Hisako Kojima

Proposal for a procedure for complete inactivation of the Creutzfeldt-Jakob disease agent

We have examined complete inactivation conditions on brain homogenates from mice affected with Creutzfeldt-Jakob disease agent, and recommend for routine use a reliable procedure first treating the affected materials with 1 N NaOH for 60 min and then autoclaving at 121 degrees C for 30 min.

Phylogenetic relation of lungfish indicated by the amino acid sequence of myelin DM20

The cDNA of lungfish Protopterus annectens myelin DM20 was cloned, and the complete amino acid sequence of Protopterusannectens DM20 was deduced. When five possible phylogenetic trees were tested for the DM20 sequences, the maximum likelihood method supported tree 1 [((tetrapods, lungfish), coelacanth), zebrafish, shark] or tree 5 [(tetrapods, lungfish), (coelacanth, zebrafish), shark]. Both tree 1 and tree 5 indicate that lungfish is phylogenetically the closest to tetrapods among the living fishes.

Alterations in the composition of brain lipids in patients with Creutzfeldt-Jakob disease ☆

Abstract Brain lipids were isolated from 3 patients with Creutzfeldt-Jakob (C-J) disease and their chemical constituents were investigated. The total lipid content increased slightly in diseased gray matter. Ganglioside was reduced to less than 20 and 50% of that of control values in gray and white matter of the Creutzfeldt-Jakob brains, respectively, on both a wet and dry weight basis. Abnormal thin-layer chromatography patterns were observed in the gangliosides from the diseased brains. The C20-sphingosine in the gangliosides from the Creutzfeldt-Jakob brains was markedly reduced. The ratio of C20 to C18-sphingosine was 0.03 to 0.23 in the diseased gray matter. An alteration in the fatty acid composition of ganglioside was observed in 1 case of C-J disease. The total cholesterol content increased both in gray and white matter of the Creutzfeldt-Jakob brain, and one-third of the total cholesterol was in the esterified form in gray matter. Major fatty acids of cholesterol ester were palmitic and oleic acids. The total phospholipids were severely decreased in the diseased brains, but the relative proportions of the individual phospholipids were constant. Polyunsaturated fatty acids in phosphatidyl ethanolamine from the diseased gray matter decreased with a moderate increase in oleic acid. Galactolipid levels and types were essentially unchanged. From the above described abnormalities of brain lipids, the pathogenesis of this disease is discussed.

Suppression of CYP3A2 mRNA expression in the warfarin-resistant roof rat, Rattus rattus: possible involvement of cytochrome P450 in the warfarin resistance mechanism

1. The continual use of warfarin as a rodenticide has caused the development of populations of warfarin-resistant roof rat. To study the biochemical mechanism of warfarin resistance, the mRNA expression levels of the major P450 forms in the warfarin-resistant and -susceptible roof rat liver following exposure to warfarin were quantified by competitive RT-PCR. 2. The constitutive levels of CYP2C11 and CYP3A2 mRNAs in the warfarin-resistant and -susceptible roof rat were extremely low compared with those in the SD rat. In response to warfarin administration, the CYP3A2 mRNA level in the warfarin-susceptible rat increased to about 3-fold of that before the treatment, whereas in the warfarin-resistant roof rat, CYP3A2 mRNA remained at a low level. 3. The present results suggest the possibility that reduced synthesis of CYP3A2 mRNA is involved in the warfarin-resistant mechanism in the roof rat.

Neonatal exposure to endocrine disruptors suppresses juvenile testis weight and steroidogenesis but spermatogenesis is considerably restored during puberty.

Neonatal exposure to endocrine disruptors induces developmental abnormalities in the male reproductive system. As to investigate whether neonatal exposure affects spermatogenesis in juvenile and pubertal testes, Sprague-Dawley rat pups were given various endocrine disruptors by a single injection on the day of birth at concentrations ranging between 4 microM and 40 mM and sacrificed on day 21 (juvenile) or 50 (puberty). The testes were weighed and examined histologically at each stage. Further, the metabolites of steroidogenesis were analyzed using normal-phase high performance liquid chromatography. Neonatal exposure significantly reduced testis weights and steroid biosynthesis of juveniles, but they were highly restored at puberty.

A monoclonal antibody against a glycolipid SEGLx from Spirometra erinaceieuropaei plerocercoid

A mouse monoclonal antibody AK97 (IgM) was established against a new type of glycosphingolipid, SEGLx, isolated from plerocercoids of tapeworm, Spirometra erinaceieuropaei. The chemical structure of SEGLx (Gal beta1-4(Fuc alpha1-3)(Glc beta1-3Gal beta1-ceramide) had been previously characterized. The specificity of AK97 was determined by thin-layer chromatography-immunostaining and enzyme-linked immunosorbent assay. AK97 was found to be directed to SEGLx and GalSEGLx (Gal beta1-4(Fuc alpha1-3)Glc beta1-3(Gal beta1-6)Gal beta1-ceramide) and also showed cross-reactivity with the stage specific embryonic antigen-1 (SSEA-1), the epitope being defined to be the non-reducing terminal trisaccharide sequence. On immunohistochemical examination, AK97 predominantly stained the tegument, the external surfaces of worms which have a brush border-like organization. Based on the immunohistochemical findings for the staining liability as to organic solvents and the results of Western blot analysis of the plerocercoid glycoproteins, it was proved that the antigens in the tapeworm were glycolipids. Considering that the tapeworm is in direct contact with its hosts tissue through the tegument, the membrane surface of which is exposed to the external environment, it is suspected that SEGLx and GalSEGLx on the tegument play functionally important roles in the host parasite interaction.

Gene Structure and Amino Acid Sequence of Latimeria chalumnae (Coelacanth) Myelin DM20: Phylogenetic Relation of the Fish

The structure of Latimeria chalumnae (coelacanth) proteolipid protein/DM20 gene excluding exon 1 was determined, and the amino acid sequence of Latimeria DM20 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 Latimeria DM20 gene is a single-copy gene. When the amino acid sequences of DM20 were compared among various species, Latimeria was 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. Latimeria was situated outside of lungfish by the maximum likelihood method. The apparent similarity of Latimeria DM20 to tetrapod proteolipid protein/DM20 is explained by the slow amino acid substitution rate of Latimeria DM20.

Subcellular Distribution of the Transmissible Agent in Creutzfeldt-Jakob Disease Mouse Brain

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.

Neutral Lipid and Sphingolipid Composition of the Brain of a Patient with Membranous Lipodystrophy

SummaryLipids were extracted from brains of a patient with membranous lipodystrophy (ML) and three normal patients and the neutral lipid and sphingolipid constituents were investigated. The storage of a large amount of free fatty acid was observed in the ML brain, but no cholesterol ester was found. Total lipid, cholesterol and cerebroside contents were slightly decreased in the white matter of the ML brain. The compositions of free and sphingolipid fatty acids did not differ between ML and normal brains.ZusammenfassungIm Gehirn einer sogenannten „Membranolipodystrophie“ (ML) und in drei normalen Gehirnen wurden Neutrallipide und Sphingolipide vergleichend analysiert. Eine große Menge Frei-Fettsäure wurde beobachtet, aber Cholesterolester fand sich weder in der grauen noch in der weißen Substanz des ML-Gehirns. In der weißen Substanz des ML-Gehirns wurde eine geringe Abnahme des Cholesterins, der Cerebroside und auch Totallipide im Vergleich mit den normalen Gehirnen beobachtet. Es gab nicht eine bedeutsame Differenz zwischen ML und den normalen in der Komposition der Frei-und Sphingolipide-Fettsäure.

Monohexosylceramides of larval and adult forms of the tapeworm,spirometra erinacei

The occurrence of glycosphingolipids with unique carbohydrate structures in different species of cestode, Platyhelminth, which had been shown, previously, prompted us to study the molecular species of the monohexosylceramides (cerebrosides) in the pseudophyllidean cestode,Spirometra erinacei. The purpose of the study was to obtain a basis for future investigations of the physiological role of glycolipids in parasitism. Cerebrosides were isolated froms. erinacei at two growth stages, i.e., from the larval form (plerocercoid) and from the adult tapeworms (intestinal form). The cerebrosides were separated into four subfractions by silica gel column chromatography, and their constituents were analyzed by gasliquid chromatography, gas chromatography/mass spectrometry, and high-performance liquid chromatography/mass spectrometry. The hexoses of the cerebrosides consisted primarily of galactose in both growth stages, while only a small amount of glucose was detected. The ceramides were composed of sphinganine (d18∶0) and phytosphingosine (t18∶0) as sphingoid bases, and of nonhydroxy fatty acids ranging from C16 to C30 and hydroxy stearic acid (18h∶0). The cerebrosides of adult tapeworms contained more 18h∶0 than those of plerocercoids. The combination of hexoses and ceramides in the cerebroside molecules was slightly different in the two growth stages: the glucocerebrosides of plerocercoids contained only d18∶0-nonhydroxy fatty acids in their ceramide moieties, whereas those of adult tapeworms contained varying ceramide moieties. Our data indicate that the molecular species of glycolipids present were essentially homeostatic throughout growth in spite of the entirely different environmental conditions, although there were slight differences in the hexose distribution in the two growth stages.