Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Conformational change of sodium- and potassium-dependent adenosine triphosphatase. Conformational evidence for the Post-Albers mechanism in Na+- and K+-dependent hydrolysis of ATP

The addition of ATP with K+ to pig kidney Na+,K+-ATPase (EC 3.6.1.3) modified with a sulfhydryl fluorescent reagent N-[p-(2-benzimidazolyl)phenyl]maleimide induced a transient decrease (t 1/2 = 0.01 s) in the fluorescence in the presence of Mg2+ with 0.64 M Na+, followed by a slow increase (t 1/2 = 0.08 s), to give a higher steady level than that observed without K+. The addition induced a transient increase (t 1/2 less than 0.02 s) in the amount of phosphoenzyme, followed by a slow decrease (t 1/2 = 0.08 s), but the addition without K+ induced a monophasic increase (t 1/2 = 0.02 s). The addition of ATP in the presence of 2 M Na+ with Ca2+ induced a monophasic decrease (t 1/2 = 0.1 s) in the fluorescence along with a much slower increase (t 1/2 = 1.2 s) in the amount of phosphoenzyme. No significant burst of acid-labile phosphate was observed. The data showed clearly the accumulation of the enzyme-ATP complex preceding the phosphoenzyme formation. Fluorescence intensity of these enzyme species and the amount of phosphoenzyme permitted the simulation using the reaction mechanism including enzyme-ATP complex, ADP-sensitive phosphoenzyme, K+-sensitive phosphoenzyme, and K+-bound enzyme. The simulation gave a good fit to the experimental data which showed that ATP is hydrolyzed in sequence through the above intermediates in the presence of both Na+ and K+.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

Trans-activation and stable integration of the maize transposable element Ds cotransfected with the Ac transposase gene in transgenic rice plants

To develop an efficient gene tagging system in rice, a plasmid was constructed carrying a non-autonomous maize Ds element in the untranslated leader sequence of a hygromycin B resistance gene fused with the 35S promoter of cauliflower mosaic virus. This plasmid was cotransfected by electroporation into rice protoplasts together with a plasmid containing the maize Ac transposase gene transcribed from the 35S promoter. Five lines of evidence obtained from the analyses of hygromycin B-resistant calli, regenerated plants and their progeny showed that the introduced Ds was trans-activated by the Ac transposase gene in rice. (1) Cotransfection of the two plasmids is necessary for generation of hygromycin B resistant transformants. (2) Ds excision sites are detected by Southern blot hybridization. (3) Characteristic sequence alterations are found at Ds excision sites. (4) Newly integrated Ds is detected in the rice genome. (5) Generation of 8 by target duplications is observed at the Ds integration sites on the rice chromosomes. Our results also show that Ds can be trans-activated by the transiently expressed Ac transposase at early stages of protoplast culture and integrated stably into the rice genome, while the cotransfected Ac transposase gene is not integrated. Segregation data from such a transgenic rice plant carrying no Ac transposase gene showed that four Ds copies were stably integrated into three different chromosomes, one of which also contained the functional hph gene restored by Ds excision. The results indicate that a dispersed distribution of Ds throughout genomes not bearing the active Ac transposase gene can be achieved by simultaneous transfection with Ds and the Ac transposase gene.     

Anthocyanin production in cultured cells of Aralia cordata Thunb.

High anthocyanin-producing cell lines, which were grown in a dark or in a light-dark regime, were selected from callus cultures initiated from stem and leaf tissues of Aralia cordata Thunb. by small-cell-aggregate selection. To verify the optimum culture conditions for anthocyanin production, cells were tested by changing the various basal media, sucrose concentration and nitrogen source and concentration. Good growth was obtained in the dark on Linsmaier-Skoog's basal medium containing 1.0 mg l^-1 2,4-d and 0.1 mg l^-1 kinetin, 2% (w/v) sucrose and full strength of nitrogen concentration. However, the highest anthocyanin yield (10.3% dry wt) was obtained in the dark on B5 medium containing 1.0 mg l^-1 2,4-d and 0.1 mg l^-1 kinetin. Our results suggested that it has became feasible to find the most effective conditions for cell growth and anthocyanin production by optimizations of the nitrogen concentration and the ratio of NH₄ ⁺ to NO₃ ^- in the medium.Abbreviations B5 Gamborg (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog (Linsmaier & Skoog 1965) - MS Murashige and Skoog (Murashige & Skoog 1962) - NN Nitsch and Nitsch (Nitsch & Nitsch 1967) - WH White (White 1963) This paper is part 81 in the series Studies on Plant Tissue Cultures. For Part 80 see Furuya T, Sakamoto K, Iida K, Asada Y, Yoshikawa T, Sakai S & Aimi N (1992) Phytochemistry 31: 3065–3068.     

Competition and Communal Regulations in the Kombu Kelp (Laminaria angustata) Harvest

Kombu is a kind of seaweed growing in northern Japan. In Hidaka District, Hokkaido Island, it comprises an important source of income. In the harvest of wild kombu, competition among the harvesters tends to be intense because of its high price and the fact that it is a limited resource. About a century ago, severe competition caused resource depletion and decline of kombu quality. Today, however, the resource is used sustainably by the villagers, who observe complex communal regulations for the use of common property. This study examines the ecological role of these regulations in the management of kombu as common property, and demonstrates that these regulations facilitate equal access to the resource, maintaining the unity of the local community, as well as efficient resource use.     

Chemical Structure of Lipid A Isolated from Flavobacterium meningosepticum Lipopolysaccharide

The chemical structure of the lipid A of the lipopolysaccharide component isolated from Flavobacterium meningosepticum IFO 12535 was elucidated. Methylation and nuclear magnetic resonance analyses showed that two kinds of hydrophilic backbone exist in the free lipid A: a β (1→6)-linked 2-amino-2-deoxy-d-glucose, which is usually present in enterobacterial lipid A’s, and a 2-amino-6-O-(2,3-diamino-2,3-dideoxy-β-d-glucopyranosyl)-2-deoxy-d-glucose, in a molar ratio of 1.00:0.35. Both backbones were α-glycosidically phosphorylated in position 1, and the hydroxyl groups at positions 4, 4′, and 6′ were unsubstituted. Liquid secondary ion-mass spectrometry revealed a pseudomolecular ion at m/z 1673 [M-H]⁻ as a major monophosphoryl lipid A component carrying five acyl groups. Fatty acid analysis showed that the lipid A contained 1 mol each of amide-linked (R)-3-OH iC_17:0, ester-linked (R)-3-OH iC_15:0, amide-linked (R)-3-O-(iC_15:0)-iC_17:0, and both amide- and ester-linked (R)-3-OH C_16:0. Fatty acid distribution analyses using several mass spectrometry determinations demonstrated that the former two constituents were distributed on positions 2 and 3 of the reducing terminal unit of the backbones and that the latter two were attached to the 2′ and 3′ positions in the nonreducing terminal residue.Lipopolysaccharide (LPS) is known to act as an endotoxin that mediates pathophysiological changes such as fever and shock which occur in the course of severe gram-negative bacterial infection (5, 18, 27). The pathophysiological activity of LPS depends on the chemical structure of the hydrophobic portion called lipid A, the biologically active center of LPS (12, 14), which generally consists of a β(1→6)-linked 2-amino-2-deoxy-d-glucose (GlcN) disaccharide carrying phosphate and fatty acid residues; many fine structural variations are observed in different bacterial families (38).Since many of the LPSs from various gram-negative bacteria cause similar endotoxic effects despite differences in chemical composition and positions of substitution, the chemical structure required for the activity does not seem to be very strict. It has been reported, however, that several lipid A forms, isolated from the LPSs of Porphyromonas gingivalis (16), Rhodobacter sphaeroides (22, 26) and Rhodobacter capsulatus (20), as well as chemically synthesized lipid A analogs (6, 12), which are structurally similar to the active-type lipid A, exhibit dramatically low endotoxicity. Biologically active lipid A has been found to be changed to completely nontoxic derivatives by simple chemical modifications (28, 29). These findings indicate that the biological activity of lipid A is controlled by the fine structural variations. The nontoxic or low-toxicity lipid A preparations are very important for the determination of the relationship between the chemical structure and biological activity of lipid A, and also for the systematic development of LPS antagonists. However, the essential structural requirements for the complete activity or nontoxicity of lipid A are still uncertain. It is, therefore, meaningful to study the chemical and biological properties of naturally occurring lipid A’s which possess a unique structure.Flavobacterium meningosepticum is an aerobic gram-negative rod which is known to cause meningitis and septicemia in newborn infants (4, 21). Interestingly, the bacterium does not induce Limulus gelation activity when tested with whole cells (32), strongly suggesting that the LPS is of low toxicity or nontoxic and that the lipid A must have a unique structure relative to other enterobacterial lipid A’s.In the present study, the chemical structure of lipid A isolated from F. meningosepticum LPS was characterized by compositional study, methylation analysis, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Landscape patterns and plant species diversity of forest reserves in the Kanto region, Japan

Plant species richness of twenty old-growth forest reserves in the cool-temperate zone in the Kanto region, Japan were investigated to detect the effect of forest fragmentation. The species richness of trees and forest floor plants were analyzed by multiple regression models relating to nine variables on the characteristics of landscape, local habitat and forest stand. The total species diversity did not have a significant correlation with any variables of landscape patterns. In this study, single large reserve in the SLOSS discussion did not seem very effective to preserve more species. However, forest reserves in large patches tend to have relatively infrequent species. Large patches of natural forests were regarded as one of the important factors to preserve infrequent species.     

Total synthesis of glycononaosyl ceramide with a sialyl dimeric Lex sequence

The first total synthesis of glycononaosyl ceramide with a sialyl dimeric Le^x sequence 1 is described. Regio- and stereo-selective glycosylations of sialyl donors 6,7,8 with the suitably protected Le^x trisaccharide acceptors 9,10 were performed to give the expected tetrasaccharides 15 and 21, which were converted into the corresponding donors 20 and 22. Boron trifluoride etherate-promoted glycosylation of 20 with pentasaccharide acceptor 11 afforded regioselectively the expected nonasaccharide 23. After replacing benzyl groups of 23 by acetyl groups, the anomeric acetate was transformed into the -trichloroacetimidate 27. The crucial coupling between 27 and (2S, 3R, 4E-3-O-benzoyl-2-N-tetracosanoylsphingenine 3 was executed to afford completely protected -glycoside 28. Finally, selective cleavage of the methyl ester and N,O-deprotection of 28 gave the target ganglioside 1.Dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.