Cloning and functional characterization of DSCAML1, a novel DSCAM-like cell adhesion molecule that mediates homophilic intercellular adhesion.

DSCAM, a conserved gene involved in neuronal differentiation, is a member of the Ig superfamily of cell adhesion molecules. Herein, we report the functional characterization of a human DSCAM (Down syndrome cell adhesion molecule) paralogue, DSCAML1, located on chromosome 11q23. The deduced DSCAML1 protein contains 10 Ig domains, six fibronectin-III domains, and an intracellular domain, all of which are structurally identical to DSCAM. When compared to DSCAM, DSCAML1 protein showed 64% identity to the extracellular domain and 45% identity to the cytoplasmic domain. In the mouse brain, DSCAML1 is predominantly expressed in Purkinje cells of the cerebellum, granule cells of the dentate gyrus, and in neurons of the cerebral cortex and olfactory bulb. Biochemical and immunofluorescence analyses indicated that DSCAML1 is a cell surface molecule that targets axonal features in differentiated PC12 cells. DSCAML1 exhibits homophilic binding activity that does not require divalent cations. Based on its structural and functional properties and similarities to DSCAM, we suggest that DSCAML1 may be involved in formation and maintenance of neural networks. The chromosomal locus for DSCAML1 makes it an ideal candidate for neuronal disorders (such as Gilles de la Tourette and Jacobsen syndromes) that have been mapped on 11q23.     

Further studies on the red cell glycolipids of various breeds of dogs. A possible assumption about the origin of Japanese dogs

Genetic polymorphism was observed in the sialic acid species constituting the terminal sugar residues of hematosides from dog erythrocytes. One was N-acetylneuraminic acid and the other phenotype was N-glycolylneuraminic acid, regulated by an autosomal dominant allele (Yasue, S., Handa, S., Miyagawa, S., Inoue, J., Hasegawa, A., & Yamakawa, T. (1978) J. Biochem. 83, 1101-1107). In this study we analyzed blood samples from 1,591 dogs of 36 breeds and demonstrated that the expression of N-glycolylneuraminic acid was limited to several breeds of oriental dogs in spite of its dominant nature. Moreover, the incidence of N-glycolylneuraminic acid was higher in native breeds of northern China, Korea and the southern part of Japan than in other oriental breeds. On the other hand, the Hokkaido-dog is unique in not expressing N-glycolylneuraminic acid. These results suggest that the native breeds in the southern part of Japan came from northern China via the Korean peninsula in contrast with indigenous breeds of the northern part of Japan.     

Two active forms of valyl-tRNA synthetase from Bacillus subtilis: alteration related to the early stages of sporulation

Two enzymatically active forms, E-I and E-II, of valyl-tRNA synthetase [EC 6.1.1.9] from cells at various stages in the life cycle of Bacillus subtilis 168 LTT, germinated cells, vegetative cells (t-0.5), sporulating cells (t0, t1, t3, and t4), forespores and mature spores, were analyzed by hydroxyapatite column chromatography. The E-II activity was detected in the main fraction of valyl-tRNA synthetase during the life cycle of B. subtilis 168 LTT. The high activity of E-II at t0 decreased rapidly in the stationary and sporulating phases. On the other hand, the E-I activity increased in the early sporulating stage and was about twofold higher at t3 than at t0. After t3, this activity also decreased rapidly and was not detected in forespores and mature spores. The relative amount of E-I at t0 was 3.4% of the total valyl-tRNA synthetase activity eluted from the hydroxyapatite column, 12.9% at t1 and 29.2% at t3, but it was less than 10% at t4 and in germinated cells. The alteration in E-I and E-II activities was also observed in cells of B. subtilis NIG 1121 (spo+), W23 and 168W, but not in any asporogenous mutant strain studied. These results show that the alteration in the valyl-tRNA synthetase activity appears only during the early stages of sporulation and is closely related to the sporulation of B. subtilis.     

Biochemical studies in cat and human gangliosidosis

The biochemical analysis of the hereditary neurological disease found in a family of Siamese cat is reported. The accumulation of GM₁ganglioside in the brain was noted. Several glycosidase activities of these cat brains were compared with that of human gangliosidoses (Tay-Sachs disease and GM₁-gangliosidosis). Glycosidase activities were estimated using ρ-nitrophenyl-glycosides, glucosyl-, galactosyl-ceramide and GM₁-ganglioside as substrates. The results indicated the defect of the β-galactosidase activities for the ρ-nitrophenyl-β-galactoside and GM₁-ganglioside in both cat and human GM₁-gangliosidoses. Glycosidase activities for glucosyl- and galactosyl-ceramide were not changed in either gangliosidoses.     

Induction of porphyrin synthesis in chick embryo liver cell culture by synthetic polychlorobiphenyl isomers

The effects of a series of synthetic di-tetra- and hexachlorobiphenyl isomers and commercial polychlorinated biphenyls on the porphyrin biosynthesis in chick embryo liver cells in culture were examined.It was found that 3,4,3′,4′-tetra- and 3,4,5,3′,4′,5′-hexachlorobiphenyl isomers were the most active inducers, which were approximately 20 times as active as 1,4-dihydro-3,5-dicarbethoxy-2,4,6-trimethylpyridine (DDC) in porphyrin production. 3,5,3′,5′-Tetra- and 2,3,4,2′,3′,4′-hexachlorobiphenyl isomers were moderate inducers, which were approximately 2.0 to 2.5 times as active as DDC. 2,4,6,2′,4′,6′-Hexachlorobiphenyl showed the same activity as DCC. Compounds such as 4,4′-di-, 2,3,2′,3′-, 2,4,2′,4′- and 2,6,2′,6′-tetrachlorobiphenyl were weak inducers and 2,5,2′,5′-tetrachloro- and decachlorobiphenyl isomers were found to be inactive. Kanechlor-400 was the strongest inducer among the commercial polychlorinated biphenyls investigated.The structural requirements for potent porphyrin-inducing activity of chlorobiphenyl isomers were found to be the para and meta substituted structure causing a more highly conjugated and nearly coplanar conformation. It was found that induction caused by some chlorobiphenyls was subject to feed-back repression by end-product heme. In addition, the metabolism of chlorobiphenyls in mice was influenced by the unsubstituted pairs of carbon atoms in the molecule. These results lead us to postulate the following hypothesis, namely, that strong inducers may displace heme directly and incorporate into a hydrophobic pocket of the apo-represor protein, thus causing an induction of δ-aminolevulinic acid synthetase.     

Prevalence of Clostridium botulinum type E and coexistence of C. botulinum nonproteolytic type B in the river soil of Japan.

Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum. Type E organism was prevalently shown throughout the whole river systems including upper part; detection rates of type E toxin in soil culture ranged from 33 to 82%. This type was also detected in soil of adjacent mountainous district. Type B and C toxins were detected at 7% and 9% of the sites examined, respectively. C. botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples. These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C. botulinum nonproteolytic type B exists in the soil of Japan.     

Conversion of deoxycorticosterone to 3-keto-4-etienic acid, catalyzed by a partially purified preparation from bovine adrenocortical mitochondria.

Bovine adrenocortical mitochondria were sonicated and subjected to extraction with sodium cholate. The extract contained not only cytochrome P-450 activities, but also an activity which catalyzed the conversion of deoxycorticosterone to an unknown steroid (designated X). The latter activity was concentrated by (NH4)2SO4 fractionation in the presence of sodium cholate, and separated from P-450 by taking advantage of their different solubilities in phosphate buffer without sodium cholate. The specific activity of the partially purified enzyme fraction was 70 times higher than that of sonicated mitochondria. The conversion of deoxycorticosterone to steroid X required NAD or NADP. The conversion rate was dependent on the concentration of deoxycorticosterone. The major product, steroid X, was isolated from the reaction mixture by means of silicic acid and Iatrobeads column chromatography. The steroid was characterized as 3-keto-4-etienic acid (3-oxoandrost-4-ene-17beta-carboxylic acid). This result suggests that an enzyme system for the conversion of deoxycorticosterone to 3-keto-4-etienic acid exists in adrenocortical mitochondria.     

Secretion of TNF-α from macrophages following induction with a lignin derivative.

Macrophages derived from rat bone marrow were treated with macrophage colony stimulating factor (M-CSF) to obtain a sufficient number of cells for the tumor necrosis factor (TNF-α) assay. The present study has been designed to investigate whether the production of TNF-α, which induces multinucleated giant cell formation, is regulated by polyanions such as lignin derivatives. ELISA for TNF-α showed that the polyanion induced TNF-α production by macrophages. The secretion of TNF-α from the cells reached a maximum at 3-6 h, and then showed a slight decline. Northern blotting of TNF-α mRNA showed that the amount of TNF-α reached a maximum within 1 h of macrophage culture in the presence of a lignin derivative. On the other hand, TNF-α mRNA was undetectable in the control cells. It was concluded that stimuli such as that provided by lignin derivatives increases the amount of TNF-α mRNA, which is then followed by translation of TNF-α.     

THE INCORPORATION OF LABELLED ACETATE INTO CEREBROSIDE AND OTHER LIPIDS OF THE DEVELOPING MOUSE BRAIN

—Cerebroside in the brain is highly localized in myelin and has a relatively slow turnover rate. The aim of this study was to evaluate the true cerebroside biosynthetic activity under conditions in which the degradation and reutilization of brain lipids were as small as possible. The 3-week-old mice were decapitated at 0·5, 1, 2·5, 5 and 15 min after the intraperitoneal injection of labelled acetate and the incorporation of radioactivity into each lipid class was examined. Even at 0·5 min, a considerable amount of radioactivity was found in simple lipids, especially in the free fatty acid fraction, and in the course of time the radioactivity of complex lipids increased. On the other hand, the incorporation of radioactivity into cerebrosides was extremely small throughout the experimental period. Results indicated that the low radioactivity of cerebroside might be due to its high content of long-chain fatty acids which were weakly labelled. The radioactivity of the sphingosine moiety was also low. In short, one of the rate-limiting steps of cerebroside synthesis in brain might exist in long-chain fatty acid and sphingosine synthesis. In addition, the incorporation curves of each component of cerebroside were compared with each other and the difference of the incorporation pattern of non-hydroxy fatty acids of cerebroside was noted.