Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett 342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.Abbreviations MAH Maackia amurensis haemagglutinin.     

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.     

Protein synthesis in the embryo ofPinus thunbergii seed III.

Dissociability of the monomer ribosomes prepared from dry and imbibed pine (Pinus thunbergii) seed embryos was analyzed in sucrose density gradient containing a high salt buffer. Abnormal dissociation into the subunits was observed with the ribosome preparation from dry seed embryos when compared with that from imbibed seed embryos, i.e. each subunit peak was broader and localized at a lower site in sucrose density gradient. This indicates some change(s) in ribosomes during imbibition of seeds. These ribosomal changes also progressedin vitro. That is, after incubation of ribosome preparation from dry seed embryos in a high salt buffer for 5 min at 30 C or in a low salt buffer for 15 hr at 0 C, complete dissociation into the normal subunits was observed. No difference was found between polyacrylamide gel electrophoresis patterns of ribosomal RNA from dry and imbibed seed embryos. These results suggest some alteration in the protein components of ribosome during imbibition of pine seeds. This paper is dedicated to Prof. Shyogo Sawamura, Utsunomiya University on his retirement in March, 1979.     

Dynamic function of the spacer region of acetogenins in the inhibition of bovine mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Studies of the action mechanism of acetogenins, the most potent and structurally unique inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), are valuable in characterizing the inhibitor binding site in this enzyme. Our previous study deepened our understanding of the dynamic function of the spacer region of bis-THF acetogenins [Abe, M., et al. (2005) Biochemistry 44, 14898-14906] but, at the same time, posed new important questions. First, while the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings) span a distance shorter than that of the extended 13 carbon atoms [-(CH 2) 13-], what is the apparent optimal length of the spacer for the inhibition of 13 carbon atoms? In other words, what is the functional role of the additional methylene groups? Second, why was the inhibitory potency of the mono-THF derivative, but not the bis-THF derivative, drastically reduced by hardening the spacer covering 10 carbon atoms into a rodlike shape [-CH 2-(C identical withC) 4-CH 2-]? This study was designed not only to answer these questions but also to further disclose the dynamic functions of the spacer. We here synthesized systematically designed acetogenins, including mono- and bis-THF derivatives, and evaluated their inhibitory effects on bovine complex I. With regard to the first question, we demonstrated that the additional methylenes enhance the hydrophobicity of the spacer region, which may be thermodynamically advantageous for bringing the polar gamma-lactone ring into the membrane-embedded segment of complex I. With regard to the second question, we observed that a decrease in the flexibility of the spacer region is more adverse to the action of the mono-THF series than that of the bis-THF series. As a cause of this difference, we suggest that for bis-THF derivatives, one of the two THF rings, being adjacent to the spacer, is capable of working as a pseudospacer to overcome the remarkable decrease in the conformational freedom and/or the length of the spacer. Moreover, using photoresponsive acetogenins that undergo drastic and reversible conformational changes with alternating UV-vis irradiation, we provided further evidence that the spacer region is free from steric congestion arising from the putative binding site probably because there is no receptor wall for the spacer region.     

Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors

We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have a functional impact on NGF-Trk-mediated intracellular signal transduction pathway.     

Formation of polar-body-like structures induced in polyspermic starfish oocytes that had been inseminated at the germinal vesicle stage

Immature starfish oocytes, which are arrested at the first meiotic prophase and contain a large nucleus called the germinal vesicle (GV), are known to accept multiple sperm on insemination. We found that if these polyspermic starfish oocytes are induced to mature, they often form small protrusion(s) adjacent to the first polar body emitted shortly earlier. We refer to these protrusion(s) as 'polar-body-like structures (PLS).' Fluorescent staining of PLS indicated that they were not merely cytoplasmic protrusions, but contained some chromatin. Maturing process of these polyspermic oocytes was examined by immnofluorescent staining, which showed that: (i) numerous sperm asters were observed after the onset of GV breakdown; (ii) before the first polar body (PB1) emission, a complex microtubular structure resembling a multipolar spindle was formed; and (iii) several isolated asters were observed after PB1 emission. These results indicate that PLS formation may be induced by interaction of meiosis-I spindle with paternal centrosomes incorporated at GV stage.     

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.