Effect of thiouracil in modifying folate function in severe vitamin B12 deficiency

The effects of thiouracil in correcting defects in folic acid function produced by B12 deficiency were studied. Addition of the thyroid inhibitor, thiouracil, to a low methionine diet containing B12, increased the oxidation of [2-14C]histidine to carbon dioxide, and increased liver folate levels. Addition of 10% pectin to the diet accentuated B12 deficiency as evidenced by a greatly decreased rate of histidine oxidation (0.19%) and an increased excretion of methylmalonic acid. Addition of thiouracil to the diet restored folate function as measured by increased histidine oxidation and increased liver folate levels similar to that produced by addition of methionine to a B12-deficient diet. Thiouracil decreased methylmalonate excretion, and increased hepatic levels of B12 in animals on both B12-deficient and -supplemented diets. Hepatic methionine synthase was increased by thiouracil, which may be the result of the elevated B12 levels. S-Adenosylmethionine and the enzyme methionine adenosyltransferase were also increased by thiouracil. Thus it is possible that the effect of thiouracil in increasing folate function consists both in the effect of thiouracil in decreasing levels of methylenetetrahydrofolate reductase, and also in its action in increasing S-adenosylmethionine which exerts a feedback inhibition of this enzyme.     

Endogenous acid ceramidase protects epithelial cells from Porphyromonas gingivalis-induced inflammation in vitro

Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival? 3D culture and OBA-9?cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9?cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9?cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9?cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9?cells produced less TNF-α, IL-6, and IL1β pro-inflammatory cytokines than observed in OBA-9?cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.     

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3′ untranslated region (UTR) but not TRAF6 3′-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from Pseudomonas sp. strain E-3

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 μmol h^–1 (mg protein)^–1 and a K _m of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7–8 and 30° C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as α-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1,10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed. Received: 21 May 1997 / Accepted: 5 September 1997     

Participation of Syndecan 2 in the Induction of Stress Fiber Formation in Cooperation with Integrin α5β1: Structural Characteristics of Heparan Sulfate Chains with Avidity to COOH-Terminal Heparin-Binding Domain of Fibronectin

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin α5β1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925–930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)–GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)–GlcNS(6OS)]₆ present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin α5β1.     

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.     

Circulating Omega-6, But Not Omega-3 Polyunsaturated Fatty Acids,Are Associated with Clinical Outcomes in Patients with Acute Decompensated Heart Failure

BackgroundCirculating polyunsaturated fatty acid (PUFA) levels are associated with clinical outcomes in cardiovascular diseases including coronary artery disease and chronic heart failure (HF). However, their clinical implications in acute decompensated HF (ADHF) remain unclear. The aim of this study was to investigate the clinical roles of circulating PUFAs in patients with ADHF.MethodsCirculating levels of PUFAs, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA) and dihomo-gamma linoleic acid (DGLA), were measured on admission in 685 consecutive ADHF patients. Adverse events were defined as all-cause death and worsening HF.ResultsDuring a median follow-up period of 560 days, 262 (38.2%) patients had adverse events. Although patients with adverse events had lower n-6 PUFA (AA + DGLA) level than those without, n-3 PUFA (EPA + DHA) level was comparable between the groups. Kaplan-Meier analyses showed that lower n-6 PUFA level on admission was significantly associated with the composite of all-cause death and worsening HF, all-cause death, cardiovascular death and worsening HF (p < 0.001, p = 0.005, p = 0.021, p = 0.019, respectively). In a multivariate Cox model, lower n-6 PUFA level was independently associated with increased risk of adverse events (HR 0.996, 95% CI: 0.993–0.999, p = 0.027).ConclusionsLower n-6 but not n-3 PUFA level on admission was significantly related to worse clinical outcomes in ADHF patients. Measurement of circulating n-6 PUFA levels on admission might provide information for identifying high risk ADHF patients.