Serum Lipoprotein(a) Dynamics before/after Menopause and Long-term Effects of Hormone Replacement Therapy on Lipoprotein(a) Levels in Middle-aged and Older Japanese Women.

AIM: To assess lipoprotein(a) Lp(a) dynamics before and after menopause and to examine long-term changes during hormone replacement therapy (HRT) in middle-aged and older Japanese women. METHODS: (1) Serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and Lp(a) concentrations of 526 patients were compared. The patients were divided into 3 groups on the basis of menopausal status (premenopause, perimenopause, postmenopause). (2) Serum markers of lipid metabolism were measured at baseline and at 6-month intervals in 161 postmenopausal women who continuously received HRT with conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) for 4 years. (3) Changes in serum concentrations of markers were compared among 120 women with hypercholesterolemia who were randomly assigned to receive HRT (CEE plus MPA, or transdermal estradiol plus MPA) or pravastatin. RESULTS: (1) Lp(a) concentrations were significantly higher in the postmenopausal women than in the premenopausal or perimenopausal women. (2) The mean Lp(a) concentration after 6 months of HRT decreased by about 19%, and similar levels were maintained for 4 years (3). The mean Lp(a) concentration after 6 months of HRT decreased by 19.9% in the CEE plus MPA group, but did not change significantly in the transdermal estradiol plus MPA group or the pravastatin group. CONCLUSION: Our results suggest that HRT with CEE plus MPA is useful for the management of elevated serum Lp(a) concentrations in middle-aged and older women. However, follow-up studies are needed to determine whether this finding is related to the future prevention of coronary heart disease events.     

Chemical and immunological studies on mycobacterial polysaccharides. 1. Purification and properties of polysaccharides from human tubercle bacilli

Defatted human tubercle bacilli, Aoyama B strain, were extracted with 0.1 n NaOH for 24 hr, and the crude polysaccharide fraction was precipitated by the addition of 5 volumes of ethyl alcohol. A yield of 17.8 g of crude polysaccharides was obtained from 800 g of bacilli. The crude polysaccharide was further fractionated into seven fractions by fractional precipitation with ethyl alcohol. Each fraction was purified by successive chromatography on Dowex 50 and diethylaminoethyl cellulose, and by gel filtration on Sephadex G-75 and G-200. Optical rotation and gas chromatographic analyses of purified polysaccharide showed that these polysaccharides contained glucan mannan, arabinomannan, and arabinogalactan. Each polysaccharide was almost completely free from nitrogen, and no tuberculin reaction was produced by 100 mug of each material. Arabinomannan and arabinogalactan showed precipitin reaction, complement fixation, and passive hemagglutination reaction with rabbit antiserum against heat-killed whole bacilli (Aoyama B). In guinea pigs sensitized with Aoyama B bacilli, arabinomannan and arabinogalactan provoked anaphylactic shock when injected intravenously, and Arthus type reaction when injected intracutaneously. With the use of rabbit antiserum, arabinomannan and arabinogalactan showed passive anaphylactic shock, passive cutaneous anaphylaxis, and Prausnitz-Küstner type reactions in guinea pigs. By immunodiffusion analysis, it was shown that the antigenic determinant of arabinomannan was different from that of arabinogalactan.     

In Planta Localization of Stilbenes within Picea abies Phloem

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutive (i.e. continuously produced) or inducible (i.e. activated as a response to insect or pathogen attack; Krokene, 2015). Many defense traits exist in both forms (Franceschi et al., 2005). For example, axial phloem parenchyma cells (or polyphenolic parenchyma) are critical in conifer bark defense. These cells regularly form in Pinaceae during annual phloem formation (Franceschi et al., 1998, 2000; Krekling et al., 2000; Jyske et al., 2015) but also are produced on invasion (Franceschi et al., 2005; Krokene, 2015). In Norway spruce (Picea abies) phloem, axial parenchyma forms distinctive, continuous tangential sheets across conducting (i.e. noncollapsed) and nonconducting (i.e. collapsed) tissue.Pioneering studies using microscopy with different dye agents and autofluorescence showed that the large vacuole is a special feature of the axial phloem parenchyma that contains phenolic substances (i.e. phenolic bodies; Franceschi et al., 1998). Microscopic imaging techniques also showed that polyphenolic content is highly dynamic (Franceschi et al., 1998, 2000, 2005) and changes seasonally (Krekling et al., 2000). Within the last 5 years, progress in laser microdissection (LMD) has facilitated the sampling of individual tissues and cells, providing information about the exact chemical composition of phenolic content. Li et al. (2012) used LMD to show that the axial parenchyma is the main site of phenolic accumulation in spruce bark, including that of stilbene compounds.Stilbenes are secondary metabolites that are composed of two phenol moieties linked by a C₂ bridge. These compounds are derived from the phenylpropanoid pathway, in which the last steps of biosynthesis are catalyzed by stilbene synthase (Chong et al., 2009). There is increasing interest in these antioxidant, antibacterial, and antiinflammatory compounds for use in healthy human diets, therapeutic approaches, and as protective agents in materials sciences (Shibutani et al., 2004; Metsämuuronen and Siren, 2014; Reinisalo et al., 2015; Hedenström et al., 2016; Sirerol et al., 2016). The tetrahydroxystilbene glucosides trans-astringin (3,3ʹ,4ʹ,5-tetrahydroxystilbene 3-O-β-d-glucoside) and trans-isorhapontin (3,4ʹ,5-trihydroxy-3ʹ-methoxystilbene 3-O-β-d-glucoside) are the most abundant constitutive stilbene compounds of Norway spruce, while the trihydroxystilbene glucoside trans-piceid (resveratrol 3-O-β-glucoside) and stilbene aglycons (i.e. without the sugar moiety) are less abundant. Stilbene synthesis in spruce probably proceeds through the formation of resveratrol (i.e. aglycon of piceid) followed by further modifications (i.e. hydroxylation, O-methylation, and O-glycosylation) to yield tetrahydroxystilbene glucosides (Hammerbacher et al., 2011). Stilbenes are assumed to provide protection against a wide variety of environmental stressors (Franceschi et al., 2005; Witzell and Martin, 2008; Chong et al., 2009). Stilbenes appear to contribute to antifungal defense in spruce (Hammerbacher et al., 2011, 2013). The fungal inoculation of spruce bark with the blue-stain fungus Endoconidiophora polonica (previously named Ceratocystis polonica; de Beer et al., 2014) causes astringin levels to decrease, in parallel with increasing dimeric stilbene glucoside levels in the LMD-isolated axial phloem parenchyma (Li et al., 2012) or increasing levels of corresponding aglycons in bulk tissue (Viiri et al., 2001). During the annual formation of phloem in Norway spruce, the accumulation of stilbene glucosides inside the newest, LMD-isolated phloem ring is preceded by the formation and cellular development of a new band of axial parenchyma (Jyske et al., 2015). These observations strongly indicate that the inducible and constitutive stilbene compounds of spruce phloem are both stored and synthesized in the axial parenchyma.New mass spectrometry imaging techniques provide significant improvements in the mapping of plant metabolites (Briggs and Seah, 1993; Vickerman and Briggs, 2001; Burrell et al., 2007; Cha et al., 2008; Lee et al., 2012; Bjarnholt et al., 2014; Aoki et al., 2016). To elucidate the synthesis, distribution, and metabolism of secondary plant metabolites, it is essential to gather positional information about them in a living state, as pretreatment of specimens, such as drying, may change the distribution and concentration features of soluble chemicals (Metzner et al., 2008; Li et al., 2012; Kuroda et al., 2013). In this study, we used a unique system of time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle (cryo-TOF-SIMS/SEM) to study the localization and accumulation patterns of stilbenes within cells and tissues of phloem. This system has been developed to study chemical distributions at high-spatial resolution (1 µm) directly from the surfaces of plant specimens in a frozen-hydrated state (i.e. in planta) representing living tissues (Kuroda et al., 2013; Aoki et al., 2016). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) directly detects organic and inorganic compounds on the specimen surface over a broad mass-to-charge ratio (m/z) range by mass spectrometry with high chemical sensitivity. Specimen surface morphology is visualized by the detection of total secondary ion content. The quality of cellular integrity may be further observed by scanning electron microscopy connected with a cryo-shuttle (cryo-SEM) imaging of the frozen surface of the same specimen. The cryo-TOF-SIMS/SEM system has still rarely been applied to the analysis of plant physiology (Metzner et al., 2008, 2010; Iijima et al., 2011; Kuroda et al., 2013; Aoki et al., 2016).Mass spectrometer imaging techniques consist of an ionizer and a mass analyzer. In the TOF-SIMS system, secondary ion mass spectrometry is used as an ionizer and time-of-flight as a mass analyzer. In another mainstream imaging mass spectrometry technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization is used as ionizer. Compared with TOF-SIMS, MALDI-MS is more quantitative and has high-M_r acceptance, but the resolution of MALDI-MS is not high enough for cell-level detection (Aoki et al., 2016). Instead, the spatial resolution of TOF-SIMS is superior to focus on cell functions. The disadvantage of TOF-SIMS is that the ionization and fragmentation phenomenon may be affected by the matrix effect, causing some degree of uncertainty. However, when time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle (cryo-TOF-SIMS) is used in combination with quantitative gas chromatography, it is very powerful to study the positional and temporal distributions of metabolites within living plants.To complement TOF-SIMS analysis, we applied quantitative chemical microanalysis methods to study the amounts of stilbene glucosides and to correlate those with the amounts of total extractives, monosaccharides and disaccharides, and water across phloem and bark. The methods include tangential cryo-sectioning of tissues and their chemical microanalysis by gas chromatography with flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).To combine the chemical information with phloem morphology, the cellular and subcellular features of the axial phloem parenchyma were analyzed by three-dimensional (3D) synchrotron radiation microtomography (µCT). µCT is a prominent tool that has gained popularity for 3D analysis of xylem structure and physiology (Brodersen, 2013; Cochard et al., 2015), but only recently has it been applied to the 3D analysis of phloem (Jyske et al., 2015). This method offers advantages over traditional light microscopic approaches, as high-throughput data at the submicrometer level can be produced from significantly larger tissue volumes. The data allow for representative volumetric analysis of cellular distributions along with 3D visualization of subcellular features.In this study, we used a novel combination of cutting-edge techniques to analyze in parallel (1) in planta cellular localization and accumulation of stilbene glucosides across phloem and bark by semiquantitative cryo-TOF-SIMS/SEM; (2) tissue-level quantitative amounts of stilbene glucosides, total extractives, and monosaccharides and disaccharides across phloem and bark by tangential cryo-sectioning and GC-FID and GC-MS; (3) 3D cell abundance distributions across phloem and bark by µCT; and (4) variation in water content across phloem and bark (Fig. 1).Open in a separate windowFigure 1.Schematic presentation of the specimen structure and preparation for different analyses. Sample blocks were taken from living tree stem (A) or stem discs (B) at 1.3 m on the stem. The blocks (C) containing outer bark (periderm), phloem, cambium, and part of the outermost xylem ring (D; transverse view of phloem and bark) were further divided into subblocks (1–3; C and E). Subblocks 1 and 2 were quick frozen, and subblock 3 was fixed chemically. Subblock 1 was used for the direct chemical mapping of stilbenes across the phloem from the cambium to the outer bark (i.e. semiquantitative analysis of stilbene localization and accumulation across transverse and radial surfaces [purple] of the tissue block by TOF-SIMS; E-1). To obtain quantitative data on the amounts of stilbenes, other extractives, and carbohydrates across phloem and bark, tangential cryo-sections (250 or 450 µm each; cut slices illustrated with purple in E-2) were cut across subblock 2 and directed for chemical microanalysis by GC-FID (E-2). Subblock 3 was divided into four to six zones, and from each zone, small cuboids (illustrated with purple in E-3) were cut and directed for morphological analysis of phloem by phase-contrast µCT (E-3). Water content across the phloem and bark was analyzed from separate fresh blocks, which were further cut tangentially into thin sections. Black arrows indicate the radial direction from the cambium toward the outer bark. Purple areas show the analyzed locations of each subblock (E). Note that schematic drawings are not to scale.     

Seroepidemiology of human parechovirus types 1, 3, and 6 in Yamagata,Japan, in 2014

To clarify the seroepidemiology of human parechovirus type 1 (HPeV1), 3 and 6, neutralizing antibodies (NT Abs) were measured in 214 serum specimens collected in 2014 in Yamagata, Japan. The seroprevalence against HPeV1 was 100% in all age groups, while that against HPeV3 and HPeV6 was 79.4% and 66.8%, respectively, overall. The geometric mean titers of NT Abs against HPeV1, 3 and 6 were 755.2, 255.0 and 55.9, respectively, overall. Our findings indicate that HPeV1 is the most prevalent HPeV circulating in Yamagata, followed by HPeV3 and HPeV6.     

Application of soft X-ray microradiography to observation of cystoliths in the leaves of various higher plants

Soft X-ray microradiography was applied to observation of the cystoliths, calcified bodies of higher plants, in the leaves ofMorus bombycis, Humulus scandens, Ficus elastica, F. retusa (Moraceae),Boehmeria platanifolia, Pilea viridissima (Urticaceae) andMomordica charantia (Cucurbitaceae). It was proved that this technique is useful for examination of the shape, size, distribution and number of cystoliths in fresh leaves. The microradiographs revealed large cigar-shaped cystoliths in the leaf ofP. viridissima, and neighbor-cystoliths in somewhat restricted areas of the leaves ofM. bombycis andH. scandens, and two to seven radially arranged cystoliths in the leaf ofM. charantia. The number of cystoliths per unit area of leaf (nos./cm²) was estimated to be from 1,090 to 3,900 by means of the microradiographs, varying from species to species. The CaCO₃ content of the leaf calculated from the volume and number of cystoliths was approximately 0.4 mg/cm² in all species exceptF. retusa. InF. retusa, it was about 1.06 mg/cm², the highest value among all species tested. Hand-sections of the leaves showed that the lithocysts were localized in the upper and/or lower epidermis, and they were associated with many photosynthetic cells in all species, suggesting some relationship between CaCO₃ deposition in cystoliths and photosynthesis. This paper is dedicated to Professor Kurazo Furuya, Department of Biology, Tokyo Gakugei University, on the occasion of his retirement (1986).     

Naturally occurring cytotoxic T lymphocyte precursors with specificity for an Ig idiotype

The humoral response to the p-azobenzenearsonate hapten in the A/J mouse includes the major cross-reactive idiotype associated with anti-p-azobenzenearsonate (CRIA) found in all immunized mice. Limiting dilution cultures of non-immunized spleen cells of A/J mice with irradiated B hybridoma cells bearing the Ig idiotype, CRIA, in the presence of T cell growth factors developed cytotoxic activity against the CRIA-bearing hybridoma; in some wells this activity was completely abrogated by an anti-idiotype mAb specific for CRIA or by a univalent hapten antigen, tyrosine-p-azobenzenearsonate, indicating the existence of cytotoxic T cell precursors (CTL-P) specific for one or more idiotopes of CRIA in normal spleen cells. The CTL clones lysed targets in a H-2D-restricted manner and were cytotoxic for CRIA-bearing hybridoma lines, but not for CRIA-non-bearing, IgG1k-bearing hybridoma lines. These CTL-P were detected at a high frequency (1/4,500 to 1/10,000) in a spleen cell population of non-immunized, relatively aged A/J mice (16 to 30 wk of age), and at a lower frequency in spleen cells of younger A/J mice (8 wk of age). However, they were not detected in normal spleen cells of B10.A (CRIA-non-producer) mice at any age (less than 1/6 x 10(5)). Normal Ighd-congenic C.AL-20 mice (16 wk of age), that are CRIA producers had as a high frequency of the CTL-P as did A/J mice, whereas normal Ighb-congenic C.B-20 mice (CRIA-non-producers) had none. In the spleen cells of the CRIA-producers, cytotoxicity of the CTL-P developed only in cultures with small numbers of seeding cells. They were completely absent in cultures with greater numbers of cells; this may be due to the presence of suppressor cells of lower frequency but greater potency. In lymph node cells or PBL of relatively aged A/J mice, the CTL-P were also detected, but only in cultures containing higher cell numbers, and at low frequency (between 1/5 x 10(5) to 1/2 x 10(6)). In thymocytes of 8-wk-old A/J mice, they were occasionally detected at very low frequency (less than or equal to 1/1 x 10(6)), but were not present in the bone marrow cells at any age. These results demonstrate the high incidence of the generation of CTL-P specific for an autologous Ag, and indicate that CRIA on B cells may induce CTL specific for CRIA. However, the development of CTL-P may be inhibited by co-existent suppressor cells under normal conditions.     

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.