Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes.

The pathogenic Listeria monocytogenes bacterium produces the flagellum as a locomotive organelle at or below 30°C outside the host, but it halts flagellar expression at 37°C inside the human host to evade the flagellum-induced immune response. Listeria monocytogenes GmaR is a thermosensor protein that coordinates flagellar expression by binding the master transcriptional repressor of flagellar genes (MogR) in a temperature-responsive manner. To understand the regulatory mechanism whereby GmaR exerts the antirepression activity on flagellar expression, we performed structural and mutational analyses of the GmaR-MogR system. At or below 30°C, GmaR exists as a functional monomer and forms a circularly enclosed multidomain structure via an interdomain interaction. GmaR in this conformation recognizes MogR using the C-terminal antirepressor domain in a unique dual binding mode and mediates the antirepressor function through direct competition and spatial restraint mechanisms. Surprisingly, at 37°C, GmaR rapidly forms autologous aggregates that are deficient in MogR neutralization capabilities.

Amino Sugar-Enriched Fraction of Korean Red Ginseng Extract Induces the Priming Step of NLRP3 Inflammasome.

Intracellular protein complexes, known as inflammasomes, activate caspase-1 and induce the secretion of pro-inflammatory cytokines, namely interleukin (IL)-1ß and -18. Korean Red Ginseng extract (RGE) is a known immunomodulator and a potential candidate for the regulation of inflammasomes. The saponins, such as ginsenosides, of RGE inhibit inflammasome signaling, while non-saponin substances containing amino sugars promote the priming step, up-regulating inflammasome components (pro-IL-1ß, NLRP3, caspase-1, and Asc). In this study, the amino sugar-enriched fraction (ASEF), which increases only non-saponin components, including amino sugars, without changing the concentration of saponin substances, was used to investigate whether saponin or non-saponin components of RGE would have a greater impact on the priming step. When murine macrophages were treated with ASEF, the gene expression of inflammatory cytokines (IL-1α, TNFα, IL-6, and IL-10) increased. Additionally, ASEF induced the priming step but did not affect the inflammasome activation step, such as the secretion of IL-1ß, cleavage of caspase-1, and formation of Asc pyroptosome. Furthermore, the upregulation of gene expression of inflammasome components by ASEF was blocked by inhibitors of Toll-like receptor 4 signaling. Maltol, the main constituent of ASEF, promoted the priming step but inhibited the activation step of the inflammasome, while arginine, sugars, arginine-fructose-glucose, and fructose-arginine, the other main constituents of ASEF, had no effect on either step. Thus, certain amino sugars in RGE, excluding maltol, are believed to be the components that induce the priming step. The priming step that prepares the NLRP3 inflammasome for activation appears to be induced by amino sugars in RGE, thereby contributing to the immune-boosting effects of RGE.

Lactoferrin-derived chimeric peptide (LFch) strongly boosts TGFß1-mediated inducible Treg differentiation possibly through downregulating TCR/CD28 signalling.

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFß receptor III (TßRIII), and this activity was further enhanced by TGFß1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17-30] and lactoferrampin [aa 265-284]), along with TGFß1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFß1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFß1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFß1 receptor III (sTßRIII). Consistent with this, the activity of LFch on TGFß1-induced Foxp3 expression was also abrogated by treatment with sTßRIII. Finally, the TGFß1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFß1-induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity.

Regulation of uterus and placenta remodeling under high estradiol levels in gestational diabetes mellitus models†.

The present study aimed to investigate the regulation of placentas and uterus remodeling and involvement of estradiol in gestational diabetes mellitus. To achieve this, we established in vitro and in vivo models for gestational diabetes mellitus placentas by culturing human placental choriocarcinoma cells (BeWo) under hyperglycemic concentration and treating pregnant rats with streptozotocin. We evaluated the expression of angiogenesis-related proteins. The expression of the anti-angiogenic factor, excess placental soluble fms-like tyrosine kinase 1 was increased in our in vitro gestational diabetes mellitus model compared with the control. Moreover, the expressions of placental soluble fms-like tyrosine kinase 1 and the von Willebrand factor were also significantly elevated in the placenta of streptozotocin-treated rats. These data indicate the disruption of angiogenesis in the gestational diabetes mellitus placentas. The expression levels of connexin 43, a component of the gap junction and collagen type I alpha 2 chain, a component of the extracellular matrix, were decreased in the gestational diabetes mellitus uterus. These results suggest that uterus decidualization and placental angiogenesis are inhibited in gestational diabetes mellitus rats. Our results also showed upregulation of the expression of genes regulating estradiol synthesis as well as estrogen receptors in vivo models. Accordingly, the concentration of estradiol measured in the culture medium under hyperglycemic conditions, as well as in the serum and placenta of the streptozotocin-treated rats, was significantly elevated compared with the control groups. These results suggest that the dysregulated remodeling of the placenta and uterus may result in the elevation of estradiol and its signaling pathway in the gestational diabetes mellitus animal model to maintain pregnancy.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

Lactoferrin Potentiates Inducible Regulatory T Cell Differentiation through TGF-ß Receptor III Binding and Activation of Membrane-Bound TGF-ß.

Lactoferrin (LF) is known to possess anti-inflammatory activity, although its mechanisms of action are not well-understood. The present study asked whether LF affects the commitment of inducible regulatory T cells (Tregs). LF substantially promoted Foxp3 expression by mouse activated CD4+T cells, and this activity was further enhanced by TGF-ß1. Interestingly, blocking TGF-ß with anti-TGF-ß Ab completely abolished LF-induced Foxp3 expression. However, no significant amount of soluble TGF-ß was released by LF-stimulated T cells, suggesting that membrane TGF-ß (mTGF-ß) is associated. Subsequently, it was found that LF binds to TGF-ß receptor III, which induces reactive oxygen species production and diminishes the expression of mTGF-ß-bound latency-associated peptide, leading to the activation of mTGF-ß. It was followed by phosphorylation of Smad3 and enhanced Foxp3 expression. These results suggest that LF induces Foxp3+ Tregs through TGF-ß receptor III/reactive oxygen species-mediated mTGF-ß activation, triggering canonical Smad3-dependent signaling. Finally, we found that the suppressive activity of LF-induced Tregs is facilitated mainly by CD39/CD73-induced adenosine generation and that this suppressor activity alleviates inflammatory bowel disease.

Genetic and immunological characterization of commercial infectious bronchitis virus vaccines used in Korea.

The aim of the study was to investigate the genetic and immunogenic features of commercial vaccines against infectious bronchitis virus (IBV), which is a major contagious pathogen of poultry. Although numerous vaccines have been developed based on the genetic characteristics of field strains, the continual emergence of variants decreases vaccine efficacy and cross-protection. To address this issue, we compared the S1 gene sequences of three IBV vaccines commercially available in Korea with those of various field isolates. Phylogenetic analysis showed that the vaccine strains clustered into two different lineages. Comparison of commercial vaccines with their parental viruses showed that most of the genetic variability occurred around hypervariable regions (HVRs). Conversely, antigenic stimulation with commercial vaccines and regional IBV variants was not sufficient to alter major immune cell phenotypes. Our study suggests that vaccines should be selected carefully based on their genetic background because genetic variability can affect the antigenicity of vaccines and host immune responses.

Absence of progesterone receptor membrane component 1 reduces migration and metastasis of breast cancer.

BACKGROUND: Progesterone receptor membrane component 1 (Pgrmc1) is a non-classical progesterone receptor associated with the development of the mammary gland and xenograft-induced breast cancer. Importantly, Pgrmc1 is associated with the expression of estrogen receptor alpha and can be used for predicting the prognosis of breast cancer. Whether the genetic deletion of Pgrmc1 affects the progression of breast cancer is still unclear. METHODS: We used MMTV-PyMT transgenic mice that spontaneously develop breast tumors. In backcrossed FVB Pgrmc1 knockout (KO) mice, we monitored the development of the primary tumor and lung metastasis. In MCF-7 and MDA-MB-231 tumor cell lines, the migratory activity was evaluated after Pgrmc1 knockdown. RESULTS: There was no significant difference in the development of breast cancer in terms of tumor size at 13 weeks of age between WT and Pgrmc1 KO mice. However, Pgrmc1 KO mice had a significantly longer survival duration compared with WT mice. Furthermore, Pgrmc1 KO mice exhibited a significantly lower degree of lung metastasis. Compared with those of WT mice, the tumors of Pgrmc1 KO mice had a low expression of focal adhesion kinase and epithelial-mesenchymal transition markers. PGRMC1 knockdown resulted in a significantly reduced migration rate in breast cancer cell lines. CONCLUSIONS: Pgrmc1 KO mice with breast cancer had a prolonged survival, which was accompanied by a low degree of lung metastasis. PGRMC1 showed a significant role in the migration of breast cancer cells, and may serve as a potential therapeutic target in breast cancer. Video Abstract.

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs.

This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

IκBζ controls NLRP3 inflammasome activation via upregulation of the Nlrp3 gene.

Inflammasome activation induces the maturation and secretion of interleukin (IL)-1ß and -18, and is dependent on NF-κB signaling to induce the transcription of the inflammasome components, called the priming step. This study elucidated the role of IκBζ, an atypical IκBs (inhibitor of κB) and a coactivator of NF-κB target genes, on the activation of inflammasome. Bone marrow-derived macrophages (BMDMs) that originated from IκBζ-encoding Nfkbiz gene depletion mice presented a defect in NLRP3 inflammasome activation. In addition, the Nfkbiz+/- and Nfkbiz-/- mice significantly attenuated serum IL-1ß secretion in response to a monosodium urate injection, a NLRP3 trigger, when compared with Nfkbiz-+/+ mice. The lack of IκBζ in BMDMs produced a disability in the expression of Nlrp3 and pro-Il1ß mRNAs during the priming step. In addition, ectopic IκBζ expression enhanced the Nlrp3 promoter activity, and Nlrp3 and pro-Il1ß transcription. Overall, IκBζ controlled the activation of NLRP3 inflammasome by upregulating the Nlrp3 gene during the priming step.

Gain-of-function Pyrin mutations induce NLRP3 protein-independent interleukin-1ß activation and severe autoinflammation in mice.

Missense mutations in the C-terminal B30.2 domain of pyrin cause familial Mediterranean fever (FMF), the most common Mendelian autoinflammatory disease. However, it remains controversial as to whether FMF is due to the loss of an inhibitor of inflammation or to the activity of a proinflammatory molecule. We generated both pyrin-deficient mice and "knockin" mice harboring mutant human B30.2 domains. Homozygous knockin, but not pyrin-deficient, mice exhibited spontaneous bone marrow-dependent inflammation similar to but more severe than human FMF. Caspase-1 was constitutively activated in knockin macrophages and active IL-1ß was secreted when stimulated with lipopolysaccharide alone, which is also observed in FMF patients. The inflammatory phenotype of knockin mice was completely ablated by crossing with IL-1 receptor-deficient or adaptor molecule ASC-deficient mice, but not NLRP3-deficient mice. Thus, our data provide evidence for an ASC-dependent NLRP3-independent inflammasome in which gain-of-function pyrin mutations cause autoinflammatory disease.

Sex hormone-binding globulin suppresses NAFLD-triggered hepatocarcinogenesis after menopause.

It is generally accepted that androgen receptors increase the risk of hepatocellular carcinoma (HCC), and that estrogen reduces risk of HCC. Many studies regarding this have involved males. We, therefore, have focused our attention on females, especially postmenopausal females, who typically have limited supplies of estrogen. By using sex hormone-binding globulin (SHBG) transgenic mice, we produced a humanoid environment, and facilitated deposition and modulation of sex hormones. After exposure to diethylnitrosamine to induce HCC and upon reaching the age of 40 weeks, mice were fed the fat-rich diet for 5 months. Fat-rich diet fed or ovariectomized (OVX) wild-type mice aged 62 weeks showed HCC progression, whereas fat-rich diet fed SHBG mice or OVX SHBG mice displayed fewer tumors. In the liver of fat-rich diet fed SHBG mice, estrogenic conditions including high levels of 17ß-estradiol and estrogen receptor alpha led to the induction of the lipogenesis inhibitor, phosphorylated acetyl-CoA carboxylase, and consequently suppressed fatty liver. The presence of plasma SHBG in HCC bearing mice suppressed the levels of steatosis and inflammation in a process mediated by estrogens and estrogen receptor alpha. Conversely, in the liver of OVX SHBG mice, lipogenic inhibition was also observed under conditions where the supply of estrogens is limited. Through in vitro experiment, it was confirmed SHBG suppresses lipogenesis via inhibition of acetyl-CoA carboxylase level. In conclusion, our results show that plasma SHBG might have a clinical impact on lipid-mediated hepatic diseases.

Dietary intake of genistein suppresses hepatocellular carcinoma through AMPK-mediated apoptosis and anti-inflammation.

BACKGROUND: Women have a lower risk of hepatocellular carcinoma (HCC) than men, and the decreased possibility of HCC in women is thought to depend on estrogen levels. As a soybean-isoflavone product, genistein has estrogenic activity in various reproductive tissues, because it mimics 17ß-estradiol and binds the estrogen receptor. Though genistein is a known liver cancer suppressor, its effects have not been studies in long-term experiment, where genistein is fed to a female animal model of HCC. METHODS: Mice were treated with diethylnitrosamine (DEN) to induce HCC at 2 weeks of age and fed with supplemental genistein for 5 months, from 40 to 62 weeks of age. RESULTS: The dietary intake of genistein decreased the incidence of HCC and suppressed HCC development. Genistein induced phospho-AMPK in total liver extracts, Hep3B cells, and Raw 264.7 cells, and phospho-AMPK promoted apoptosis in liver and Hep3B cells. Moreover, phospho-AMPK down-regulated pro-inflammatory responses and ameliorated liver damage. A suppressed pro-inflammatory response with increased mitochondrial respiration was concomitantly observed after genistein treatment. CONCLUSIONS: Genistein-mediated AMPK activation increases hepatocyte apoptosis through energy-dependent caspase pathways, suppresses the inflammatory response in resident liver macrophages by increased cellular respiration, and consequently inhibits the initiation and progression of HCC.

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts.

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP.

Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1ß (IL-1ß) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1ß production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.

Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs.

This study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4 µg/mL demecolcine, 2 mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.

Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth.

Mechanism underlying the suppressor activity of retinoic acid on IL4-induced IgE synthesis and its physiological implication.

The present study extends an earlier report that retinoic acid (RA) down-regulates IgE Ab synthesis in vitro. Here, we show the suppressive activity of RA on IgE production in vivo and its underlying mechanisms. We found that RA down-regulated IgE class switching recombination (CSR) mainly through RA receptor α (RARα). Additionally, RA inhibited histone acetylation of germ-line ε (GL ε) promoter, leading to suppression of IgE CSR. Consistently, serum IgE levels were substantially elevated in vitamin A-deficient (VAD) mice and this was more dramatic in VAD-lecithin:retinol acyltransferase deficient (LRAT-/-) mice. Further, serum mouse mast cell protease-1 (mMCP-1) level was elevated while frequency of intestinal regulatory T cells (Tregs) were diminished in VAD LRAT-/- mice, reflecting that deprivation of RA leads to allergic immune response. Taken together, our results reveal that RA has an IgE-repressive activity in vivo, which may ameliorate IgE-mediated allergic disease.

Structural and biochemical characterization of the Bacillus cereus 3-hydroxyisobutyrate dehydrogenase.

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) family catalyzes the NAD(+)- or NADP(+)-dependent oxidation of various ß-hydroxyacid substrates into their cognate semialdehydes for diverse metabolic pathways. Because HIBADH group members exhibit different substrate specificities, the substrate-recognition mode of each enzyme should be individually characterized. In the current study, we report the biochemical and structural analysis of a HIBADH group enzyme from Bacillus cereus (bcHIBADH). bcHIBADH mediates a dehydrogenation reaction on S-3-hydroxyisobutyrate substrate with high catalytic efficiency in an NAD(+)-dependent manner; it also oxidizes l-serine and 3-hydroxypropionate with lower activity. bcHIBADH consists of two domains and is further assembled into a functional dimer rather than a tetramer that has been commonly observed in other prokaryotic HIBADH group members. In the bcHIBADH structure, the interdomain cleft forms a putative active site and simultaneously accommodates both an NAD(+) cofactor and a substrate mimic. Our structure-based comparative analysis highlights structural motifs that are important in the cofactor and substrate recognition of the HIBADH group.

Sulforaphane attenuates activation of NLRP3 and NLRC4 inflammasomes but not AIM2 inflammasome.

Sulforaphane (SFN), a compound within the isothiocyanate group of organosulfur compounds originating from cruciferous vegetables, has gained attention for its antioxidant, anti-inflammatory, and cancer chemopreventive properties. However, the effects of SFN on inflammasomes, which are multi-protein complexes that induce maturation of interleukin (IL)-1ß, have been poorly studied. In this study, we investigated the effects of SFN on the assembly of NLRP3, NLRC4, and AIM2 inflammasomes as well as on the priming step of NLRP3 inflammasome in murine macrophages. In our results, SFN attenuated activation of NLRP3 and NLRC4 inflammasomes but not AIM2 inflammasome. In addition, SFN blocked expression of the NLRP3 gene and pro-IL-1ß during the priming step. SFN further attenuated IL-1ß secretion of monosodium uric acid-induced peritonitis in mice. Lastly, SFN inhibited generation of mitochondrial reactive oxygen species, which trigger NLRP3 inflammasome activation. Thus, SFN is suggested as an anti-inflammasome molecule for NLRP3 and NLRC4 inflammasome activation.