols, five monophenols and six sugars. After incubation at 80°C with an imidazole buffer (pH 9.5) the substances were detected by peroxyoxalate chemiluminescence. The polyphenols tested (e.g., pyrogallol, purpurogallin, and dopamine) showed the strongest light emission. The sugars with hydroxyl groups (e.g., fructose and lactose) and the monophenols (e.g., phenol, serotonin, and β-estradiol) produced only a weak light emission. Reaction of hydroxyl compounds and imidazole generated hydrogen peroxide. Imidazole served two roles, it catalysed the reaction with the hydroxyl compound and initiated peroxyoxalate chemiluminescence on-line. A novel reactor formed by packing glass beads into a flow cell (Teflon) of a chemiluminometer improved the sensitivity of light detection.     

A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently

Synthetic short peptides containing only the nuclear localization signal (NLS) direct the transport of nonnuclear proteins into the nucleus. As a conjugate of the synthetic peptide with immunoglobulin M (IgM) did not enter the nucleus, there was believed to be a size limit for nuclear transport of NLS-conjugated proteins. However, we found that IgM conjugated with purified nucleoplasmin, a nuclear protein of Xenopus oocytes, rapidly accumulated in the nucleus. For direct comparison with the short peptide, we prepared a long peptide containing the NLS and its flanking sequences of SV40 large T-antigen and its mutated long peptide, in which possible phosphorylation sites located at the amino terminal of the NLS were changed to alanine. Kinetic experiments showed that wild-type long peptide-IgM conjugates were almost entirely taken up into the nucleus within 30 min after their injection, whereas almost 60 min was required for the mutated long peptide-IgM conjugates to enter the nucleus of all the cells examined, and there was no apparent accumulation of short peptide-IgM conjugates in the nucleus within 60 min. These results indicate that even when the kinetics of transport are affected by amino acid substitutions, the long peptide directs the transport of large molecules such as IgM into the nucleus.     

1H NMR study on the conformation of bacitracin A in aqueous solution.

The conformation of bacitracin A, a widely used cyclic dodecapeptide antibiotic in aqueous solution, has been investigated using 500 MHz 1H NMR and molecular modeling. Findings revealed that a region (residues 1-6) is folded over the cyclic ring, resulting in metal coordination sites, a thiazoline ring, and Glu4 and His10 being proximate to each other.     

Industrial production of disodium 5′-guanylate

A process for manufacturing disodium 5′-guanylate was devised. 5′-Amino 4-imidazole carboxamide riboside (AICA-R) was accumulated with an amount over 100 times those reported in the literature by fermentation of D -glucose with a non-exacting purineless mutant derived from Bacillus megaterium JAM 1245) by x-ray irradiation. The influence of RNA, amino acids, and salts on AICA-R accumulation was clarified. Appropriate aeration and agitation was found necessary. The (60-hr, cultivation of the medium containing 8% of D -glucose gave AICA-R in the concentration above, 11 g/l. AICA-R thus accumulated was separated from the fermentation broth by ion-exchange technique and subjected to synthetic processes to yield disodium 5′-guanylatc with the yield over 40%, based on AICA-R.     

Structure, tissue distribution, and pharmacological characterization of Xenopus orexins

We isolated the Xenopus gene encoding prepro-orexin to predict the structures of orexins in submammalian chordates. Putative mature Xenopus orexin-A and -B are highly similar to each mammalian counterpart. Especially, the C-terminal 10 residues were highly conserved among these species and isopeptides. Immunohistochemical examination of Xenopus brain revealed that orexin-containing neurons were highly specifically localized in the ventral hypothalamic nucleus. A rich network of immunoreactive fibers was found in various regions of the Xenopus brain. The distribution was similar to that of mammalian orexins. Xenopus orexin-A and -B specifically bind and activate human orexin receptors expressed in Chinese hamster ovary cells. Of interest, Xenopus orexin-B had several-fold higher affinity to human OX2R compared with human orexins. These results suggest that Xenopus orexin-B might be a useful pharmacological tool as an OX2R selective high-affinity agonist.