Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.     

1,4-Dioxane degradation potential of members of the genera <Emphasis Type="Italic">Pseudonocardia</Emphasis> and <Emphasis Type="Italic">Rhodococcus</Emphasis>

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855^T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343^T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707^T and Pseudonocardia asaccharolytica JCM 10410^T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.     

Diets and body condition of polar cod (<Emphasis Type="Italic">Boreogadus saida</Emphasis>) in the northern Bering Sea and Chukchi Sea

To understand trophic responses of polar cod Boreogadus saida (a key species in Arctic food webs) to changes in zooplankton and benthic invertebrate communities (prey), we compared its stomach contents and body condition between three regions with different environments: the northern Bering Sea (NB), southern Chukchi Sea (SC), and central Chukchi Sea (CC). Polar cod were sampled using a bottom trawl, and their potential prey species in the environment were sampled using a plankton net and a surface sediment sampler. Polar cod fed mainly on appendicularians in the NB and SC where copepods were the most abundant in the environment, while they fed on copepods, euphausiids, and gammarids in the CC where barnacle larvae were the most abundant species in plankton samples on average. The stomach fullness index of polar cod was higher in the NB and SC than CC, while their body condition index did not differ between these regions. The lower lipid content of appendicularians compared to other prey species is the most plausible explanation for this inconsistency.     

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.     

Interaction of calcium ion and maleic acid copolymer

Interaction between Ca2+ ion and poly(styrene-alt-maleic acid) was studied by using a Ca2+ ion sensitive electrode. The Ca2+ activity had a peak at a degree of neutralization of 0.5 and decreased with increasing Ca(OH)2 concentration beyond it when the polymer solution was neutralized with Ca(OH)2. The decrease in the Ca2+ activity was not observed when the polymer concentration was very low. The counter ion condensation theory did not hold for this solution except in the case of an extremely dilute solution. The additivity rule for Ca2+ was confirmed for this solution. When the maleic acid copolymer was neutralized with both Ca(OH)2 and KOH, the Ca2+ activity had a peak at a degree of neutralization of 0.5 when neutralization with KOH was less than 0.3 and the Ca2+ activity decreased more drastically than that neutralized with only Ca(OH)2. The appearance of the peak of the Ca2+ activity at a degree of neutralization of 0.5 was independent of the ratio of Ca2+ concentration to polymer concentration or absolute Ca2+ concentration, but depended on the degree of ionization, i.e., linear electric charge density on the polymer because of ionization of the carboxyl groups. Interpretations of the behavior of the Ca2+ activity are discussed.     

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.     

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.