Effect of Macrolide Antibiotics on Macrophage Functions

Macrolide antibiotics have a variety of actions other than antimicrobial activities. Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effects of macrolide antibiotics on macrophage functions. For the macrophage, we used the mouse macrophage cell line J774.1. The following effects of macrolide antibiotics on macrophage functions were evaluated: the effect of macrolide antibiotics on macrophage growth; the phagocytosis of beads; cytocidal activity against Candida albicans; and chemotaxis to lipopolysaccharide (LPS). Macrolide antibiotics except for azithromycin significantly stimulated the growth of the macrophage. In addition, pretreatment with macrolide antibiotics except for roxithromycin significantly stimulated the macrophage phagocytosis of beads, macrophage chemotaxis to LPS, and macrophage cytocidal activity against Candida albicans. These results suggest that macrolide antibiotics stimulate macrophage functions.     

The relationship between Japanese cedar pollinosis and air pollutants deposited on the pollen

In this study, air pollutants such as NO ₃ ^– and SO ₄ ^–2 which were found deposited on Japanese cedar pollen in a mountainous area (Tsukechi-cho), and in an urban area (Mizuho Ward, Nagoya City) were measured by ion-chromatography in March 1987 and again in March 1991. An investigation of the pollinosis rate in about 400 Junior High School students in both areas was undertaken during the same periods through both a questionnaire study, and a physical examination. Information was also gathered on the number of vehicles passing through the corresponding areas. The results of this research are as follows: (1) The quantity of NO ₃ ^– deposited on the pollen of Japanese cedar was about 2.3 and 2.0 times greater in the urban area than in the mountainous area (1987 and 1991, respectively). The concentration of SO ₄ ^–2 was about 7.8 and 3.6 times higher in the urban area than in the mountainous area (1987 and 1991, respectively). (2) The results of a questionnaire study taken at the two Junior High Schools from the different districts showed a lower incidence of nasal symptoms in the mountainous area than in the urban area for 1987. The percentage of pollinosis shown by physical examination in the mountainous area rose from 20.1% in 1987 to 30.0% in 1991, and this corresponds to a rise in the number of motor vehicles in the area. Comparatively, in the urban area, the questionnaire results and physical examinations revealed no changes from 1987 to 1991.     

Anti-apoptotic genes, bag-1 and bcl-2, enabled hybridoma cells to survive under treatment for arresting cell cycle

Hybridoma 2E3-O cells were transfected with bcl-2 alone or with bcl-2 and bag-1 in combination. The bcl-2/bag-1 transfectant survived maintaining viability above 75% for almost 5 days when the cells were treated with excess (30 mM) thymidine for arresting cell cycle, whereas the mock transfectant survived for only 2 days, and the bcl-2 alone transfectant lived for 4 days. Owing to this extended viable culture period, the bcl-2/bag-1 transfectant produced twofold amount of antibody in comparison with the mock transfectant in non-proliferating state prepared by the excess thymidine treatment. When their proliferation was arrested by serum limitation, the bcl-2/bag-1 transfectant and the bcl-2 alone transfectant survived for 3 days maintaining viability above 75% while the mock transfectant survived only 1 day. The bcl-2/bag-1 transfectans produced the antibody at the rate three times as high as the bcl-2 alone transfectant and the mock transfectant in non-proliferating state established by serum limitation. Such genetic engineering of hybridoma cells for improving survival in the non-proliferating state will be useful for using nutrients in culture medium efficiently to produce antibody, since nutrients could be diverted from cell proliferation to antibody production in such non-proliferating viable cell culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and properties of polyvinyl alcohol oxidase with broad substrate range obtained from Pseudomonas vesicularis var. povalolyticus PH

Extracellular PVA oxidase produced by Pseudomonas vesicularis var. povalolyticus PH was purified to homogeneity by ammonium sulphate fractionation followed by successive column chromatography, and a study made of its characteristics. The molecular weight of the purified enzyme was estimated to be 75,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consists of monomeric protein. Its isoelectric point was 5.7. The purified enzyme was colourless, and contained one atom of iron per molecule. It exhibited a broad pH activity profile with maximum activity at pH 10.0, and was stable between pH 6.0 and 10.0. The optimum temperature for enzyme activity was 40°C, with stability up to 45°C. The enzyme activity was inhibited strongly by Fe2+, Hg2+ and Sn2+, and weakly by Cu2+, EDTA, thiourea and IAA. The enzyme exhibited activity toward several secondary alcohols, suggesting that it was a secondary alcohol oxidase. In particular, the enzyme exhibited strong activity towards the larger secondary alcohols such as 2-octanol and 4-decanol, and relatively strong activity towards cyclohexanol and benzyl alcohol.     

Lupin alkaloids from flowers of Echinosophora koreensis

(?)-Methyl 12-cytisineacetate (2) was isolated from methanol extracts of fresh flowers of Echinosophora koreensis together with seven known lupin alkaloids. Ethyl 12-cytisineacetate (3) was also isolated from ethanol extracts of the same flowers. Compounds 2 and 3 were artifacts and (?)-12-cytisineacetic acid (4) is assumed to be the principal source of 2 and 3. The variations in alkaloid content during growth of the flowers and the seedlings were also examined.     

Carbohydrate regulation of attachment, encystment, and appressorium formation by Pythium porphyrae (Oomycota) zoospores on Porphyra yezoensis (Rhodophyta)

Pythium porphyrae Takahashi et Sasaki, a facultative parasite of Porphyra spp., is the common microbial agent responsible for red rot disease of this red alga in Japan. Host infection by this species and other plant parasitic members of the Pythiaceae is initiated by motile biflagellate zoospores. Factors regulating host specificity and the initial steps involved in the infection process, consisting of attachment, encystment and appressorium formation, are not known. Zoospore encystment and appressorium formation of P. porphyrae were monitored by staining of the fungal cell walls using calcofluor. The zoospores infected only Porphyra spp. and Bangia atropurpurea (Roth) C. Agardh thalli, although they attached to, and encysted on, many other members of the Rhodophyceae (Stylonema alsidii[Zanardini] Drew, Gelidium elegans Kützing, Pterocladiella capillacea[Gmelin] Santelices et Hommersand, Carpopeltis affinis[Harvey] Okamura, Gloiosiphonia capillaris[Hudson] Carmichael in Berkeley, Grateloupia turuturu Yamada, Callophyllis adhaerens Yamada, Gracilaria spp., Lomentaria hakodatensis Yendo, Rhodymenia intricata[Okamura] Okamura, Griffithsia subcylindrica Okamura, Wrangelia tanegana Harvey, and Polysiphonia morrowii Harvey). No attachment or encystment was observed on the red alga Kappaphycus striatum (Schmitz) Doty ex Silva in Silva et al., the brown algae Undaria pinnatifida (Harvey) Suringar, Scytosiphon sp., and Sargassum thunbergii (Mertens ex Roth) Kuntze as well as members of the Ulvaceae (green algae). Sequential extraction of carbohydrates from Porphyra yezoensis Ueda thalli and the addition of diverse monosaccharides, polysaccharides, and amino acids to zoospore suspensions indicated that encystment and appressorium formation were induced only by sulfated galactans (porphyran, commercial agar, agarose, and carrageenans). Zoospore attachment and encystment on thalli of P. yezoensis was abolished by periodate oxidation of the thallus surface and was reduced by 80–90% after enzymatic removal of sulfated galactan (porphyran). It appears that the interaction of zoospore surface receptors with sulfated galactan (porphyran) determinants on the thallus surface induced specific attachment and encystment on Porphyra spp. thalli. Zoospores encysted, germinated, and formed appressoria on sulfated galactan films and in suspensions of this carbohydrate. Attachment and encystment were induced on commercial agar and agarose films, but appressoria were not induced on agarose films. Supplementation of agarose media with both cold and hot water fractions and with porphyran from P. yezoensis–induced appressoria implicated sulfated galactans (porphyran) in appressorium formation.     

Cloning and Inactivation of Genes Encoding Ferredoxin- and NADH-Dependent Glutamate Synthases in the Cyanobacterium Plectonema boryanum. Imbalances in Nitrogen and Carbon Assimilations Caused by Deficiency of the Ferredoxin-Dependent Enzyme

Glutamate synthase (GOGAT) is a key enzyme in the assimilation of inorganic nitrogen in photosynthetic organisms. We found that, like higher plants, the facultative heterotrophic cyanobacterium Plectonema boryanum had ferredoxin (Fd)- and NADH-dependent GOGATs. The genes glsF, gltB, and gltD were cloned, and structural analyses and target mutageneses demonstrated that glsF encoded Fd-GOGAT and that gltB and gltD encoded the two subunits of NADH-GOGAT. All three mutants lacking one of the GOGAT genes were able to grow photosynthetically and heterotrophically. However, the Fd-GOGAT mutant exhibited a phenotype of marked nitrogen deficiency when grown under conditions of saturating illumination and CO₂ supply. In these conditions the rate of the ammonia uptake from the culture medium was slower in the Fd-GOGAT mutant than in the wild type or in the NADH-GOGAT mutant, but no significant differences were found in the rate of the CO₂ fixation-dependent O₂ evolution among these strains. Our results suggest that, although both Fd- and NADH-GOGATs were operative in the cells growing in light, the contribution of Fd-GOGAT, which directly utilizes photoreducing power for the catalytic reaction, is essential for balancing photosynthetic nitrogen and carbon assimilation.     

Effects of space flight on the histological characteristics of the aortic depressor nerve in the adult rat: electron microscopic analysis.

The effects of microgravity on the histological characteristics of the aortic depressor nerve, which is the afferent of the aortic baroreflex arc, were determined in 10 female adult rats. The rats were assigned for nursing neonates in the Space Shuttle Columbia or in the animal facility on the ground (NASA Neurolab, STS-90), and were housed for 16 days under microgravity in space (microg, n=5) or under one force of gravity on Earth (one-g, n=5). In the Schwann cell unit in which the axons of unmyelinated fibers are surrounded by one Schwann cell, the average number of axons per unit in the microg group was 2.1 +/- 1.6 (mean +/- SD, n=312) and significantly less than that in the one-g group (3.0 +/- 2.9, n=397, p<0.05). The proportion of unmyelinated fibers in the aortic depressor nerve in the microg group was 64.5 +/- 4.4% and significantly less than that in the one-g group (74.0 +/- 7.3%, p<0.05). These results show that there is a decrease in the number of high-threshold unmyelinated fibers in the aortic depressor nerve in adult rats flown on the Shuttle Orbiter, suggesting that the aortic baroreflex is depressed under microgravity during space flight.     

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid,a histone deacetylase inhibitor

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.     

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.Starch is the most important carbohydrate storage material and contains the Glc polymers amylose and amylopectin. At least four classes of enzymes, ADP-Glc pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), are necessary for efficient starch biosynthesis in storage tissues.SSs (EC 2.4.1.21) play a central role in starch synthesis during α-glucan elongation by adding Glc residues from ADP-Glc to the nonreducing ends via α-1,4-glucosidic linkages. Rice (Oryza sativa) contains 11 SS genes that are grouped into six classes, SSI to SSV and granule-bound starch synthase (GBSS; Supplemental Fig. S1; Hirose and Terao, 2004; Ohdan et al., 2005). Every class contains multiple isozymes, except for SSI and SSV; SSI, SSIIa, SSIIIa, and GBSSI are highly expressed in developing rice endosperm (Hirose and Terao, 2004; Ohdan et al., 2005). SSI elongates short amylopectin chains with degree of polymerization (DP) from 6 or 7 to DP 8 to 12 (Fujita et al., 2006). SSIIa elongates amylopectin from DP 6 to 12 to DP 13 to 24 (Umemoto et al., 2002; Nakamura et al., 2005), and SSIIIa elongates long amylopectin chains with DP 33 or greater (Fujita et al., 2007). GBSSI synthesizes amylose and extra-long amylopectin chains (Sano, 1984; Takeda et al., 1987; Hizukuri, 1995). The functions of other SS isozymes, such as SSIIb, SSIIc, SSIIIb, SSIVa, SSIVb, SSV, and GBSSII, remain largely unknown due to the lack of respective mutant lines. It is not clear how SS isozymes contribute to starch granule formation.Rice endosperm amyloplasts produce characteristic compound-type starch granules, which consist of dozens of polyhedral, sharp-edged granules (Matsushima et al., 2010). Compound-type starch granules are the most common type in endosperm of Poaceae species (Tateoka, 1962; Grass Phylogeny Working Group, 2001; Prasad et al., 2011; Matsushima et al., 2013). Simple-type starch granules (one starch granule per amyloplast) are produced in some species of the Bambusoideae, Pooideae, Micrairoideae, Chloridoideae, and Panicoideae subfamilies. The taxonomic relationships in the Poaceae do not enable an accurate prediction of granule morphology (Tateoka 1962; Shapter et al., 2008; Matsushima et al., 2013).Two studies that changed starch granule shape from simple type to compound type have been reported (Suh et al., 2004; Myers et al., 2011). A hull-less cultivar of cv Betzes barley (Hordeum vulgare), cv Nubet, contains simple-type and bimodal starch granules, which are typical of wild-type barley. Chemical mutagenesis of cv Nubet produced a mutant called franubet, which contains compound-type starch granules (Suh et al., 2004). In the maize monogalactosyldiacylglycerol synthase-deficient mutant opaque5, simple-type granules are replaced by compound-type granules separated by a membranous structure (Myers et al., 2011). The molecular mechanisms that control starch granule morphology in cereal endosperm are largely unknown, although an alteration in membrane lipid synthesis may be involved (Myers et al., 2011).A structural model for the compound-type amyloplast is shown Figure 1. The amyloplast envelope contains an outer envelope membrane (OEM), inner envelope membrane (IEM), and intermembrane space (IMS). Each starch granule is enclosed by an IEM, and granules are separated by a septum-like structure (SLS; Yun and Kawagoe, 2010). In this model, the IMS and SLS are directly connected, and fluorescent proteins such as GFP and Cherry can move freely between the two (Fig. 1; Kawagoe, 2013). The chloroplast envelope membrane contains little protein compared with the thylakoid membrane (Heber and Heldt, 1981). The endosperm amyloplast envelope membrane contains even less protein. Low protein content could be a major reason why the amyloplast envelope in rice endosperm is difficult to observe using high-resolution electron microscopy. In transgenic rice, a fluorescent protein fused to an IEM protein, the ADP-Glc transporter BRITTLE1, visualized the amyloplast IEM (Yun and Kawagoe, 2010). Fluorescent proteins fused to the chloroplast OEM protein OEP7 visualized the amyloplast OEM in endosperm (Kawagoe, 2013). These studies revealed that the outermost membranes of rice amyloplasts are OEM and contain intraamyloplast compartments. Starch is synthesized within the amyloplast compartments and is ultimately formed as compound-type granules that are individually wrapped in IEM (Yun and Kawagoe, 2010; Kawagoe, 2013).Open in a separate windowFigure 1.Structural model of the wild-type amyloplast in developing rice endosperm. The OEM is in black, the IEM is in magenta, the IMS is in green, and the SLS is in blue. G, Starch granules.Confocal microscopy analyses of the rice IEM protein, BRITTLE1, revealed that an SLS, or cross wall, divides starch granules in the amyloplast (Yun and Kawagoe, 2010). A model for the synthesis of compound-type starch granules consisting of polyhedral, sharp-edged granules proposed that the SLS functions as a mold that casts growing granules into a characteristic shape (Yun and Kawagoe, 2010; Kawagoe, 2013). The model postulates a central role for the SLS in producing characteristic compound-type granules, although neither the SLS components nor the enzymes affecting its properties have been characterized.Arabidopsis (Arabidopsis thaliana) SS genes are grouped into six classes. Leaf transitory starch biosynthesis has been investigated in single mutants of SSI, SSII, SSIII, and SSIV and in various double and triple SS mutants (Ral et al., 2004; Delvallé et al., 2005; Zhang et al., 2005, 2008; Szydlowski et al., 2009, 2011). Starch granules in leaf chloroplasts are reduced in number but enlarged in the ssIV mutant (Roldán et al., 2007; Crumpton-Taylor et al., 2013) and in the ssIV double and triple mutants (Szydlowski et al., 2009). Immature ssIV leaves have no starch granules but accumulate the starch synthase substrate ADP-Glc at high concentrations. Starch granules are flattened and discoid in wild-type leaves but are rounded in mature leaves of ssIV, suggesting that SSIV is essential for coordinating granule formation with chloroplast division during leaf expansion (Crumpton-Taylor et al., 2013). The ssIII ssIV double mutant does not accumulate measurable amounts of starch in the leaves, despite the presence of SSI and SSII activity (Szydlowski et al., 2009), implying that Arabidopsis SSIII and SSIV are involved in the initiation of starch granule formation and that either SSIII or SSIV is sufficient. Overexpression of AtSSIV increases the starch level in Arabidopsis leaves and potato (Solanum tuberosum) tubers (Gámez-Arjona et al., 2011). In transgenic plants, the AtSSIV-GFP fusion protein is enriched in specific regions at the edge of granules in Arabidopsis chloroplasts and potato tuber amyloplasts. In rice, SSIVa and SSIVb are expressed in the endosperm and other organs at an early developmental stage (Hirose and Terao, 2004; Ohdan et al., 2005).In this study, two rice allelic SSIVb-deficient mutant lines (ss4b) were generated by insertion of the retrotransposon Tos17 and crossed with the SSIIIa null mutant (ss3a). Surprisingly, the ss3a ss4b endosperm produced spherical starch granules that were separated from each other within amyloplasts, whereas the single mutants produced compound-type polyhedral starch granules. The SSIVb and GBSSI enzymes were localized to distinct compartments in developing amyloplasts. We discuss the changes in rice starch structure due to the deficiency of both SSIIIa and SSIVb, the alteration in starch granule morphology, and possible unconventional functions of SSIIIa and SSIVb. We also present a model of how spherical granules are produced in ss3a ss4b rice endosperm.