Salt tolerance and glycerol accumulation of a respiration-deficient mutant isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii

A respiration-deficient (RD) mutant was isolated from the petite-negative, salt-tolerant yeast Zygosaccharomyces rouxii. One strain among sixteen glycerol-non-utilizing mutants exhibited vigorous liberation of CO2 but no uptake of O2. Furthermore, this strain lacked cytochrome aa3 and had a reduced level of cytochrome b. The few mitochondria found in cells of this strain contained few or no cristae. Salt tolerance and intracellular accumulation of glycerol by the RD strain were almost equal to that of the wild-type strain in media containing NaCl up to 2.5 M. In media with more than 3 M NaCl, the growth of the RD mutant was retarded and the intracellular accumulation of glycerol was depressed in spite of ample production.     

DEVELOPMENTAL STUDIES IN PORPHYRA (BANGIOPHYCEAE). II. CHARACTERIZATIO OF THE MAJOR LECTIN BINDING GLYCOPROTEINS IN DIFFERENTIATED REGIONS OF THE PORPHYRA PERFORATA THALLUS1

Detergent soluble extracts of differentiated regions of the Porphyra perforata J. Ag. thallus (holdfast, rhizoidal, vegetative and reproductive cells) were fractionated on sodium dodecyl sulfate polyacrylamide gels. Glycoproteins were identified by their lectin affinity. Extracts from all areas of the thallus contained glycoproteins, but the staining patterns were different for each region with each of the lectins tested: concanavalin A, Ulex europeaus agglutinin, Ricinus communis agglutinin, soybean agglutinin and peanut agglutinin. These data indicate that the morphologically distinct regions of the thallus also differ biochemically. Analysis of the lectin blots revealed the presence of tissue-specific glycoproteins in the five thallus areas. Such unique glycoproteins could be used as markers of differentiation in this species.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Mutagenesis of human granulocyte colony stimulating factor

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.     

Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and