Unexpected A-form formation of 4'-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance

Fully modified 4′-thioDNA, an oligonucleotide only comprising 2′-deoxy-4′-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4′-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4′-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4′-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3′-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4′-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4′-thioDNA showed that partially modified 4′-thioDNA that contained some 2′-deoxy-4′-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4′-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4′-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.     

Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies

Aerobic phototrophic bacterium Roseobacter denitrificans has a nitric oxide reductase (NOR) homologue with cytochrome c oxidase (CcO) activity. It is composed of two subunits that are homologous with NorC and NorB, and contains heme c, heme b, and copper in a 1:2:1 stoichiometry. This enzyme has virtually no NOR activity. Electron paramagnetic resonance (EPR) spectra of the air-oxidized enzyme showed signals of two low-spin hemes at 15 K. The high-spin heme species having relatively low signal intensity indicated that major part of heme b₃ is EPR-silent due to an antiferromagnetic coupling to an adjacent Cu_B forming a Fe-Cu binuclear center. Resonance Raman (RR) spectrum of the oxidized enzyme suggested that heme b₃ is six-coordinate high-spin species and the other hemes are six-coordinate low-spin species. The RR spectrum of the reduced enzyme showed that all the ferrous hemes are six-coordinate low-spin species. ν(Fe-CO) and ν(C-O) stretching modes were observed at 523 and 1969 cm⁻¹, respectively, for CO-bound enzyme. In spite of the similarity to NOR in the primary structure, the frequency of ν(Fe-CO) mode is close to those of aa₃- and bo₃-type oxidases rather than that of NOR.     

Biotransformation of (+)-catechin into taxifolin by a two-step oxidation: primary stage of (+)-catechin metabolism by a novel (+)-catechin-degrading bacteria, Burkholderia sp. KTC-1, isolated from tropical peat

Peat contains various persistent compounds derived from plant materials. We isolated a novel (+)-catechin-degrading bacterium, Burkholderia sp. KTC-1 (KTC-1), as an example of a bacterium capable of degrading persistent aromatic compounds present in tropical peat. This bacterium was isolated by an enrichment technique and grew on (+)-catechin as the sole carbon source under acidic conditions. The reaction of a crude enzyme extract and a structural study of its products showed that (+)-catechin is biotransformed into taxifolin during the preliminary stages of its metabolism by KTC-1. HPLC analysis showed that this transformation occurs in two oxidation steps: 4-hydroxylation and dehydrogenation. Furthermore, both (+)-catechin 4-hydroxylanase and leucocyanidin 4-dehydrogenase were localized in the cytosol of KTC-1. This is the first report on biotransformation of (+)-catechin into taxifolin via leucocyanidin by an aerobic bacterium. We suggest that tropical peat could become a unique resource for microorganisms that degrade natural aromatic compounds.     

Recovery plan for an exploited species, southern bluefin tuna

Southern bluefin tuna (SBT) were heavily depleted in the mid-1980s, and the fishing quota has been restricted since 1985. As a result of this restriction and protection of immature individuals, spawning stock biomass (SSB) recently has shown a slight increase. The Commission for the Conservation of Southern Bluefin Tuna (CCSBT) has the target of recovering SSB to the 1980 level by 2020. We investigated whether SBT populations will recover and reach the target level set by CCSBT. Our projection shows that the SSB of the SBT will temporarily decrease again after 1999. This temporary decrease of SSB does not always mean failure of the recovery plan, because the SSB trend is highly vulnerable to age-composition dynamics. The SBT is an example of this. The number of mature SBT was small during the 1980s because of overfishing. Thus, the number of eggs that were spawned by these mature SBT was small in these years, and when these small numbers of immature fish become mature, the SSB will decrease again. We call this effect the inverse baby-boom effect. The inverse baby-boom effect may be common for managed bioresources that have once been overexploited. We also examine the use of spawning potential (SP) and SSB as an index of stock recovery. Received: August 17, 2000 / Accepted: March 19, 2001     

Immunohistochemical recognition of human follicular dendritic cells (FDCs) in routinely processed paraffin sections.

A number of monoclonal antibodies (MAbs) that recognize human follicular dendritic cells (FDCs) have been identified. Although some of them have already been applied individually in routine immunolabeling using formalin-fixed paraffin sections for diagnostic and experimental purposes, many antibodies are still employed only for immunolabeling using cryostat sections or particularly processed sections because they have been thought unsuitable for routine sections. A comprehensive examination re-evaluating their suitability in paraffin sections has not been reported. Accordingly, there is limited ability to examine the immunopathological contribution or diagnostic value of FDCs using routinely processed specimens or archived materials. In this study a broad panel of antibodies was systematically applied to the immunolabeling of paraffin sections of reactive tonsils or lymph nodes, in combination with advanced antigen retrieval (AR) techniques. Several antibodies, including Ki-M4p, X-11, 12B1, CNA.42, 1F8/BU32 (anti-CD21), BU38/1B12 (anti-CD23), Ber-MAC-DRC/To5 (anti-CD35), 1.4C3 (anti-CD106), NGFR5 (anti-nerve growth factor receptor p75), IIH6 (anti-CD55), 55K-2 (anti-fascin), and anti-S100 protein alpha-chain, were found to label FDCs in routine sections when combined with suitable AR techniques. Our results are easily adaptable for routine practice and provided useful suggestions concerning the immunopathological behavior and diversity of the particular cells.     

Behaviors of mice given forced-swimming.

Behaviors of mice in the forced swimming test are motionlessness, climbing and the other stereotypical behaviors. We observed these behaviors in different ages and sex and in repeated forced swimming trials. The findings were 1) quantities of the climbing and the other behaviors were different with the age and sex, 2) repeated per day forced swimming remarkably increased motionlessness and motionlessness is memorized for at least 14 days, and 3) climbing is the typical opposite behavior of motionlessness and was related to adrenergic but not serotonergic neuronal activity. When these behaviors are recognized as adaptation behaviors, we conclude that mice given repeated forced swimming, but not mice given one trial of forced swimming, can be considered as a model of human depression relating to adrenaline neuronal activity.     

Phytoestrogens from the roots of Polygonum cuspidatum (Polygonaceae): structure-requirement of hydroxyanthraquinones for estrogenic activity.

The methanolic extract from the roots of Polygonum (P.) cuspidatum was found to enhance cell proliferation at 30 or 100 microg/mL in MCF-7, an estrogen-sensitive cell line. By bioassay-guided separation from P. cuspidatum with the most potent activity, emodin and emodin 8-O-beta-D-glucopyranoside were isolated as active principles. The methanolic extracts from Polygonum, Cassia, Aloe, and Rheum species, which were known to contain anthraquinones, also showed the MCF-7 proliferation. As a result of the evaluation of various anthraquinones from plant sources and synthetic anthraquinones, aloe-emodin, chrysophanol, chrysophanol 8-O-beta-D-glucopyranoside, and 1,8-dihydroxyanthraquinone showed weak activity. On the other hand, alizalin and 2,6-dihydroxyanthraquinone as well as emodin having the 2- and/or 6-hydroxyl groups showed potent activity. These results show that the unchelated hydroxyl group is essential for strong activity. Emodin and 2,6-dihydroxyanthraquinone also inhibited 17beta-estradiol binding to human estrogen receptors (ERs) with K(i) values of 0.77 and 0.31microM for ERalpha and 1.5 and 0.69 microM for ERbeta. These findings indicate that hydroxyanthraquinones such as emodin are phytoestrogens with an affinity to human estrogen receptors.