Distinctive duodenal microbiomes and bile acid profiles in duodenal tumor patients revealed by prospective observational study.

The incidence of duodenal tumors (DTs) is increasing. However, the mechanisms underlying its development remain unclear. Environmental factors, including the microbiome and bile acids (BAs), are believed to influence tumor development. Therefore, we conducted a single-center, prospective, observational study to investigate the potential differences between patients with DTs and healthy controls (HCs) based on these factors. In addition, the BAs in the duodenal fluid were measured using liquid chromatography-tandem mass spectrometry. We recruited 41 patients and performed 16S rRNA-seq. There was no difference in the observed ASVs or PCoA plot of Bray-Curtis dissimilarity between the DTs and HCs. The lithocholic acid concentration was significantly lower in the DT group than in the control group. The ratio of CDCA to LCA was significantly higher in patients with DTs. No significant differences in microbiota were observed between DTs and HCs. In patients with DTs, the lithocholic acid concentration in duodenal was significantly lower than in HCs.

The tryptophan metabolic pathway of the microbiome and host cells in health and disease.

The intricate and dynamic tryptophan (Trp) metabolic pathway in both the microbiome and host cells highlights its profound implications for health and disease. This pathway involves complex interactions between host cellular and bacteria processes, producing bioactive compounds such as 5-Hydroxytryptamine (5-HT) and kynurenine (Kyn) derivatives. Immune responses to Trp metabolites through specific receptors have been explored, highlighting the role of the aryl hydrocarbon receptor (AHR) in inflammation modulation. Dysregulation of this pathway is implicated in various diseases, such as Alzheimer's and Parkinson's diseases, mood disorders, neuronal diseases, autoimmune diseases such as multiple sclerosis (MS), and cancer. In this article, we describe the impact of the 5-HT, Trp, indole, and Trp metabolites on health and disease. Further, we review the impact of microbiome-derived Trp metabolites that affect immune responses and contribute to maintaining homeostasis, especially in an experimental autoimmune encephalitis (EAE) model of MS.

Double-bundle anterior cruciate ligament reconstruction in patients aged 60 years and older.

Background: This study aimed to examine the clinical outcomes of double-bundle (DB) anterior cruciate ligament (ACL) reconstruction in patients aged ≥60 years. Methods: Anatomical DB-ACL reconstruction using hamstring tendon autografts was performed in 13 patients aged ≥60 years at our institution between June 2012 and May 2018. The patients included seven men and six women, and the mean age at surgery was 65.0 years (range, 60-73 years). The mean time from injury to surgery was 80.5 months (range, 1-480 months), and the mean follow-up time was 26.2 months (range, 24-42 months). All patients were assessed based on physical examination findings, clinical scores, Kellgren-Lawrence grades preoperatively and at the final postoperative follow-up, intraoperative meniscal or chondral lesions, and perioperative complications. Status of returning to sports for all patients was assessed at the final follow-up. Results: The mean side-to-side differences by arthrometer improved from 4.3 mm (range, 2-8 mm) to 0.9 mm (range, 0-2 mm), and the positive pivot-shift test decreased from 100% to 8%. The mean extensor muscle strength was 93.3% (range, 74-116%) postoperatively. The mean Lysholm score improved from 71.1 (range, 27-85) to 95.2 (range, 89-100). Ten of the 13 patients (77%) returned to their pre-injury level of sports performance, and one patient (8%) returned to sports with less intensity. Intraoperatively, meniscal tears were observed in 10 patients (77%), and chondral lesions >grade 2 were observed in 11 (85%). One patient developed perioperative complications. At the final follow-up, the Kellgren-Lawrence grade worsened in only one patient. No re-injury or infection was observed, and revision surgery was not required for any patients. Conclusions: Anatomical DB-ACL reconstruction could provide satisfactory clinical outcomes and knee function restoration in patients aged ≥60 years. Level of evidence: A retrospective study, case series (IV).

The gut microbiota-induced kynurenic acid recruits GPR35-positive macrophages to promote experimental encephalitis.

The intricate interplay between gut microbes and the onset of experimental autoimmune encephalomyelitis (EAE) remains poorly understood. Here, we uncover remarkable similarities between CD4+ T cells in the spinal cord and their counterparts in the small intestine. Furthermore, we unveil a synergistic relationship between the microbiota, particularly enriched with the tryptophan metabolism gene EC:1.13.11.11, and intestinal cells. This symbiotic collaboration results in the biosynthesis of kynurenic acid (KYNA), which modulates the recruitment and aggregation of GPR35-positive macrophages. Subsequently, a robust T helper 17 (Th17) immune response is activated, ultimately triggering the onset of EAE. Conversely, modulating the KYNA-mediated GPR35 signaling in Cx3cr1+ macrophages leads to a remarkable amelioration of EAE. These findings shed light on the crucial role of microbial-derived tryptophan metabolites in regulating immune responses within extraintestinal tissues.

Single-cell transcriptomics of human gut T cells identifies cytotoxic CD4+CD8A+ T cells related to mouse CD4 cytotoxic T cells.

Cytotoxic CD4+ T cells (CD4-CTLs) show the presence of cytolytic granules, which include the enzymes granzyme and perforin. The cells have a pathogenic and protective role in various diseases, including cancer, viral infection, and autoimmune disease. In mice, cytotoxic CD4+ T cells express CD8αα+ and reside in the intestine (mouse CD4+CTLs; mCD4-CTLs). The population of cytotoxic CD4+ T cells in the human intestine is currently unknown. Moreover, it is unclear how cytotoxic CD4 T cells change in patients with inflammatory bowel disease (IBD). Here, we aimed to identify cytotoxic CD4+ T cells in the human intestine and analyze the characteristics of the population in patients with IBD using single-cell RNA-seq (scRNA-seq). In CD4+ T cells, granzyme and perforin expression was high in humanMAIT (hMAIT) cells and hCD4+CD8A+ T cell cluster. Both CD4 and CD8A were expressed in hTreg, hMAIT, and hCD4+CD8A+ T cell clusters. Next we performed fast gene set enrichment analysis to identify cell populations that showed homology to mCD4CTLs. The analysis identified the hCD4+CD8A+ T cell cluster (hCTL-like population; hCD4-CTL) similar to mouse CTLs. The percentage of CD4+CD8A+ T cells among the total CD4+ T cells in the inflamed intestine of the patients with Crohn's disease was significantly reduced compared with that in the noninflamed intestine of the patients. In summary, we identified cytotoxic CD4+CD8+ T cells in the small intestine of humans. The integration of the mouse and human sc-RNA-seq data analysis highlight an approach to identify human cell populations related to mouse cell populations, which may help determine the functional properties of several human cell populations in mice.

Aryl hydrocarbon receptor signals in epithelial cells govern the recruitment and location of Helios+ Tregs in the gut.

CD4+Foxp3+ regulatory T cells (Tregs) are essential for homeostasis in the colon, but the mechanism by which local environmental cues determine the localization of colonic Tregs is unclear. Here, we administer indigo naturalis (IN), a nontoxic phytochemical aryl hydrocarbon receptor (AhR) agonist used for treating patients with ulcerative colitis (UC) in Asia, and we show that IN increases Helios+ Tregs and MHC class II+ epithelial cells (ECs) in the colon. Interactions between Tregs and MHC class II+ ECs occur mainly near the crypt bottom in the steady state, whereas Tregs dramatically increase and shift toward the crypt top following IN treatment. Moreover, the number of CD25+ T cells is increased near the surface of ECs in IN-treated UC patients compared with that in patients treated with other therapies. We also highlight additional AhR-signaling mechanisms in intestinal ECs that determine the accumulation and localization of Helios+ Tregs in the colon.

Promethazine Downregulates Wnt/ß-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing.

BACKGROUND: Wnt/ß-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. HYPOTHESIS: We hypothesized that the inhibition of Wnt/ß-catenin signaling would accelerate tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/ß-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. RESULTS: The amount of ß-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/ß-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased ß-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/ß-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased ß-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. CONCLUSION: IWR-1 and PH suppressed Wnt/ß-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. CLINICAL RELEVANCE: PH is a candidate repositioned drug that potentially accelerates tendon repair.

Role of CC chemokine receptor 9 in the progression of murine and human non-alcoholic steatohepatitis.

BACKGROUND & AIMS: The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. METHODS: Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9-/- mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. RESULTS: Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9-/- mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9+CD11b+ inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9-/- mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. CONCLUSIONS: These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. LAY SUMMARY: Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.

Diversity of Gut Microbiota Affecting Serum Level of Undercarboxylated Osteocalcin in Patients with Crohn's Disease.

Several reports have indicated a possible link between decreasing plasma levels of vitamin K and bone mineral density. It has been suggested that intestinal bacteria contribute to maintenance of vitamin K. Several factors are involved in the reduction of vitamin K in patients with Crohn's disease (CD). We aimed to assess the relationship between gut microbiota and alternative indicators of vitamin K deficiency in patients with CD. We collected the feces of 26 patients with clinically inactive CD. We extracted 16S rRNA from the intestinal bacteria in the feces and amplified it by polymerase chain reaction. The generated polymerase chain reaction product was analyzed using a 16S metagenomic approach by Illumina Miseq platform. Serum undercarboxylated osteocalcin concentration was used as an alternative indicator of vitamin K deficiency. There was a significant negative correlation between serum undercarboxylated osteocalcin and mean Chao1 index in cases of low activity. The diversity of the gut microbiota was significantly lower, and Ruminococcaceae and Lachnospiraceae were significantly decreased in the vitamin K-deficient group in comparison to the vitamin K-normal group. Taken together, these data suggested the significance of investigating the gut microbiota even in patients with clinically inactive CD for improving patients' vitamin K status.

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (TH17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

Toll-Like Receptor 7 Agonist-Induced Dermatitis Causes Severe Dextran Sulfate Sodium Colitis by Altering the Gut Microbiome and Immune Cells.

Background & Aims: Psoriasis and inflammatory bowel disease (IBD) are both chronic inflammatory diseases occurring in the skin and gut, respectively. It is well established that psoriasis and IBD have high concordance rates, and similar changes in immune cells and microbiome composition have been reported in both conditions. To study this connection, we used a combination murine model of psoriatic dermatitis and colitis in which mice were treated topically with the Toll-like receptor 7 agonist imiquimod (IMQ) and fed dextran sulfate sodium (DSS). Methods: We applied IMQ topically to B6 mice (IMQ mice) and subsequently fed them 2% DSS in their drinking water. Disease activity and immune cell phenotypes were analyzed, and the microbial composition of fecal samples was investigated using 16S ribosomal RNA sequencing. We transplanted feces from IMQ mice to germ-free IQI/Jic (IQI) mice and fed them DSS to assess the effect of the gut microbiome on disease. Results: We first confirmed that IMQ mice showed accelerated DSS colitis. IMQ mice had decreased numbers of IgD+ and IgM+ B cells and increased numbers of non-cytokine-producing macrophages in the gut. Moreover, the gut microbiomes of IMQ mice were perturbed, with significant reductions of Lactobacillus johnsonii and Lactobacillus reuteri populations. Germ-free mice transplanted with feces from IMQ mice, but not with feces from untreated mice, also developed exacerbated DSS colitis. Conclusions: These results suggest that skin inflammation may contribute to pathogenic conditions in the gut via immunologic and microbiological changes. Our finding of a novel potential skin-gut interaction provides new insights into the coincidence of psoriasis and IBD.

Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/ß-catenin signaling.

Abnormal activation of the Wnt/ß-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/ß-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/ß-catenin activity in the presence of 10 µM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/ß-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/ß-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total ß-catenin and a LiCl-induced decrease of phosphorylated ß-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of ß-catenin with Axin1, which is a scaffold protein forming the degradation complex for ß-catenin. Fluoxetine suppressed LiCl-induced ß-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed ß-catenin accumulation.

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation.

BACKGROUND: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments. RESULTS: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heat-killed Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified. CONCLUSION: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen.

Molecular hydrogen suppresses activated Wnt/ß-catenin signaling.

Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/ß-catenin signaling by promoting phosphorylation and degradation οf ß-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of ß-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the ß-catenin degradation complex, minimized the suppressive effect of H2 on ß-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of ß-catenin, as well as the ß-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/ß-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating ß-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/ß-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.

Anatomical reconstruction of the patellar tendon using the fascia lata attached to the iliac bone following resection for soft tissue sarcoma: a case report.

A new reconstruction of the patellar tendon was performed in a 43-year-old patient who lost tendon and tibial tuberosity after a wide tumor resection for low-grade myofibroblastic sarcoma of the parapatellar tendon. In this technique, the patellar tendon was anatomically reconstructed using a fascia lata attached to the iliac bone. The iliac bone was fixed to the tibial bony trough with absorbable screws, and the fascia lata was fashioned into three branches: the central branch was folded through the tunnel in the patella, and the medial and lateral branches were tagged to the medial and lateral retinaculum, respectively, around the patella. The skin defect was covered by the bilateral head of the gastrocnemius flap and a split-thickness skin graft. At the 3-year follow-up, the active range of motion of the knee joint was 0 to 110 degrees. The functional result according to the Musculoskeletal Tumor Society scoring system was 97%. Radiographs showed that the grafted bone was united well to the tibial bone, and the grafted fascia was confirmed as a dark band on MRI. There was no evidence of disease and no complaint of the donor site. This procedure allows for the reconstruction of the patellar tendon in the original location. To our knowledge, this reconstructive procedure of the patellar tendon using the fascia lata attached to the iliac bone has never been reported in English literature.

Characterization of TRIM31, upregulated in gastric adenocarcinoma, as a novel RBCC protein.

To explore the molecules associated with gastric adenocarcinoma, we used the gene expression profile database of various human tissues and identified TRIM31 upregulated in both patients with chronic gastritis and stomach cancer. TRIM31 is a new member of RBCC proteins composed of RING finger, B-box and coiled-coil domains. We characterized TRIM31 biochemically and found it possess properties in common with other RBCC proteins, such as occurrence of alternative splicing transcripts, in vitro autoubiquitylating activity and a tendency to homo-oligomerize. The primary localization site of TRIM31 is the cytoplasm but some fraction is potentially associated with the mitochondria. TRIM31 overexpression suppresses colony formation of HCT116 cells while knockdown of its expression with short interfering RNAs (siRNAs) consistently tends to enhance growth of AsPC-1 cells slightly. Thus, TRIM31 is a characteristic RBCC protein with the ability to regulate cell proliferation negatively and may be a potential biomarker of gastric cancer as it is overexpressed from the early stage of gastric carcinogenesis.

Proteomic identification of a PSF/p54nrb heterodimer as RNF43 oncoprotein-interacting proteins.

RNF43 is an oncogenic RING finger protein overexpressed in colorectal cancer. To dissect its biological functions, we explored RNF43-interacting proteins by pull-down assay and MS. We identified a heterodimer, p54nrb and PSF, as RNF43's binding partners and confirmed their physical interaction in vivo by the co-immunoprecipitation experiment. Immunofluorescence analysis revealed that co-expression of PSF relocates RNF43 from the nuclear periphery to the nucleoplasm. Thus, proteomic identification of RNF43-associated proteins sheds light on its dynamic interaction network in nuclear events.

A cancer-associated RING finger protein, RNF43, is a ubiquitin ligase that interacts with a nuclear protein, HAP95.

RNF43 is a recently discovered RING finger protein that is implicated in colon cancer pathogenesis. This protein possesses growth-promoting activity but its mechanism remains unknown. In this study, to gain insight into the biological action of RNF43 we characterized it biochemically and intracellularly. A combination of indirect immunofluorescence analysis and biochemical fractionation experiments suggests that RNF43 resides in the endoplasmic reticulum (ER) as well as in the nuclear envelope. Sucrose density gradient fractionation demonstrates that RNF43 co-exists with emerin, a representative inner nuclear membrane protein in the nuclear subcompartment. The cell-free system with pure components reveals that recombinant RNF43 fused with maltose-binding protein has autoubiquitylation activity. By the yeast two-hybrid screening we identified HAP95, a chromatin-associated protein interfacing the nuclear envelope, as an RNF43-interacting protein and substantiated this interaction in intact cells by the co-immunoprecipitation experiments. HAP95 is ubiquitylated and subjected to a proteasome-dependent degradation pathway, however, the experiments in which 293 cells expressing both RNF43 and HAP95 were treated with a proteasome inhibitor, MG132, show that HAP95 is unlikely to serve as a substrate of RNF43 ubiquitin ligase. These results infer that RNF43 is a resident protein of the ER and, at least partially, the nuclear membrane, with ubiquitin ligase activity and may be involved in cell growth control potentially through the interaction with HAP95.

RING finger, B-box, and coiled-coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica.

An RBCC (RING finger, B-box, and coiled-coil) protein was identified that belongs to the superfamily of zinc-binding proteins and is specifically expressed in the gill of eel, Anguilla japonica. Euryhaline fishes such as eels can migrate between freshwater and seawater, which is considered to be accomplished by efficient remodeling of the architecture and function of the gill, a major osmoregulatory organ. To identify molecules involved in such adaptive changes, we performed differential display using mRNA preparations from freshwater and seawater eel gills and obtained an RBCC clone among several differentially expressed clones. The clone encoded a protein of 514 amino acid residues with structural features characteristic of the RBCC protein; we therefore named it eRBCC (e for eel). eRBCC mRNA was specifically expressed in the gills with a greater extent in the gills of freshwater eels. Immunohistochemistry revealed that the expression of eRBCC is confined to particular epithelial cells of the gills including freshwater-specific lamellar chloride cells. The RING finger of eRBCC was found to have a ubiquitin ligase activity, suggesting an important regulatory role of eRBCC in the remodeling of branchial cells.