We report the case of a Japanese family suffering from familial juvenile hyperuricemic nephropathy (FJHN) due to a rare missense mutation of the uromodulin (UMOD) gene. An 18-year-old male presented with gout, hyperuricemia, and stage 3 chronic kidney disease. Mostly, FJHN is caused by a mutation altering the cystine residue of UMOD/Tamm-Horsfall protein. However, in the present case, a T688C mutation was identified in exon 4, resulting in amino acid substitution with arginine replacing tryptophan at position 230 (Trp230Arg). This mutation was also found in his brother and father with the same phenotype, indicating autosomal dominant inheritance. The affected amino acid was conserved in 200 healthy Japanese controls. Therefore, mutation T688C most likely causes rare structural and/or functional abnormalities in UMOD/Tamm-Horsfall protein.
Chronic kidney disease; Familial juvenile hyperuricemic nephropathy; Gene; mutation; Uromodulin
We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.
The child’s brain is more malleable or plastic than that of adults and this accounts for the ability of children to learn new skills quickly or recovery from brain injuries. Several mechanisms contribute to this ability including overproduction and deletion of neurons and synapses, and activity-dependent stabilization of synapses. The molecular mechanisms for activity dependent synaptic plasticity are being discovered and this is leading to a better understanding of the pathogenesis of several disorders including neurofibromatosis, tuberous sclerosis, Fragile X syndrome and Rett syndrome. Many of the same pathways involved in synaptic plasticity, such as glutamate-mediated excitation, can also mediate brain injury when the brain is exposed to stress or energy failure such as hypoxia-ischemia. Recent evidence indicates that cell death pathways activated by injury differ between males and females. This new information about the molecular pathways involved in brain plasticity and injury are leading to insights that will provide better therapies for pediatric neurological disorders.
Plasticity; Injury; Fragile X Syndrome; Rett Syndrome; Hypoxia-Ischemia; NMDA; AMPA; Periventricular Leukomalacia
Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.
Thrombin and angiotensin II (angII) have trophic properties as mediators of vascular remodeling. Focal adhesions and actin cytoskeleton are involved in cell growth, shape, and movement and may be important in vascular remodeling. To characterize mechanisms by which thrombin and angII modulate vessel structure, we studied the effects of these G protein–coupled receptor ligands on focal adhesions in vascular smooth muscle cells (VSMCs). Both thrombin and angII stimulated bundling of actin filaments to form stress fibers, assembly of focal adhesions, and protein tyrosine phosphorylation at focal adhesions, such as p130Cas, paxillin, and tensin. To test whether c-Src plays a critical role in focal adhesion rearrangement, we analyzed cells with altered c-Src activity by retroviral transduction of wild-type (WT) and kinase-inactive (KI) c-Src into rat VSMCs, and by use of VSMCs from WT (src+/+) and Src-deficient (src–/–) mice. Tyrosine phosphorylation of Cas, paxillin, and tensin were markedly decreased in VSMCs expressing KI-Src and in src–/– VSMCs. Expression of KI-Src did not inhibit stress fiber formation by thrombin. Surprisingly, actin bundling was markedly decreased in VSMCs from src–/– mice both basally and after thrombin stimulation, compared with src+/+ mice. We also studied the effect of KI-Src and WT-Src on VSMC spreading. Expression of KI-Src reduced the rate of VSMC spreading on collagen, whereas WT-Src enhanced cell spreading. In conclusion, c-Src plays a critical role in agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in VSMCs. c-Src kinase activity is required for the cytoskeletal turnover that occurs in cell spreading, whereas c-Src appears to regulate actin bundling via a kinase-independent mechanism.
Interactions between biofilm cells of Pseudomonas aeruginosa and levofloxacin were studied. P. aeruginosa incubated for 6 days with Teflon sheets formed a biofilm on its surface. Against the biofilm bacteria, levofloxacin at an MIC determined by the standard method for the strain was highly bactericidal whereas gentamicin, ceftazidime, and ciprofloxacin showed no significant killing activity. Levofloxacin, ciprofloxacin, and gentamicin, but not ceftazidime, exhibited killing activity against nongrowing cells of the strain incubated in phosphate buffer. In addition, levofloxacin, ciprofloxacin, and ceftazidime, but not gentamicin, showed the ability to penetrate an agar containing alginate. These findings may explain the efficacy of levofloxacin and the ineffectiveness of gentamicin and ceftazidime against biofilm bacteria; however, the cause of the ineffectiveness of ciprofloxacin still remains to be determined. In experimental pneumonia in guinea pigs, in which the biofilm mode of growth of the strain was observed in the lung, only levofloxacin exhibited substantial therapeutic efficacy. These findings suggest the significant role of levofloxacin in therapy of biofilm bacterium-associated infectious diseases.
Contemporary hunter–gatherer groups are often thought to serve as models of an ancient lifestyle that was typical of human populations prior to the development of agriculture. Patterns of genetic variation in hunter–gatherer groups such as the !Kung and African Pygmies are consistent with this view, as they exhibit low genetic diversity coupled with high frequencies of divergent mtDNA types not found in surrounding agricultural groups, suggesting long-term isolation and small population sizes. We report here genetic evidence concerning the origins of the Mlabri, an enigmatic hunter–gatherer group from northern Thailand. The Mlabri have no mtDNA diversity, and the genetic diversity at Y-chromosome and autosomal loci are also extraordinarily reduced in the Mlabri. Genetic, linguistic, and cultural data all suggest that the Mlabri were recently founded, 500–800 y ago, from a very small number of individuals. Moreover, the Mlabri appear to have originated from an agricultural group and then adopted a hunting–gathering subsistence mode. This example of cultural reversion from agriculture to a hunting–gathering lifestyle indicates that contemporary hunter–gatherer groups do not necessarily reflect a pre-agricultural lifestyle.
Genes, language and culture reveal that the Mlabri reverted from an agricultural to a hunter-gatherer lifestyle, suggesting that hunter-gatherer groups might not always represent the pre- agricultural lifestyle of humans
C/EBPα plays important roles in metabolism as well as in the maintenance of energy homeostasis. Here we describe loss of the circadian oscillation of C/ebpα expression in liver of Clock mutant mice. Reporter assays indicate Clock and Bmal significantly induced C/ebpα gene expression whereas Cry suppressed. Real time reporter assays showed that two mutated E-boxes disrupted C/ebpα promoter dependent-oscillation. Chromatin immunoprecipitation suggests Clock can bind to two E-boxes in the C/ebpα promoter with a circadian manner in vivo. Thus, C/ebpα gene transcription is under circadian control of a core clock component, Clock. The data suggests that circadian disturbances may affect metabolic abnormalities through the C/ebpα pathway in liver.
Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.
The navel orangeworm, Amyelois transitella is a major agricultural pest causing large losses in a variety of tree crops. Control of this insect pest may be achieved by interfering with olfactory pathways to block detection of female-produced sex pheromones and consequently, disrupt mating. The first component of this pathway is the pheromone-binding protein AtraPBP1, which recognizes the pheromone and presents it to the odorant receptor housed in a sensory neuron of the male antennae. Release of the ligand depends on a pH-induced conformational change associated with the acidity of the membrane surface. To characterize this conformational change and to understand how pheromones bind, we have determined the high resolution crystal structures of AtraPBP1 in complex with two main constituents of the sex pheromone, i.e., (11Z,13Z)-hexadecadienal and (11Z,13Z)-hexadecadienol. Comparison with the structure of the unliganded form demonstrates a large ∼90° movement of the C-terminal helix which is observed in other pheromone- or odorant-binding proteins accompanied by an unpredicted 37° displacement of the N-terminal helix. Molecular dynamic trajectories suggest that the conformational change of the α1 helix facilitates the movement of the C-terminal helix.
Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7–1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae.
Estrogen, a class of female sex steroids, is neuroprotective. Estrogen is synthesized in specific areas of the brain. There is a possibility that the de novo synthesized estrogen exerts protective effect in brain, although direct evidence for the neuroprotective function of brain-synthesized estrogen has not been clearly demonstrated. Methylmercury (MeHg) is a neurotoxin that induces neuronal degeneration in the central nervous system. The neurotoxicity of MeHg is region-specific, and the molecular mechanisms for the selective neurotoxicity are not well defined. In this study, the protective effect of de novo synthesized 17β-estradiol on MeHg-induced neurotoxicity in rat hippocampus was examined.
Neurotoxic effect of MeHg on hippocampal organotypic slice culture was quantified by propidium iodide fluorescence imaging. Twenty-four-hour treatment of the slices with MeHg caused cell death in a dose-dependent manner. The toxicity of MeHg was attenuated by pre-treatment with exogenously added estradiol. The slices de novo synthesized estradiol. The estradiol synthesis was not affected by treatment with 1 µM MeHg. The toxicity of MeHg was enhanced by inhibition of de novo estradiol synthesis, and the enhancement of toxicity was recovered by the addition of exogenous estradiol. The neuroprotective effect of estradiol was inhibited by an estrogen receptor (ER) antagonist, and mimicked by pre-treatment of the slices with agonists for ERα and ERβ, indicating the neuroprotective effect was mediated by ERs.
Hippocampus de novo synthesized estradiol protected hippocampal cells from MeHg-induced neurotoxicity via ERα- and ERβ-mediated pathways. The self-protective function of de novo synthesized estradiol might be one of the possible mechanisms for the selective sensitivity of the brain to MeHg toxicity.
Statins are frequently administered to reduce low-density lipoprotein cholesterol (LDL-C) and vascular inflammation, because LDL-C and high sensitive C-reactive protein (hs-CRP) are associated with high risk for cardiovascular events. When statins do not reduce LDL-C to desired levels in high-risk patients with coronary artery disease (CAD), ezetimibe can be added or the statin dose can be increased. However, which strategy is more effective for treating patients with CAD has not been established. The present study compares anti-inflammatory effects and lipid profiles in patients with CAD and similar LDL-C levels who were treated by increasing the statin dose or by adding ezetimibe to the original rosuvastatin dose to determine the optimal treatment for such patients.
46 patients with high-risk CAD and LDL-C and hs-CRP levels of >70 mg/dL and >1.0 mg/L, respectively, that were not improved by 4 weeks of rosuvastatin (2.5 mg/day) were randomly assigned to receive 10 mg (R10, n = 24) of rosuvastatin or 2.5 mg/day of rosuvastatin combined with 10 mg/day of ezetimibe (R2.5/E10, n = 22) for 12 weeks. The primary endpoint was a change in hs-CRP.
Baseline characteristics did not significantly differ between the groups. At 12 weeks, LDL-C and inflammatory markers (hs-CRP, interleukin-6, tumour necrosis factor-alpha and pentraxin 3) also did not significantly differ between the two groups (LDL-C: R10 vs. R2.5/E10: -19.4 ± 14.2 vs. -22.4 ± 14.3 mg/dL). However, high-density lipoprotein cholesterol (HDL-C) was significantly improved in the R10, compared with R2.5/E10 group (4.6 ± 5.9 vs. 0.0 ± 6.7 mg/dL; p < 0.05).
Both enhanced therapies exerted similar anti-inflammatory effects under an equal LDL-C reduction in patients with high-risk CAD despite 2.5 mg/day of rosuvastatin. However, R10 elevated HDL-C more effectively than R2.5/E10.
Statin; Secondary prevention; Coronary artery disease
To assess the potential acute effects regarding the immunogenicity and safety of non-adjuvanted influenza A H1N1/2009 vaccine in patients with mixed connective tissue disease and healthy controls.
Sixty-nine mixed connective tissue disease patients that were confirmed by Kasukawa's classification criteria and 69 age- and gender-matched controls participated in the study; the participants were vaccinated with the non-adjuvanted influenza A/California/7/2009 (H1N1) virus-like strain. The percentages of seroprotection, seroconversion, geometric mean titer and factor increase in the geometric mean titer were calculated. The patients were clinically evaluated, and blood samples were collected pre- and 21 days post-vaccination to evaluate C-reactive protein, muscle enzymes and autoantibodies. Anti-H1N1 titers were determined using an influenza hemagglutination inhibition assay. ClinicalTrials.gov: NCT01151644.
Before vaccination, no difference was observed regarding the seroprotection rates (p = 1.0) and geometric mean titer (p = 0.83) between the patients and controls. After vaccination, seroprotection (75.4% vs. 71%, p = 0.7), seroconversion (68.1% vs. 65.2%, p = 1.00) and factor increase in the geometric mean titer (10.0 vs. 8.0, p = 0.40) were similar in the two groups. Further evaluation of seroconversion in patients with and without current or previous history of muscle disease (p = 0.20), skin ulcers (p = 0.48), lupus-like cutaneous disease (p = 0.74), secondary Sjögren syndrome (p = 0.78), scleroderma-pattern in the nailfold capillaroscopy (p = 1.0), lymphopenia ≤1000/mm3 on two or more occasions (p = 1.0), hypergammaglobulinemia ≥1.6 g/d (p = 0.60), pulmonary hypertension (p = 1.0) and pulmonary fibrosis (p = 0.80) revealed comparable rates. Seroconversion rates were also similar in patients with and without immunosuppressants. Disease parameters, such as C-reactive protein (p = 0.94), aldolase (p = 0.73), creatine phosphokinase (p = 0.40) and ribonucleoprotein antibody levels (p = 0.98), remained largely unchanged pre and post-vaccination. No severe side effects were reported.
The non-adjuvanted influenza A/H1N1 vaccination immune response in mixed connective tissue disease patients is adequate and does not depend on the disease manifestations and therapy.
Mixed Connective Tissue Disease; Influenza A Virus; H1N1 Subtype; Influenza Vaccine
Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, especially in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1– deficient mice by crossing mcl-1flox/flox mice and AlbCre mice and further crossed them with bcl-xflox/flox mice, giving Mcl-1/Bcl-xL– deficient mice. The mcl-1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased number of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL)-positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte-specific Bcl-xL– deficient mice. Although mcl-1flox/+
AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-xflox/+
AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL– deficient or Mcl-1– deficient mice. In contrast, bcl-xflox/flox mcl-1flox/+
mcl-1flox/flox AlbCre, and bcl-xflox/flox mcl-1flox/flox AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl-1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner.
Bcl-2 homology domain 3 (BH3)-only protein Bid is posttranslationally cleaved by caspase-8 into its truncated form (tBid) and couples with stress signals to the mitochondrial cell death pathway. However, the physiological relevance of Bid is not clearly understood. Hepatocyte-specific knockout (KO) of Bcl-xL leads to naturally-occurring apoptosis despite co-expression of Mcl-1, which shares a similar anti-apoptotic function. We generated Bcl-xL KO, Bcl-xL/Bid double KO, Bcl-xL/Bak double KO, Bcl-xL/Bax double KO, and Bcl-xL/Bak/Bax triple KO mice and found that hepatocyte apoptosis caused by Bcl-xL deficiency was completely dependent on Bak and Bax, and surprisingly on Bid. This indicated that, in the absence of Bid, Bcl-xL is not required for the integrity of differentiated hepatocytes, suggesting a complicated interaction between core Bcl-2 family proteins and BH3-only proteins even in a physiological setting. Indeed, a small but significant level of tBid was present in wild-type liver under physiological conditions. tBid was capable of binding to Bcl-xL and displacing Bak and Bax from Bcl-xL, leading to release of cytochrome c from wild-type mitochondria. Bcl-xL–deficient mitochondria were more susceptible to tBid-induced cytochrome c release. Finally, administration of ABT-737, a pharmacological inhibitor of Bcl-2/Bcl-xL, caused Bak/Bax-dependent liver injury, but this was clearly ameliorated with a Bid KO background.
Bid, originally considered to be a sensor for apoptotic stimuli, is constitutively active in healthy liver cells and is involved in the Bak/Bax-dependent mitochondrial cell death pathway. Healthy liver cells are addicted to a single Bcl-2–like molecule because of BH3 stresses, and therefore special caution may be required for the use of the Bcl-2 inhibitor for cancer therapy.
The Jaynes-Cummings model, describing the interaction between a single two-level system and a photonic mode, has been used to describe a large variety of systems, ranging from cavity quantum electrodynamics, trapped ions, to superconducting qubits coupled to resonators. Recently there has been renewed interest in studying the quantum strong-coupling (QSC) regime, where states with photon number greater than one are excited. This regime has been recently achieved in semiconductor nanostructures, where a quantum dot is trapped in a planar microcavity. Here we study the quantum strong-coupling regime by calculating its photoluminescence (PL) properties under a pulsed excitation. We discuss the changes in the PL as the QSC regime is reached, which transitions between a peak around the cavity resonance to a doublet. We particularly examine the variations of the PL in the time domain, under regimes of short and long pulse times relative to the microcavity decay time.
Selective Alzheimer’s disease (AD) indicator 1 (Seladin-1) has been identified as a gene down-regulated in the degenerated lesions of AD brain. Up-regulation of Seladin-1 reduces the accumulation of β-amyloid and neuronal death. Thyroid hormone (TH) exerts an important effect on the development and maintenance of central nervous systems. In the current study, we demonstrated that Seladin-1 gene and protein expression in the forebrain was increased in thyrotoxic mice compared with that of euthyroid mice. However, unexpectedly, no significant decrease in the gene and protein expression was observed in hypothyroid mice. Interestingly, an agonist of liver X receptor (LXR), TO901317 (TO) administration in vivo increased Seladin-1 gene and protein expression in the mouse forebrain only in a hypothyroid state and in the presence of mutant TR-β, suggesting that LXR-α would compensate for TR-β function to maintain Seladin-1 gene expression in hypothyroidism and resistance to TH. TH activated the mouse Seladin-1 gene promoter (−1936/+21 bp) and site 2 including canonical TH response element (TRE) half-site in the region between −159 and −154 bp is responsible for the positive regulation. RXR-α/TR-β heterodimerization was identified on site 2 by gel-shift assay, and chromatin immunoprecipitation assay revealed the recruitment of TR-β to site 2 and the recruitment was increased upon TH administration. On the other hand, LXR-α utilizes a distinct region from site 2 (−120 to −102 bp) to activate the mouse Seladin-1 gene promoter. Taking these findings together, we concluded that TH up-regulates Seladin-1 gene expression at the transcriptional level and LXR-α maintains the gene expression.
The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of l-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-d-glucitol and d-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of l-gulonate in an extended (or all-trans) conformation; l-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.