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The structure of Latimeria chalumnae(coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of LatimeriaDM20 corresponding to exons 2–7 was deduced. The nucleotide sequence of exon 3 suggests that only DM20 isoform is expressed in Latimeria.The structure of proteolipid protein/DM20 gene is well preserved among human, dog, mouse, and Latimeria.Southern blot analysis indicates that LatimeriaDM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeriawas more similar to tetrapods than other fishes including lungfish, confirming the previous finding by immunoreactivity (Waehneldt and Malotka 1989 J. Neurochem. 52:1941–1943). However, when phylogenetic trees were constructed from the DM20 sequences, lungfish was clearly the closest to tetrapods. Latimeriawas situated outside of lungfish by the maximum likelihood method. The apparent similarity of LatimeriaDM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of LatimeriaDM20.

Abstract:In mammalian brain, two 2',3'-cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) isoforms, CNP1 and CNP2, are translated, respectively, from the two mRNAs, which have been transcribed and processed by alternative use of the two transcription start points and by differential splicing. In the present study, the cDNAs encoding chicken CNP2 and bullfrog CNP1, respectively, were isolated, and the amino acid sequences of chicken CNP2 and bullfrog CNP1 were deduced. Western blot analysis showed that chicken brain contains a major CNP2-type protein together with a minor unidentified isoform, and bullfrog brain contains only a CNP1-type protein. All available amino acid sequences of vertebrate 2',3'-cyclic-nucleotide 3'-phosphodiesterases were aligned and compared. Three conserved motif sequences were noted: (a) an ATP-binding site near the amino terminus, (b) an isoprenylation site at the carboxyl terminus, and (c) a probable catalytic site resembling the active site of ß-ketoacyl synthase (EC 2.3.1.41). The second and the third motifs are conserved also in goldfish RICH (regeneration-induced 2',3'-cyclic-nucleotide 3'-phosphodiesterase homologue), which has been shown recently to have 2',3'-cyclic-nucleotide 3'-phosphodiesterase activity. The third motif (probably catalytic site) was assigned for the first time in the present report.

To determine the intracellular localization of the Creutzfeldt‐Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka‐1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.

We have examined the expression of GDP-fucose: glycosphingolipid fucosyltransferase activity in PC12 cells and PC12 sublines in relation to the neuronal differentiation induced by nerve growth factor (NGF) or dexamethasone. Transfer of fucose to paragloboside (nLc4Cer) yielded a product which was determined to be a blood group H1antigen (Fucα1-2Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer) by gas chromatography/mass spectrometry analysis and enzymatic hydrolysis, suggesting that PC12 cells have an α1,2-fucosyltransferase. Lactosylceramide was also fucosylated at a reduced rate. When the differentiation of PC12 cells and PC12 subline cells, PC12D and MR31, was induced by exposure to either NGF or dexamethasone, the fucosyltransferase activity for nLc4Cer was found to decrease in both cell lines, suggesting the association with cell differentiation. This is the first report of the presence of an α1,2-fucosyltransferase in cultured neuronal cell lines which catalyses thein vitrobiosynthesis from nLc4Cer of a type-2 chain glycosphingolipid having the blood group H1determinant. The disaccharides, β-lactose andN-acetyllactosamine, were also fucosylated by PC12 cell enzyme, although the specificity for the carbohydrate structure was different from that for glycosphingolipids.

Rough-surfaced and light and heavy smooth-surfaced microsomes were isolated from rat brain by means of discontinuous sucrose gradient centrifugation. Electron microscopically, the rough-surfaced microsomes were characterized by vesicles with ribosomes and the light and heavy smooth-surfaced microsomes by fairly homogeneous membrane features without ribosomes. The rough-surfaced microsomal membranes were distinguished by the absence of glycolipids, such as ganglioside, cerebroside, and sulfatide. Cerebroside was exclusively recovered in the light smooth-surfaced microsomal membranes. Ganglioside and Na,K-ATPase were contained in larger amounts in the heavy smooth-surfaced microsomal membranes than in the light smooth-surfaced microsomal membranes in terms of protein content. Among the three submicrosomal membranes, cholesterol and phospholipid were found in the largest amounts in the light smooth-surfaced microsomal membranes, where the molar ratio of cerebroside-cholesterol-phospholipid was about 1:10:10. The membranes of rough- and smooth-surfaced microsomes were very similar in regards to the composition of phospholipid classes, although the fatty acid composition of the former contained a greater proportion of unsaturated fatty acids than that of the latter. When the membrane proteins were analyzed by sodium dodecyl sulfate gel electrophoresis, some differences were observed between the light and heavy smooth-surfaced microsomal membranes.

We have examined the distribution and composition of gangliosides in central and peripheral nervous tissues from two patients with Creutzfeldt-Jakob disease (CJD). There were marked decreases in total ganglioside levels in CJD, with reductions in the cerebral cortex and cerebellum in the order of 20–30% and 50% of control values, respectively, though in spinal cord and sciatic nerve total gangliosides were not significantly altered. The percentage distribution of individual gangliosides was characterized by marked increases in GD3 and GD2, contrasting with severe decreases in GD1a, GD1b, GT1b and GQ1b; such changes were found throughout the patients' nervous tissues. An abnormal long-chain base composition was detected with the d20:1 component being decreased to less than 50% of control values, in the cerebrum, cerebellum and spinal cord. Changes in gangliosides occurred even in those tissues not severely affected neuropathogically. These ganglioside abnormalities are discussed in relationship to the pathogenesis of CJD.

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