Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergenCry j I: relationship between the structures and antigenic epitopes of plantN-linked complex-type glycans

The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz¹H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing 1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.Abbreviations Cry j I a major allergenic glycoprotein ofCryptomeria japonica - B-SJA-II Sophora japonica bark lectin II - CTA Clerodendron trichotomum lectin - TFMS trifluoromethanesulfonic acid - HRP horseradish peroxidase     

The Composition of Metals Bound to Class III Metallothionein (Phytochelatin and Its Desglycyl Peptide) Induced by Various Metals in Root Cultures of Rubia tinctorum

The induction of phytochelatins (PCs) and their desglycyl peptides (both are referred to as class III metallothionein [CIIIMT]) by exposure to various metals (Ag+, As3+, As5+, Cd2+, Cu2+, Ga3+, Hg2+, In3+, Ni2+, Pb2+, Pd2+, Se4+, and Zn2+) and the metal composition in the CIIIMTs were investigated in root cultures of Rubia tinctorum L. All of these metal species induced PCs to various degrees when analyzed by the postcolumn derivatization high-performance liquid chromatography method. The desglycyl peptides of PCs often were also present. However, only Ag, Cd, and Cu were bound to the CIIIMTs that they induced when analyzed by the high-performance liquid chromatography-inductively coupled plasma-atomic emission spectrometry method. Cu was also bound to the CIIIMTs induced by Ag+, As3+, and Cd2+. After Ag+ exposure, an Fe peak that may be of Fe-CIIIMT was also observed. However, most of the metal species studied were not bound to the CIIIMTs that they induced.     

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.     

A novel complex mutation in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region

A novel complex mutation with the presence of both deletion and insertion in very close proximity in the same region was detected in exon 8 of the LDL receptor gene from two apparently unrelated Japanese families with familial hypercholesterolemia (FH). In this mutant LDL receptor gene, the nine bases from nucleotide (nt) 1115 to nt 1123 (AGGGTGGCT) were replaced by six different bases (CACTGA), and consequently the four amino acids from codon 351 to 354, Glu-Gly-Gly-Tyr, were replaced by three amino acids, Ala-Leu-Asn, in the conserved amino acid region of the growth factor repeat B of the LDL receptor. The nature of the amino acid substitution and data on the families suggest that this mutation is very likely to affect the LDL receptor function and cause FH. The generation of this complex mutation can be explained by the simultaneous occurrence of deletion and insertion through the formation of a hairpin-loop structure mediated by inverted repeat sequences. Thus this mutation supports the hypothesis that inverted repeat sequences influence the stability of a given gene and promote human gene mutations.     

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.     

A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Metamorphic Changes in Glycolipids and Myelin Proteins and 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Bullfrog and Axolotl Brains

Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed.     

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.