Spinal cord stimulation for the treatment of pain and toe ulceration associated with systemic sclerosis: a case report.

Systemic sclerosis is a complex disease characterized by extensive fibrosis, microvascular alterations, and additional sequelae. Microvascular alterations can cause painful ulcers and necrosis; however, conservative or surgical treatment is often challenging in terms of healing. The study aimed to describe a toe ulcer with systemic sclerosis and its' successful treatment with spinal cord stimulation. An 83-year-old woman, who was diagnosed with systemic sclerosis over the past decade, was distressed by a non-healing toe ulcer for an extended period of time. The patient underwent spinal cord stimulation treatment with the expectation of pain relief and an improvement in microcirculatory insufficiency. Her pain scales and microcirculation improved, and the toe ulcer healed. Furthermore, the frequency of Raynaud's symptoms was reduced, and the patient's pain decreased. There was no recurrence of the ulcer and she no longer needed a cane for walking.

Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages.

Microtubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.

OCT3/4-binding sequence-dependent maintenance of the unmethylated state of CTCF-binding sequences with DNA demethylation and suppression of de novo DNA methylation in the H19 imprinted control region.

DNA demethylation and suppression of de novo DNA methylation are activities that maintain an unmethylated state. However, the strength of these two activities at the same locus has not been estimated separately. Furthermore, the association between these two activities and the unmethylated state remains unclear. Octamer-binding transcription factor-binding sequences (OBSs) and CCCTC-binding factor-binding sequences (CBSs) within the mouse H19-imprinted control region (ICR) are involved in the induction of DNA demethylation and maintenance of the unmethylated state in mouse undifferentiated embryonic cell lines. To reveal the association between the two cis-elements and the two unmethylated state maintenance activities in maintaining the unmethylated state of the ICR, we evaluated the altered DNA methylation levels at sites that were initially methylated or unmethylated using a stable transfection-based assay, and estimated the strength of the two unmethylated state maintenance activities separately via a Poisson process model that described the DNA methylation state regulatory process. Although DNA demethylation depending on OBSs affected almost the entire ICR, DNA demethylation depending on CBSs occurred near CBSs, resulting in redundant demethylation of CBS regions. Detailed analysis of the CBS4 region suggested that OBSs were required to induce unmethylated state maintenance activities, and that CBSs-dependent activities contributed, but diminished, during incubation when protection of the CBS4 region by OBSs-dependent activities was absent. Analysis via the Poisson process model indicated that the unmethylated state at the CBS4 region was maintained by OBSs-dependent suppression of de novo DNA methylation rather than DNA demethylation. We propose that the hierarchical regulation of redundant protection of the CBS region via cooperation between the two unmethylated state maintenance activities is a potential function of the ICR that effectively maintains allele-specific methylation status in the same DNA sequence.

Oct motif variants in Beckwith-Wiedemann syndrome patients disrupt maintenance of the hypomethylated state of the H19/IGF2 imprinting control region.

The methylation status of imprinting control center 1 (IC1) regulates the monoallelic transcription of H19 and Igf2 in mammalian cells. Several single nucleotide variants in Oct motifs within IC1 occur in patients with Beckwith-Wiedemann syndrome (BWS) who have hypermethylated maternal IC1. However, the importance of Oct motifs in the regulation of IC1 methylation status remains unclear. Here, we demonstrate that three variants found in BWS (BWS variants) suppress intensive induction of DNA demethylation, whereas consensus disruption of motifs unrelated to BWS only slightly affects the induction of demethylation. BWS variants reduce DNA demethylation levels and trigger the accumulation of DNA methylation downstream of the IC1 transgenes. Thus, the risk of IC1 hypermethylation is associated with inhibitory levels of Oct motif-dependent hypomethylation maintenance activities.

Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages.

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ-activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane-localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane-localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection.

The paternal-allele-specific methylation of the Igf2/H19 imprinting control region (ICR) is established during gametogenesis and maintained throughout development. To elucidate the requirement of the germline passage in the maintenance of the imprinting methylation, we established a system introducing a methylated or unmethylated ICR-containing DNA fragment (ICR-F) into the paternal or maternal genome by microinjecting into the paternal or maternal pronucleus of fertilized eggs, and traced the methylation pattern in the ICR-F. When the ICR-F was injected in a methylated form, it was demethylated approximately to half degree at blastocyst stage but was almost completely remethylated at 3 weeks of age. In the case of the unmethylated form, the ICR-F remained unmethylated at the blastocyst stage, but was almost half-methylated at 3 weeks of age. Interestingly, the paternally injected ICR-F was highly methylated compared with maternally injected ICR-F at 3 weeks of age, partially mimicking the endogenous methylation pattern. Moreover, introduction of mutations in the CTCF (CCCTC binding factor) binding sites of the ICR-F, which are known to be important for the maintenance of hypomethylated maternal ICR, induced hypermethylation of the mutated ICR-F in both paternal and maternal pronuclear injected 3-week-old mice. Our results suggest the presence of a protection-against-methylation activity of the CTCF binding site in establishing the preferential paternal methylation during post-fertilization development and the importance of germline passage in the maintenance of the parental specific methylation at H19 ICR.

Quantitative analysis of phagosome formation and maturation using an Escherichia coli probe expressing a tandem fluorescent protein.

Phagosome formation and maturation are essential innate immune mechanisms to engulf and digest foreign particles. To analyze these processes quantitatively, we established a specific Escherichia coli probe expressing a tandem fluorescent protein, comprising glutathione S-transferase fused with monomeric Cherry (mCherry) and monomeric Venus (mVenus). We demonstrated that mVenus was more susceptible to bleaching in an acidic environment than mCherry, and that the mVenus:mCherry fluorescence intensity ratio can be used to monitor phagosomal pH changes during maturation. Using this probe, we revealed that synaptosomal-associated protein of 23 kDa, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, actively regulated phagocytosis of E. coli and subsequent phagosome maturation in macrophages. Our results indicated that this probe has the potential to be a powerful tool in understanding the molecular mechanisms of phagosome formation and maturation.

Induction of DNA demethylation depending on two sets of Sox2 and adjacent Oct3/4 binding sites (Sox-Oct motifs) within the mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region.

DNA demethylation is used to establish and maintain an unmethylated state. The molecular mechanisms to induce DNA demethylation at a particular genomic locus remain unclear. The mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region (ICR) is a methylation state-sensitive insulator that regulates transcriptional activation of both genes. The unmethylated state of the ICR established in female germ cells is maintained during development, resisting the wave of genome-wide de novo methylation. We previously demonstrated that a DNA fragment (fragment b) derived from this ICR-induced DNA demethylation when it was transfected into undifferentiated mouse embryonal carcinoma cell lines. Moreover, two octamer motifs within fragment b were necessary to induce this DNA demethylation. Here, we demonstrated that both octamer motifs and their flanking sequences constitute Sox-Oct motifs (SO1 and SO2) and that the SO1 region, which requires at least four additional elements, including the SO2 region, contributes significantly to the induction of high-frequency DNA demethylation as a Sox-Oct motif. Moreover, RNAi-mediated inhibition of Oct3/4 expression in P19 cells resulted in a reduced DNA demethylation frequency of fragment b but not of the adenine phosphoribosyltransferase gene CpG island. The Sox motif of SO1 could function as a sensor for a hypermethylated state of the ICR to repress demethylation activity. These results indicate that Sox-Oct motifs in the ICR determine the cell type, DNA region, and allele specificity of DNA demethylation. We propose a link between the mechanisms for maintenance of the unmethylated state of the H19/Igf2 ICR and the undifferentiated cell-specific induction of DNA demethylation.

Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction.

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.

Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS.

Previous work from this laboratory has reported the biotransformation of bile acids (BA) into the thioester-linked glutathione (GSH) conjugates via the intermediary metabolites formed by BA:CoA ligase and shown that such GSH conjugates are excreted into the bile in healthy rats as well as rats dosed with lithocholic acid or ursodeoxycholic acid. To examine whether such novel BA-GSH conjugates are present in human bile, we determined the concentration of the GSH conjugates of the five BA that predominate in human bile. Bile was obtained from three infants (age 4, 10, and 13 months) and the BA-GSH conjugates quantified by means of liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS) in negative-ion scan mode, monitoring characteristic transitions of the analytes. By LC/ESI-MS, only primary BA were present in biliary BA, indicating that the dehydroxylating flora had not yet developed. GSH conjugates of chenodeoxycholic and lithocholic acid were present in concentrations ranging from 27 to 1120 pmol/ml, several orders of magnitude less than those of natural BA N-acylamidates. GSH conjugates were not present, however, in the ductal bile obtained from 10 adults (nine choledocholithiasis, one bile duct cancer). Our results indicate that BA-GSH conjugates are formed and excreted in human bile, at least in infants, although this novel mode of conjugation is a very minor pathway.

Identification of bile acid S-acyl glutathione conjugates in rat bile by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry.

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to acylate the thiol group of glutathione (GSH); the reaction is catalyzed by glutathione S-transferase (GST) and the product is a thioester-linked BA-GSH conjugate. Such GSH conjugates are present in bile in lithocholic acid and ursodeoxycholic acid dosed-rats. To determine whether such novel BA-GSH conjugates are present in the bile of normal rats, we first synthesized the GSH conjugates of the major and minor biliary BAs of the rat and defined their MS and proton NMR properties. We then analyzed the BA-GSH composition in the bile of anesthetized biliary fistula rats by means of liquid chromatographic separation and electrospray ionization-linear ion trap mass spectrometric detection in negative- and positive-ion scan modes, monitoring characteristic transitions of the analytes. GSH conjugates of cholic, ω-muricholic, hyodeoxycholic, deoxycholic, 12-oxolithocholic, and lithocholic acids were present with concentrations in the range of 1.4-2.8 nmol/ml, some four orders of magnitude less than those of natural BA N-acyl amidates. Our results indicate that BA-GSH conjugates are formed and excreted in bile in the healthy rat, although this novel mode of BA conjugation is a very minor pathway.

Isolation, expression, and characterization of the human ZCRB1 gene mapped to 12q12.

While isolating morphine-dependence-related genes with differential display, we cloned a novel human gene, zinc finger CCHC-type and RNA-binding motif 1 (ZCRB1, alias MADP-1) encoding a nuclear protein (217 residues). The ZCRB1 gene consists of eight exons and seven introns. It is mapped to 12q12, which is within a locus reported for Parkinson disease (M. Funayama et al., Ann. Neurol. 51 (2002) 296-301). The 5'-flanking region contains an enhancer core motif and binding sites for AP-1, AP-2, and LF-A1. ZCRB1 is characterized by an RNA-binding motif and a CCHC zinc finger motif. The latter overlaps the C..C...GH....C core nucleocapsid motif. ZCRB1 is conserved from zebrafish to human and shares homology with cold-inducible RNA-binding protein. Transfection assay showed that ZCRB1 is located in the nucleoplasm, but outside the nucleolus. ZCRB1 gene expression was stimulated by morphine, inhibited by 30-36 degrees C, and up-regulated by 39 degrees C incubation in SH-SY5Y neural cells. Zcrb1 gene expression is highest in the heart and testes, lower in the cerebellum, and lowest in the liver in mice. ZCRB1 mRNA expression is specifically elevated in hepatocarcinoma HepG2 cells. These data provide new clues for further understanding of morphine dependence, heat shock, and hepatocarcinoma.

CCAAT/enhancer binding protein-beta (C/EBP-beta), a pivotal regulator of the TATA-less promoter in the rat catalase gene.

The rat catalase gene carries a TATA-less promoter and its transcriptional mechanism is interesting because of downregulation in liver injury. We characterized the core element in the promoter and found that C/EBP-beta binding downstream of the transcription initiation site plays a crucial role for transcription. The multiple complexes binding to the promoter were composed of homodimers and heterodimers of C/EBP-beta isoforms. Transduction of the C/EBP-beta gene showed complete reconstitution of multiple binding complexes in HeLa cells, similar to normal liver. Furthermore, C/EBP-beta was observed to bind to the endogenous catalase promoter. These data suggest that multiple complex formation of C/EBP-beta regulates transcription in the TATA-less catalase promoter.

A dyad oct-binding sequence functions as a maintenance sequence for the unmethylated state within the H19/Igf2-imprinted control region.

DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages.

Identification of differentially expressed genes in rat hepatoma cell lines using subtraction and microarray.

Rat hepatoma Fao is a well-differentiated cell line that expresses numerous liver-specific enzymes and liver-enriched transcription factors, whereas the C2 cell line derived from Fao exhibits dedifferentiated phenotypes and fails to express these differentiated traits. Hence, to identify the gene related to the differentiation status in these cells, we analyzed differentially expressed genes between Fao and C2 cells using a microarray of 1,000 identified cDNA clones, and isolated a cDNA clone from subtractive library constructed by suppression subtractive hybridization. D6.1A, a member of the tetraspanin protein superfamily, was identified as a gene enhanced in C2 cells compared with Fao cells. Interestingly, expression of this gene was also induced following liver injury by CCl(4) and in liver regeneration following partial hepatectomy. These results suggest that the D6.1A gene is related to stimulation of cell proliferation and differentiation status in C2 hepatoma cells.