Hideharu Taira

Comparisons of several biological and physicochemical properties of human leukocyte interferons produced by human leukocytes and by E. coli

Human interferon (IFN) prepared from virus-induced human leukocyte suspensions (leukocyte-derived interferon) was compared to the IFN extracted from Escherichia coli harboring a human interferon-alpha cDNA hybrid plasmid (Hif-SN35-AH-L6). E coli-derived IFN was 20 to 50 times more active than leukocyte-derived IFN on heterologous bovine, feline, murine and guinea pig cells, relative to the activity on human cells. After partial purification by affinity chromatography on an anti-human lymphoblastoid IFN antibody column, the IFN was analyzed by SDS-polyacrylamide gel electrophoresis. While leukocyte-derived IFN gave a heterogeneous pattern with major peaks of activity of 24000 and 19000 daltons, E. coli-derived IFN gave a heterogeneous peak of activity at about 17-18000 daltons. The leading edge of leukocyte-derived IFN in SDS-polyacrylamide gels was significantly more active on bovine cells than on human cells and coincided in mobility with E. coli-derived IFN, which was also much more active on bone than on human cells. After reduction with mercaptoethanol in SDS, the E. coli-derived IFN lost no activity, whereas the leukocyte-derived IFN lost about 90% of its activity. After reduction, E. coli-derived IFN migrated in SDS-polyacrylamide gels as a single peak at 24000 daltons, as did the residual activity of reduced leukocyte-derived interferon. Out data suggest that the interferon produced by the E. coli harboring the clone Hif-SN35-AH-L6 is analogous in size and cross-species activity to one of the molecular species of leukocyte-derived interferon.

The Biological Function of Cauxin, a Major Urinary Protein of the Domestic Cat (Felis catus)

A major protein component of domestic cat urine is the carboxylesterase family member termed cauxin. Cauxin is secreted into the urine from the proximal straight tubular cells of the kidney, and the level of cauxin excretion is species-, sex-, and age-dependent. Cauxin is excreted in large amounts in the closely related members of the Felidae lineage, the cat (Felis catus), bobcat (Lynx rufus), and lynx (Lynx lynx). Male and female immature cats begin excreting cauxin about 2.5 months after birth, and excretion levels increase with age. In mature cats, cauxin excretion is significantly higher in intact males than in castrated males or female cats. The physiological function of cauxin is to provide species-, sex-, and age-dependent regulation of 2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid (felinine) production. Cauxin hydrolyzes the peptide bond of the felinine precursor, 3-methylbutanol-cysteinylglycine, to produce felinine and glycine. The sulfur-containing volatile compounds, 3-mercapto-3-methyl-1-butanol, 3-mercapto-3-methylbutyl formate, 3-methyl-3-methylthio-1-butanol, and 3-methyl-3-(2-methyl-disulfanyl)-1-butanol, are identified as species-specific odorants and candidates of felinine derivatives from the headspace gas of cat urine. These cat-specific volatile compounds may represent pheromones used as territorial markers for conspecific recognition or reproductive purposes by mature cats. The elucidation of cauxin-dependent felinine production provides new evidence for the existence of species-specific odorants and pheromones produced by species-specific biosynthetic mechanisms in mammalian species.

The Roles of Individual Cysteine Residues of Sendai Virus Fusion Protein in Intracellular Transport

Abstract The role of intramolecular disulfide bonds in the fusion (F) protein of Sendai virus was studied. The 10 cysteine residues were changed to serine residues using site-directed mutagenesis. None of the cysteine mutant F proteins reacted with a monoclonal antibody specific for the mature conformation of the F protein, but eight of ten mutants reacted with an immature conformation-specific monoclonal antibody. The transport of these mutant proteins to the cell surface was drastically reduced. All of the cysteine mutant F proteins remained sensitive to endoglycosidase H (endo H) for 3 h after their synthesis. Moreover, cell surface transport of the hemagglutinin-neuraminidase (HN) protein co-expressed with each of these cysteine mutant F proteins was also reduced. These results suggest that all cysteine residues participate in the formation of intramolecular disulfide bonds, that co-translational disulfide bond formation is crucial to the correct folding and intracellular transport of the F protein, and that interaction of the F and HN proteins takes place intracellulary.

Construction of expression plasmids for the fusion protein of Sendai virus, and their expression in E. coli cells and eucaryotic cells

To examine the properties and the role of the fusion protein (F) of Sendai virus at the molecular level, a plasmid, pUC‐F, was constructed by inserting cDNA for the F protein into a pUC vector. Upon induction of E. coli cells transformed with pUC‐F, a new protein was obtained, which was identified as Fo on Western blot analysis. The cDNA fragment for the F gene was excised from pUC‐F and inserted into an eucaryotic expression vector, pSVL, to yield pSVL‐F. COS‐1 cells transfected with pSVL‐F gave a band on SDS‐gel electrophoresis which corresponded to the size of the Fo protein.

Interaction between Elongation Factors 1β and 1γ fromBombyx moriSilk Gland

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: α (51 kDa), β (26 kDa), γ (49 kDa), and δ (33 kDa). The EF-1α subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1α-bound GDP is then exchanged for GTP by the EF-1βγδ complex. To facilitate analysis of the roles of the individual EF-1β, γ, and δ subunits in GDP/GTP exchange on EF-1α, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1β, EF-1γ, and the carboxyl-terminal half of EF-1δ were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1β and γ subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1γ relative to EF-1β was also detected. The amino-terminal region of EF-1β (amino acid residues 1-129) was sufficient for binding to EF-1γ. The carboxyl-terminal half of EF-1δ did not appear to form a complex with EF-1γ.