Identification of ABCG2 as an Exporter of Uremic Toxin Indoxyl Sulfate in Mice and as a Crucial Factor Influencing CKD Progression.

Chronic kidney disease (CKD) patients accumulate uremic toxins in the body, potentially require dialysis, and can eventually develop cardiovascular disease. CKD incidence has increased worldwide, and preventing CKD progression is one of the most important goals in clinical treatment. In this study, we conducted a series of in vitro and in vivo experiments and employed a metabolomics approach to investigate CKD. Our results demonstrated that ATP-binding cassette transporter subfamily G member 2 (ABCG2) is a major transporter of the uremic toxin indoxyl sulfate. ABCG2 regulates the pathophysiological excretion of indoxyl sulfate and strongly affects CKD survival rates. Our study is the first to report ABCG2 as a physiological exporter of indoxyl sulfate and identify ABCG2 as a crucial factor influencing CKD progression, consistent with the observed association between ABCG2 function and age of dialysis onset in humans. The above findings provided valuable knowledge on the complex regulatory mechanisms that regulate the transport of uremic toxins in our body and serve as a basis for preventive and individualized treatment of CKD.

Reversible logic gate using adiabatic superconducting devices.

Reversible computing has been studied since Rolf Landauer advanced the argument that has come to be known as Landauer's principle. This principle states that there is no minimum energy dissipation for logic operations in reversible computing, because it is not accompanied by reductions in information entropy. However, until now, no practical reversible logic gates have been demonstrated. One of the problems is that reversible logic gates must be built by using extremely energy-efficient logic devices. Another difficulty is that reversible logic gates must be both logically and physically reversible. Here we propose the first practical reversible logic gate using adiabatic superconducting devices and experimentally demonstrate the logical and physical reversibility of the gate. Additionally, we estimate the energy dissipation of the gate, and discuss the minimum energy dissipation required for reversible logic operations. It is expected that the results of this study will enable reversible computing to move from the theoretical stage into practical usage.

Factors associated with inappropriate weight loss attempts by early adolescent girls in Japan.

Attempting to lose weight by normal or underweight adolescent girls is a serious issue in many countries. It has been reported that the mode of attempted weight loss does not differ between normal weight and overweight girls. These inappropriate weight loss attempts (IWLA) by normal or underweight adolescent girls is associated with various health issues, but factors associated with IWLA have only been marginally elucidated. In this study, we applied a single multivariate regression analysis to clarify independent factors for IWLA. Study subjects were 134 pairs of early adolescent girls (aged 12-15) and their mothers. In addition to IWLA, many factors including height, weight, body image, perceived weight status, depressive symptoms, media influence and self-esteem were surveyed in both mothers and daughters and subjected to multivariate analysis. Approximately half of girls surveyed had IWLA, even though all were of normal weight and 62.9% knew that they were of normal weight. IWLA were independently associated with depressive symptoms (OR (95% CI); 2.80 (1.21-6.50), p=0.016) independent of actual or perceived weight status. Factors significantly associated with IWLA by the girls were percentage deviation of weight from standard weight (%DW) and media influence on the girls themselves, and media influence on and self-esteem of their mothers. IWLA, which were frequently observed among early adolescent girls even among those of normal weight, were closely related to depressive status. IWLA were significantly associated with not only factors related to the girls (1.09 (1.04-1.14), p=0.001), but also with maternal psychological factors (1.06 (1.00-1.13), p=0.035) conveyed by the media. Future prospective or interventional studies are required to clarify whether these factors could be targeted in an effort to prevent IWLA.

Dok-7/MuSK signaling and a congenital myasthenic syndrome.

Skeletal muscle contraction is controlled by motor neurons, which contact the muscle at the neuromuscular junction (NMJ). The formation and maintenance of the NMJ, which includes the aggregation of densely packed clusters of acetylcholine receptor (AChR) opposite the motor nerve terminal, is orchestrated by muscle-specific receptor tyrosine kinase, MuSK. Recently, a MuSK-interacting cytoplasmic adaptor-like protein Dok-7 was identified and its localization at the postsynaptic region of the NMJ was revealed. Mice lacking Dok-7 have a phenotype indistinguishable from MuSK-deficient mice, and fail to form both AChR clusters and NMJs. In cultured myotubes, Dok-7 is required for MuSK activation and AChR clustering. Thus, Dok-7 is essential for neuromuscular synaptogenesis and it appears that the regulatory interaction of Dok-7 with MuSK is integrally involved in this process. In humans there are both autoimmune and genetic causes of defective neuromuscular transmission that gives rise to the fatigable muscle weakness known as myasthenia. DOK7 has been found to be a major locus for mutations that underlie a genetic form of myasthenia with a characteristic 'limb girdle' pattern of muscle weakness (DOK7 CMS). Patients with DOK7 CMS have small, simplified NMJs but normal AChR function. The most common mutation causes a COOH-terminal truncation, which greatly impairs Dok-7's ability to activate MuSK. Recently, a series of differing DOK7 mutations have been identified, which affect not only the COOH-terminal region but also the NH2-terminal moiety. The study of these mutations may help understand the underlying pathogenic mechanism of DOK7 CMS.

Domain-dependent function of the rasGAP-binding protein p62Dok in cell signaling.

p62Dok, the rasGAP-binding protein, is a common target of protein-tyrosine kinases. It is one of the major tyrosine-phosphorylated molecules in v-Src-transformed cells. Dok consists of an amino-terminal Pleckstrin homology domain, a putative phosphotyrosine binding domain, and a carboxyl-terminal tail containing multiple tyrosine phosphorylation sites. The importance and function of these sequences in Dok signaling remain largely unknown. We have demonstrated here that the expression of Dok can inhibit cellular transformation by the Src tyrosine kinase. Both the phosphotyrosine binding domain and the carboxyl-terminal tail of Dok (in particular residues 336-363) are necessary for such activity. Using a combinatorial peptide library approach, we have shown that the Dok phosphotyrosine binding domain binds phosphopeptides with the consensus motif of Y/MXXNXL-phosphotyrosine. Furthermore, Dok can homodimerize through its phosphotyrosine binding domain and Tyr(146) at the amino-terminal region. Mutations of this domain or Tyr(146) that block homodimerization significantly reduce the ability of Dok to inhibit Src transformation. Our results suggest that Dok oligomerization through its multiple domains plays a critical role in Dok signaling in response to tyrosine kinase activation.

Role of the rasGAP-associated docking protein p62(dok) in negative regulation of B cell receptor-mediated signaling.

Antigenic stimulation of the B-cell receptor (BCR) is a central event in the immune response. In contrast, antigen bound to IgG negatively regulates signals from the BCR by cross-linking it to the inhibitory receptor FcgammaRIIB. Here we show that upon cross-linking of BCR or BCR with FcgammaRIIB, the rasGAP-associated protein p62(dok) is prominently tyrosine phosphorylated in a Lyn-dependent manner. Inactivation of the dok gene by homologous recombination has shown that upon BCR cross-linking, p62(dok) suppresses MAP kinase and is indispensable for FcgammaRIIB-mediated negative regulation of cell proliferation. We propose that p62(dok), a downstream target of many PTKs, plays a negative role in various signaling situations.

Tyrosine phosphorylation of p62(Dok) induced by cell adhesion and insulin: possible role in cell migration.

Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok as a signal transducer that potentially links, through its interaction with NCK or rasGAP, cell adhesion and insulin receptors to the machinery that controls cell motility.

Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis.

The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr-378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.

Identification of the Abl- and rasGAP-associated 62 kDa protein as a docking protein, Dok.

A 62 kDa protein is highly phosphorylated in many cells containing activated tyrosine kinases. This protein, characterized mainly by its avid association with rasGAP, has proved elusive. Anti-phosphotyrosine antibody was used to purify p62. From peptide sequence, molecular cloning revealed a cDNA encoding a novel protein, p62dok, with little homology to others but with a prominent set of tyrosines and nearby sequences suggestive of SH2 binding sites. In cells, v-Abl tyrosine kinase binds and strongly phosphorylates p62dok, which then binds rasGAP. A monoclonal antibody, 2C4, to the rasGAP-associated p62 reacts with p62dok. Thus, p62dok appears to be the long-sought major substrate of many tyrosine kinases.

Thy-1 molecule associates with protein tyrosine kinase(s) in rat mesangial cells.

Glycophosphatidylinositol (GPI)-linked Thy-1 molecules, well known cell surface markers of murine T cells, are present on the glomerular mesangial cells of the rat kidney. The administration of anti-Thy-1.1 MoAbs 1-22-3 and OX-7 to rats induces severe and mild complement-dependent mesangial proliferative glomerulonephritis, respectively. In order to determine whether protein-tyrosine kinase (PTK) activity is associated with Thy-1 molecules on rat mesangial cell surface, we performed an immune complex kinase assay, using anti-Thy-1 MoAbs 1-22-3 and OX-7, followed by reimmunoprecipitation with anti-phosphotyrosine, anti-fyn, anti-lck and anti-lyn antibodies. Physical association of PTK, p59fyn and p56/53lyn with Thy-1 molecules was demonstrated in cultured rat mesangial cells. The activities of these kinases detected in MoAb 1-22-3 precipitates were higher than those in MoAb OX-7 precipitates. These results suggest that Thy-1 molecule transduces some signals also in rat mesangial cells.

Physical and functional association of cortactin with Syk in human leukemic cell line K562.

Human leukemic cell line K562 is induced to differentiate into the megakaryocytic lineage by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate here that TPA stimulation increases tyrosine phosphorylation of an 80-kDa protein at an early stage of megakaryocytic differentiation and that this 80-kDa protein is identical with cortactin. Since tyrosine kinase Syk was activated by TPA stimulation, we examined the possibility that cortactin is a potential substrate of Syk in K562 cells. TPA-induced tyrosine phosphorylation of cortactin was decreased profoundly by overexpression of dominant-negative Syk. Furthermore, cortactin was associated with Syk even before TPA stimulation. Since cortactin was previously referred as an 80/85-kilodalton pp60src substrate, we examined the association between Src and cortactin, whereas its association could not be detected. These data suggest that Syk phosphorylates cortactin in K562 cells upon TPA treatment.

Involvement of Fyn tyrosine kinase in progression of cytokinesis of B lymphocyte progenitor.

We analyzed the role of Fyn tyrosine kinase in cell cycle progression of B lymphocyte progenitor (pro B cell). Whereas there were no substantial defects in the intramarrow B cell genesis in the fyn(-) mouse, and long-term proliferation of fyn(-) pro B cells was maintained in vitro under a serum containing culture condition, the cell cycle was arrested at G2/M upon serum deprivation. Morphological analyses demonstrated that the cytokinesis of fyn(-) pro B cells was retarded in the presence of serum and that the entry of fyn(-) pro B cells into late telophase was completely blocked under the serum-free condition. In contrast, the earlier phases of mitosis of fyn(-) pro B cells proceeded normally without FCS. This failure to initiate late telophase resulted in the accumulation of elliptical binucleated cells that might be the outcome of the nuclear division without cytokinesis. Consistent with this defect in the progression of cytokinesis, Fyn was localized in the midspace of dividing pro B cells at anaphase. These results suggested that Fyn localizes at the midspace of dividing pro B cells and regulates the progression of cytokinesis.

Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice.

The Src family protein-tyrosine kinase Lyn associates physically with the BCR and has been suggested to play an important role in BCR-mediated signaling. Studies with lyn-/- mice showed that the number of B cells decreased by half in their peripheral tissues. In addition, these B cells do not respond normally to a number of stimuli, including BCR cross-linking and CD40 ligand. Induction of tyrosine phosphorylation on a variety of cellular proteins, such as Vav, Cbl, and HS1, upon BCR cross-linking was also abolished in these B cells. Despite the impaired BCR-mediated signaling, concentrations of IgM and IgA in sera were remarkably elevated, and production of autoantibodies was detected in lyn-/- mice. Histological study showed splenomegaly and enlargement of lymph nodes that became evident with age in the mutant mice. The spleen contained significant number of plasma cells as well as unusual lymphoblast-like cells carrying Mac1 antigen and cytoplasmic IgM. These cells spontaneously secreted a large amount of IgM in vitro. Finally, significant number of lyn-/- mice show glomerulonephritis, an indication of autoimmune disease. From these data, we conclude that Lyn plays a role in signal transduction for not only clonal expansion and terminal differentiation of peripheral B cells but also elimination of autoreactive B cells.

Syk activation by the Src-family tyrosine kinase in the B cell receptor signaling.

Signaling through the B cell antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Src-protein-tyrosine kinase [PTK]; Lyn, Fyn, Blk, or Lck) and Syk kinase. We have investigated the interaction between the Src-PTK and the Syk kinase in the BCR signaling. In contrast to wild-type B cells, BCR-mediated tyrosine phosphorylation of Syk and activation of its in vitro kinase activity were profoundly reduced in lyn-negative cells. The requirement of the Src-PTK to induce tyrosine phosphorylation and activation of Syk was also demonstrated by cotransfection of syk and src-PTK cDNAs into COS cells. These results suggest that the Src-PTK associated with BCR phosphorylates the tyrosine residue(s) of Syk upon receptor stimulation, enhancing the activity of Syk.

Down-regulation of membrane immunoglobulin-associated proteins, MB-1, B29 and Lyn, in AIDS-lymphomas and related conditions.

B-lymphocytes infected with Epstein-Barr virus (EBV) can proliferate in immunocompromised hosts to form lymphomas (MLs). Similar MLs are produced in mice with severe combined immune deficiency (SCID) by transfusion of human lymphocytes infected with EBV (SCID-EBV-positive BML). Mb-1 and B29 are recently found transmembrane proteins associated with membrane immunoglobulins (mIg) on the surface of B cells. Lyn is a src family gene product expressed in B cells submembranously, in association with mIg, possibly through Mb-1/B29 heterodimer. These mIg-associated proteins (Mb-1, B29 and Lyn) are known to mediate antigenic stimulation through mIgs. We noted recently that Lyn is decreased selectively in around a half of SCID-EBV-positive BMLs. We extended this line of investigation to other mIg-associated proteins. Five acquired immunodeficiency syndrome (AIDS)-MLs and ten SCID-EBV-positive BMLs were first analysed by immunohistochemistry for the expression of Mb-1, B29 and Lyn. It was found that in AIDS-MLs, all the mIg-associated proteins were heavily down-regulated. In SCID-EBV-positive BMLs, Mb-1 was down regulated in six of ten, B29 in nine of ten and Lyn in six of ten, whereas no down-regulation was noted in eight EBV-free B MLs that were also maintained in SCID mice. An additional flow-cytometric study of two SCID-EBV-positive and two EBV-negative BMLs showed similar down-regulation in the former cases exclusively. Whereas mIg was also decreased in three of five SCID-EBV positive BMLs, it did not necessarily match the decrease of mIg-associated proteins, which contrasts with the recent finding that mIgs coexist with Mb-1 or B29. Some EBV-encoded proteins may activate host molecules located downwardly; this, in turn, may lead to the suppression of these upwardly-located mIg-associated proteins.

Expression of Lyn protein on human malignant lymphomas.

BACKGROUND: lyn is one of the src family genes encoding protein-tyrosine kinases, expressed preferentially in B lymphocytes and monocytes/macrophages. Its gene product, Lyn protein, is thought to participate in cell membrane-associated signal transduction on B lymphocytes by associating physically and functionally with membrane-bound IgM. EXPERIMENTAL DESIGN: To investigate the expression of Lyn on human malignant lymphomas (MLs), 50 ML biopsies, 12 ML samples maintained in mice with severe combined immunodeficiency, and 4 African Burkitt type cell lines were studied with the use of immunohistology, immunochemistry, and Southern blot analysis. RESULTS: Among biopsy specimens, 27 of 27 B-MLs, 5 of 21 T-MLs, and 2 of 2 null-MLs were stained. In severe combined immunodeficiency mouse-maintained B-MLs, unlike biopsied B-MLs, 4 of 11 were found to be unstained. Further analysis disclosed that all 4 of these unstained B-MLs were the Epstein Barr virus transformed B cells proliferating in severe combined immunodeficiency mice and not the original ML cells, suggesting the presence of a specific mechanism down-regulating the Lyn protein in this group. One IgA+ IgM- B-ML and one IgG+ IgM- B-ML were stained by the antibody, indicating the possible existence of molecular mechanisms other than membrane-bound IgM that facilitate Lyn protein expression. Decrease of Lyn expression was also noted in 3 of 4 Epstein-Barr virus-positive African Burkitt's ML lines. Complementary Western blot analysis of 8 immunostained and 4 unstained MLs confirmed the immunohistologic findings. However Southern blot analysis showed that the lyn gene in Lyn-positive and -negative cases were apparently unchanged. CONCLUSIONS: The level of Lyn expression in MLs reflects mainly their normal counterpart, whereas it can be expressed somewhat differently in some cases, especially in Epstein Barr virus-transformed MLs that occur in immunocompromised hosts, on which it is often down-regulated. This is the first report of Lyn expression on human MLs.

Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling.

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins and rapid activation of Src-like kinases. The amino acid sequences of five proteolytic peptides of p75, a major substrate of protein-tyrosine(s) in the signaling, showed that p75 is the human HS1 gene product. The HS1 gene is expressed specifically in hematopoietic cells and encodes p75HS1, which carries both helix-turn-helix and Src homology 3 motifs. p75HS1 showed rapid tyrosine phosphorylation and association with a Src-like kinase, Lyn, after crosslinking of membrane-bound IgM. Thus, p75HS1 may be an important substrate of Lyn and possibly other protein-tyrosine kinases upon B-cell antigen receptor-mediated signaling.

Association of Src-family kinase Lyn with B-cell antigen receptor.

Antigen is thought to cross-link membrane-bound immunoglobulins (Igs) of B cells, causing proliferation and differentiation or the inhibition of growth. Compelling evidence suggests that protein-tyrosine phosphorylation is involved in signal transduction for cell proliferation and differentiation. Indeed cross-linking of membrane-bound IgM (mIgM) induced a rapid increase in tyrosine phosphorylation of at least 10 distinct proteins in B cells. The Src-family protein tyrosine kinase Lyn (p56lyn and p53lyn) is expressed preferentially in B cells. The Lyn protein and its kinase activity could be coimmunoprecipitated with both IgM and IgD from detergent lysates. Cross-linking of membrane-bound IgM with antibody induced down-regulation of the Lyn protein. From these data we concluded that Lyn is physically associated with mIgs. Further evidence showed that cross-linking of mIgM induced rapid increase in the kinase activity of Lyn and association of Lyn with 85-kDa noncatalytic subunit of phosphatidylinositol 3-kinase. Thus, Lyn is likely to participate in B-cell antigen receptor-mediated signaling. As a novel signaling molecule downstream of Lyn, we identified src homology 3-containing, transcription factor-like molecule p75HS1.

Characterization of the promoter region of the src family gene lyn and its trans activation by human T-cell leukemia virus type I-encoded p40tax.

The src family gene lyn is expressed preferentially in B lymphocytes but very little in normal T lymphocytes. Transcription of the lyn gene in T lymphocytes was shown to be induced by the p40tax protein encoded by human T-cell lymphotropic virus type I. For determination of the mechanism of p40tax-mediated trans activation, the transcriptional promoter region of the lyn gene was characterized. By endonuclease S1 mapping, the transcriptional initiation sites were identified within the 770-bp EcoRI-SacI fragment of the 5'-terminal portion of the human lyn gene. This fragment showed promoter activity when placed upstream of the bacterial chloramphenicol acetyltransferase gene and transfected into various cell lines. Nucleotide sequence analysis revealed that the lyn promoter region contained four GC box-like sequences but not a TATA or CCAAT box. In addition, it contained sequences characteristic of a cyclic AMP-responsive element, octamer-binding motif, PEA3-like motifs, and NF kappa B-binding motif-like sequence. Mutational analysis suggested that the octamer-binding motif sequence is of primary importance for the lyn promoter activity but that the other elements are not. Cotransfection of various chloramphenicol acetyltransferase constructs containing different length of the lyn promoter together with p40tax expression plasmids into Jurkat T cells showed that the sequence responsible for p40tax-induced transcription is present around the transcription initiation sites.

Tyrosine protein kinase is involved in anti-IgM-mediated signaling in BAL17 B lymphoma cells.

BAL17 B lymphoma cells, representing mature B lymphocytes, were used to analyze the role of tyrosine kinase in B cell activation. Anti-IgM-induced tyrosine phosphorylation was inhibited by preincubation of cells with tyrosine kinase inhibitor herbimycin A. Enzymatic activity of lyn protein was also inhibited by this drug, accompanied by down-regulation of p53lyn and p56lyn. However, a protein kinase C-mediated event was intact in the herbimycin A-pretreated cells, suggesting that the inhibitor acts selectively on tyrosine kinase. Anti-IgM failed to stimulate herbimycin A-pretreated cells to induce increases in inositol phospholipid metabolism or increased [Ca2+]i, whereas aluminum fluoride-induced metabolism was not altered. Moreover, membrane IgM density as revealed by flow cytometry was not changed by herbimycin A. These results indicate that tyrosine kinase(s) participates in the coupling of an Ag receptor cross-linkage to phospholipase C activation through a phosphorylation event in B lymphoma cells.