Structure and Chromosomal Assignment of the Human S1-5 Gene (FBNL) That Is Highly Homologous to Fibrillin

The human S1-5 gene (fibrillin-like; FBNL) was originally isolated from a subtractively enriched cDNA library established from a subject with Werner syndrome (WS). We isolated genomic clones containing the entire S1-5 gene and determined its genomic structure including the exon–intron organization. The gene spanned approximately 18 kb of genomic DNA and consisted of 12 exons. Its expression was abundant in all tissues examined except brain and peripheral leukocytes, where it was undetectable. In addition, we have mapped S1-5 by fluorescencein situhybridization to chromosome 2p16, a position that excludes it as a candidate for WS. Our data should facilitate an understanding of the function and regulation of S1-5 in human tissues.     

Antioxidative Activity of 5,6,7,8-Tetrahydrobiopterin and its Inhibitory Effect on Paraquat-Induced Cell Toxicity in Cultured Rat Hepatocytes

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (MξPMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the MξPMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Characterization of a Chlamydia pneumoniae Strain Isolated from a 57-Year-Old Man

The isolation of Chlamydia pneumoniae, especially from elderly persons, is generally not easy. Recently, we succeeded in isolating a chlamydial strain, which was designated KKpn-15, from a 57-year-old man suffering from acute bronchitis. It was compared with well established strains of C. pneumoniae, C. trachomatis and C. psittaci, and its biological properties, such as the morphology of elementary bodies (EBs) and inclusions, and the immunochemistry of EB proteins, were investigated. Based on the results obtained in the present study, it was confirmed that the new chlamydial strain, KKpn-15, is a member of the C. pneumoniae strain and that the organisms of KKpn-15 are useful as an antigen for the serodiagnosis and epidemiology of C. pneumoniae infection.     

Study on measurement of δ-aminolevulinic acid in plasma by high-performance liquid chromatography

A method for the determination of δ-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent δ-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of δ-aminolevulinic acid in plasma was 0.01 μg/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of δ-aminolevulinic acid injected from 0.01 to 0.5 μg/ml (r = 0.999). The recovery of 0.05 and 0.1 μg/ml of δ-aminolevulinic acid added to plasma with various concentrations of δ-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of δ-aminolevulinic acid in plasma.     

The giant panda is closer to a bear,judged by α- and β-hemoglobin sequences

Summary Using - and -hemoglobin sequences, we made a maximum likelihood inference as to the phylogenetic relationship among carnivores, including the two pandas, giant and lesser. Molecular phylogenetic studies up to 1985 had consistently indicated that the giant panda is more closely related to the bear than to the lesser panda. In 1986, however, a contradictory tree was constructed, using hemoglobins and so on, in which the two pandas link together (Tagle et al. 1986). In contrast to that tree, our conclusion supports the close relationship between bear and giant panda. The surface impression of a close relationship between the two pandas drawn from pairwise amino acid differences is explained by a rapid rate of hemoglobin evolution in the bear compared to that in the two pandas.Offprint requests to: T. Hashimoto     

Glycosphingolipids of Human Peripheral Nervous System Myelins Isolated from Cauda Equina

Abstract: Compositions of neutral and sulfated glucuronyl glycosphingolipids purified from human motor and sensory nerves and myelins were studied. Higher neutral glycosphingolipids (fraction B), which were separated from GazlCer (fraction A), were analyzed by TLC and TLC-immunostaining. Both nerve myelins contained paragloboside (nLc₄Cer) and nLc₆Cer dominantly as major higher glycosphingolipids and very little globoside (Gb₄Cer), whereas both nerves contained Gb₄Cer and nLc₄Cer. Besides these major glycosphingolipids, a neutral glycolipid containing asialoGMI (Gg₄Cer) epitope and other minor components such as ceramide trihexoside and ceramide dihexoside were detected in both nerves and their myelins. Furthermore, sulfated glucuronyl nLc₄Cer and n Lc₆Cer, which were monoclonal antibody HNK-1 reactive glycolipids, were detected in both nerves and myelins.