Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

The beta subunit of tryptophan synthase. Clarification of the roles of histidine 86, lysine 87, arginine 148, cysteine 170, and cysteine 230

Our studies, which are aimed at understanding the catalytic mechanism of the beta subunit of tryptophan synthase from Salmonella typhimurium, use site-directed mutagenesis to clarify the functional roles of several putative active site residues. Although previous chemical modification studies have suggested that histidine 86, arginine 148, and cysteine 230 are essential residues in the beta subunit, our present findings that beta subunits with single amino acid replacements at these positions have partial activity show that these 3 residues are not essential for catalysis or substrate binding. These conclusions are consistent with the recently determined three-dimensional structure of the tryptophan synthase alpha 2 beta 2 complex. Amino acid substitution of lysine 87, which forms a Schiff base with pyridoxal phosphate in the wild type beta subunit, yields an inactive form of the beta subunit which binds alpha subunit, pyridoxal phosphate, and L-serine. We also report a rapid and efficient method for purifying wild type and mutant forms of the alpha 2 beta 2 complex from S. typhimurium from an improved enzyme source. The enzyme, which is produced by a multicopy plasmid encoding the trpA and trpB genes of S. typhimurium expressed in Escherichia coli, is crystallized from crude extracts by the addition of 6% poly(ethylene glycol) 8000 and 5 mM spermine. This new method is also used in the accompanying paper to purify nine alpha 2 beta 2 complexes containing mutant forms of the alpha subunit.     

Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar).

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.     

Electron microscopic radioautographic study of the localization of an anti-allergic agent, Tranilast, in rat mast cells.

We have previously reported that Tranilast, an anti-allergic agent, was rapidly taken into the cytoplasm of rat mast cells in vitro by means of light microscopic radioautography. The present study was performed at the electron microscopic level to elucidate the fine localization of this agent in the mast cells. The results revealed that the number of radioautographic silver grains in the cells increased by the incubation with 3H-labelled Tranilast for 0 to 60 min. and that many silver grains were localized on the specific granules, especially on the perigranular membranes. These results suggest that the mode of inhibitory action of mast cell degranulation by Tranilast is related to the specific localization of this agent on the perigranular membranes.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures I. A PKH26 Labeling Study in Microexplant and Organotypic Cultures

Granule cells were dissociated from early postnatal mouse cerebella and labeled with a fluorescent dye probe PKH26. Small number of the labeled cells were mixed with cerebellar cortical microexplant cultures or transplanted into cerebellar cortical organotypic explants, and their time-dependent morphological changes during cultures were examined with fluorescence microscopy. Granule cell neurons first extended asymmetrical short bipolar processes in both cultures, and migrated actively in microexplant cultures. After elongation of symmetrically bipolar long and thin neurites, they sprouted short thick processes from cell bodies and migrated perpendicular to neurite bundles that were devoid of glia in microexplant cultures, or migrated vertically inward into the internal granular layer in the organotypic explant. During such migrations, they extended short thick processes in front and thin processes behind the cell body. The latter processes were connected to thin long neurites with T- or Y-shaped junctions in both cultures. Finally, they extended many short thick processes from cell bodies in both cultures. Such behaviors of granule cell neurons in microexplant cultures were, thus, similar to those in organotypic explant cultures despite of the absence of Bergmann glial cells. These migration patterns may be closely related to migration of granule cells in histogenesis of the cerebellar cortex.     

Rhodium-catalyzed synthesis of 2(5H)-furanones from terminal alkynes and non-substituted alkynes under water-gas shift reaction conditions

Rhodium-catalyzed synthesis of 2(5H)-furanones from alkynes under water-gas shift reaction conditions was studied. By improving the reaction conditions for internal alkynes reported previously, the reaction could be extended to terminal alkynes. Terminal alkynes are selectively converted into 3- and 4-substituted 2(5H)-furanones (2 and 3). When acetylene itself is used, 2(5H)-furanone (2n) is obtained in a good yield. Examination of reaction solutions by IR spectroscopy and some other experimental findings suggest that the active species would be an alkyne-coordinated monomeric rhodium anion. A new reaction path is proposed.     

Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

Effects of body and head positions on bilateral difference in tympanic temperatures

We have examined the nonparallel changes in tampanic membrane temperatures (T _ty) from the two ears in response to various changes in body and head positions. Upon assuming a lateral recumbent position, the T _ty on the lower side increased while that on the upper side decreased. Pressure application over a wide area of the lateral chest only caused inconsistent and obscure asymmetric changes in T _ty. A lateral flexion of the head with the subject sitting upright and a rotation of the head to the side in a supine position induced an increase in the T _ty on the lower side compared to that on the upper side. The temperature and blood flow of the forehead often decreased on the lower side and increased on the upper side, although such responses were not always concomitant with the asymmetric changes in T _ty. A dorsal flexion of the head with the subject in a reclining position caused a slight increase in the T _ty, whereas raising the head upright induced a slight decrease in them. Two additional experiments were carried out with single photon emission computed tomography using ^99mTc-hexamethylpropyleneamine oxime as tracer, and a slight, relative decrease in counts was noted in the right hemisphere during rotation of the head to the right. These results would strongly suggest that unilateral increases and decreases in T _ty could have been caused by one-sided decreases and increases, respectively, in blood flow to the brain and/or the tympanic membrane, induced by a vasomotor reflex involving vestibular stimulation.     

The structure of a local population and dispersal pattern in the Styan's grasshopper warbler,Locustella pleskei

The breeding sites of Styan's grasshopper warblers are restricted to small islands and distributed discretely. The study on the marked population was conducted on two islets, Okitsu-jima and Ohtsukue-jima, located near Fukuoka, Japan, from 1981 to 1989. About 20 and 70 birds bred on the two islets, respectively, and the mean density of breeding pairs was 25.5 ha⁻¹. The dispersal pattern and population, structure were examined. Adult birds returned to the same breeding site. Males always reoccupied the same territory as in previous years but females not. Males were more philopatric and only female yearlings dispersed over long distances and moved between the breeding sites in Hakata Bay. The returning rate was 0.57 for adults, and 0.21 for juveniles. The age distribution of birds at Ohtsukue-jima was almost stable, while that at Okitsu-jima was unstable because it was a marginal habitat for the birds. Estimating from the dispersal pattern and age structure of this warbler the population near Fukuoka consists of only four islets and is almost stable.