Cytokine Expression and Macrophage Localization in Xenograft and Allograft Tumor Models Stimulated with Lipopolysaccharide.

T cell-deficient mice such as nude mice are often used to generate tumor xenograft for the development of anticancer agents. However, the functionality of the other immune cells including macrophages, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) in the xenograft are largely unknown. Macrophages and dendritic cells (DCs) acquire functionally distinct properties in response to various environmental stimuli; the interaction of these cells with MDSCs in tumor microenvironments regulates cancer progression. Nude mice are less likely to reject human cancer cells because of major histocompatibility complex (MHC) mismatches. The tumor microenvironment in a xenograft, comprising human and mouse cells, exhibits more complex bidirectional signaling and function than that of allograft. Here, we evaluated the differences of myeloid cells between them. Plasma interferon-γ and interleukin-18 concentrations in the xenograft tumor model after lipopolysaccharide (LPS) administration were significantly higher than those in the allograft tumor model. MHC class I, II, and CD80 expression levels were increased in CD11b⁺ and MDSC populations after LPS administration in the spleen of a xenograft tumor model but not in that of an allograft tumor model. Additionally, the number of CD80- and mannose receptor C type 1 (MRC1)-expressing cells was decreased upon LPS administration in the tumor of the xenograft tumor. These results suggest that functions of macrophages and DCs are sustained in the xenograft, whereas their functions in response to LPS were suppressed in the allograft. The findings will encourage the consideration of the effects of myeloid cells in the xenograft for drug development.

Targeting Glioblastoma Cells Expressing CD44 with Liposomes Encapsulating Doxorubicin and Displaying Chlorotoxin-IgG Fc Fusion Protein.

We recently have established a successful xenograft model of human glioblastoma cells by enriching hyaluronic acid-dependent spheroid-forming populations termed U251MG-P1 cells from U251MG cells. Since U251MG-P1 cells have been confirmed to express CD44 along with principal stemness marker genes, OCT3/4, SOX2, KLF4 and Nanog, this CD44 expressing population appeared to majorly consist of undifferentiated cells. Evaluating the sensitivity to anti-cancer agents, we found U251MG-P1 cells were sensitive to doxorubicin with IC50 at 200 nM. Although doxorubicin has serious side-effects, establishment of an efficient therapy targeting undifferentiated glioblastoma cell population is necessary. We previously designed a chlorotoxin peptide fused to human IgG Fc region without hinge sequence (M-CTX-Fc), which exhibited a stronger growth inhibitory effect on the glioblastoma cell line A172 than an original chlorotoxin peptide. Combining these results together, we designed M-CTX-Fc conjugated liposomes encapsulating doxorubicin and used U251MG-P1 cells as the target model in this study. The liposome modified with M-CTX-Fc was designed with a diameter of approximately 100-150 nm and showed high encapsulation efficiency, adequate loading capacity of anticancer drug, enhanced antitumor effects demonstrating increasing uptake into the cells in vitro; M-CTX-Fc-L-Dox shows great promise in its ability to suppress tumor growth in vivo and it could serve as a template for targeted delivery of other therapeutics.

Evaluation of glycosylated docetaxel-encapsulated liposomes prepared by remote loading under solubility gradient.

Docetaxel comprises one of the most effective anti-cancer drugs despite of serious side effects. Liposomes encapsulation is practically feasible to deliver the drug. However, due to the significant hydrophobicity, docetaxel will be integrated into the lipid bilayer resulting in poor encapsulation capacity. Here, we evaluated a remote loading strategy using a solubility gradient made between the two solvents for 7-glucosyloxyacetyldocetaxel, which has enhanced water solubility of docetaxel with a coupled glucose moiety. Therefore, 7-glucosyloxyacetyldocetaxel was more effectively encapsulated into liposomes with 71.0% of encapsulation efficiency than docetaxel. While 7-glucosyloxyacetyldocetaxel exhibited 90.9% of tubulin stabilisation activity of docetaxel, 7-glucosyloxyacetyldocetaxel encapsulated in liposomes significantly inhibited the growth of tumour in vivo with side effects less than unencapsulated drug. Collectively, the encapsulation of 7-glucosyloxyacetyldocetaxel into liposomes by remote loading under the solubility gradient is considered to be a promising application to prepare practical drug delivery system.

Eosinophil Cationic Protein Shows Survival Effect on H9c2 Cardiac Myoblast Cells with Enhanced Phosphorylation of ERK and Akt/GSK-3ß under Oxidative Stress.

Eosinophil cationic protein (ECP) is well known as a cationic protein contained in the basic granules of activated eosinophils. Recent studies have reported that ECP exhibits novel activities on various types of cells, including rat neonatal cardiomyocytes. Here we evaluated the effects of ECP on rat cardiac myoblast H9c2 cells. Our results showed that ECP enhanced the survival of the cells, in part by promoting the ERK and Akt/GSK-3ß signaling pathways. ECP attenuated the cytotoxic effects of H2O2 on H9c2 cells as well as the production of reactive oxygen species, the number of apoptotic cells and caspase 3/7 activity in the cells. In conclusion, ECP activated the ERK and Akt/GSK-3ß pathways, resulting in anti-oxidative effects on H9c2 cells that attenuated apoptosis.

Cancer stem cells maintain a hierarchy of differentiation by creating their niche.

The self-renewal and differentiation properties of cancer stem cells (CSCs) are regulated and maintained by the CSC niche. However, the mechanism of this maintenance, especially the maintenance contributed by differentiated cancer cells, remains to be fully elucidated. Recently, we have established a model of CSCs, miPS-LLCcm, from mouse induced pluripotent stem cells (miPSCs). In vitro cultured miPS-LLCcm cells were autonomously balanced with stem-like cells and differentiated cells including vascular endothelial cells. Under these conditions, the CSC properties appeared to be stable in the presence of the factor(s) secreted by the differentiated cells. The factor(s) activated Notch signaling and promoted self-renewal of CSCs. In addition, the secreted factor(s) appeared to regulate the differentiation lineage of CSCs. Our results indicate that the differentiated progenies of CSCs containing vascular endothelium play important roles for regulating the CSC's properties. Therefore, miPS-LLCcm cells create their own in vitro niche to maintain themselves in the hierarchy of differentiating CSCs.

Eosinophil cationic protein enhances stabilization of ß-catenin during cardiomyocyte differentiation in P19CL6 embryonal carcinoma cells.

Prior to gastrulation, the Wnt signaling pathway through stabilized ß-catenin enhances the differentiation of mouse ES cell into cardiomyocytes. We have recently shown that cardiomyocyte differentiation is enhanced by eosinophil cationic protein (ECP) through accelerated expression of marker genes of early cardiac differentiation. Furthermore, ECP enhanced the expression of Wnt3a in P19CL6 cells which were stimulated to differentiate into cardiomyocytes by DMSO. Following these findings, we evaluated in this study the potential of ECP to activate the Wnt/ß-catenin signaling pathway during cardiomyocyte differentiation. Analysis by real time qPCR revealed that ECP increased the expression of Frizzled genes such as Frizzled-1, -2, -4 and -10 in P19CL6 cells in the presence of DMSO. The increased expression of those Wnt receptors was found to inhibit the phosphorylation of ß-catenin resulting in the stabilization and translocation of ß-catenin into the nucleus of P19CL6 cells during the early stages of cardiomyocyte differentiation. When assessed for ß-catenin/TCF transcriptional activity with a TCF-luciferase (TOP/FOP) assay, ECP enhanced luciferase activity in P19CL6 cells during 48 h after transfection with TOP/FOP flash reporter in a stoichiometric manner. Collectively, this suggests that ECP can activate a canonical Wnt/ß-catenin signaling pathway by enhancing the stabilization of ß-catenin during cardiomyocyte differentiation.

Eosinophil cationic protein enhances cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells by stimulating the FGF receptor signaling pathway.

We investigated the functional role of eosinophil cationic protein (ECP) in regulating cardiomyogenesis using mouse P19CL6 embryonic carcinoma cells. ECP was confirmed to accelerate the cardiomyocyte differentiation of P19CL6 cells by enhancing the rate and area size of beating of cardiomyocyte and by facilitating the expression of cardiomyocyte-specific genes, such as GATA4 and α-MHC. Since cardiomyocyte differentiation in vivo is considered to follow mesoderm induction, the induction of Brachyury, a marker of mesoderm, was assessed. Brachyury expression was found to be enhanced after the addition of ECP. This enhancement was due to the stimulation of extracellular signal-regulated kinase (ERK)1/2 phosphorylation by ECP. In this context, treatment with SU5402, an inhibitor of fibroblast growth factor (FGF) receptor 1, suppressed Brachyury expression, phosphorylation of ERK1/2, and cardiomyocyte differentiation induced by ECP. We concluded that ECP might induce mesoderm differentiation through FGF signaling pathway and enhance subsequent cardiomyocyte differentiation in concert with dimethyl sulfoxide in P19CL6 cells. ECP may be a novel factor for cardiomyocyte differentiation, which should be very useful to prepare adequate numbers of cardiomyocytes for therapeutic cell transplantation.

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white.

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower ß1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

Transcription factor YY1 interacts with retroviral integrases and facilitates integration of moloney murine leukemia virus cDNA into the host chromosomes.

Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and in vitro pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the in vitro integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration in vivo, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events in vivo. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.

Daunorubicin can eliminate iPS-derived cancer stem cells via ICAD/CAD-independent DNA fragmentation.

Aim: To identify a drug that can effectively eliminate these cancer stem cells (CSCs) and determine its mode of action. Methods: CSCs were obtained from mouse induced pluripotent stem cells (miPSCs) using cancer cell-conditioned media. Drug screening was performed on these cells or after transplantation into mice. Apoptosis was analyzed by flow cytometry and western blotting. Results: Drug screening studies showed that daunorubicin, a topoisomerase II inhibitor, is specifically cytotoxic to miPS-CSCs. Daunorubicin-induced apoptosis was found to be associated with p53 accumulation, activation of the caspase cascade, and oligonucleosomal DNA fragmentation. Treatment with the caspase inhibitor abolished daunorubicin-induced DNA fragmentation and was therefore considered to act downstream of caspase activation. This was also suppressed by treatment with a Ca2+-specific chelator, which suggested that CAD endonuclease does not contribute. Moreover, no obvious ICAD reduction/degradation was detected. Conclusion: Daunorubicin effectively eliminated CSCs, which are dependent on the p53/caspase signaling cascade. The current findings provided the basis for further studies on CSC-targeted drugs for the development of cancer treatment strategies.

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment.

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.

Suppression effect on IFN-γ of adipose tissue-derived mesenchymal stem cells isolated from ß2-microglobulin-deficient mice.

Administration of bone marrow-derived mesenchymal stem cells (MSCs) is a possible treatment for graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation and other inflammatory conditions. To address the mechanism of immunosuppression by MSCs, in particular those derived from adipose tissue (AMSCs), AMSCs were isolated from three different mouse strains, and the suppressive capacity of the AMSCs thus obtained to suppress interferon (IFN)-γ generation in mixed lymphocyte reaction cultures serving as an in vitro model of GVHD were assessed. It was revealed that the AMSCs had a potent capacity to suppress IFN-γ production regardless of their strain of origin and that such suppression was not associated with production of interleukin-10. In addition, the results demonstrated that ß2-microglobulin (ß2m)-deficient AMSCs from ß2m-/- mice were also potent suppressor cells, verifying the fact that the mechanism underlying the suppression by AMSCs is independent of major histocompatibility complex (MHC) class I expression or MHC compatibility. As AMSCs appear to have immunosuppressive properties, AMSCs may be a useful source of biological suppressor cells for the control of GVHD in humans.

Practical Liposomal Formulation for Taxanes with Polyethoxylated Castor Oil and Ethanol with Complete Encapsulation Efficiency and High Loading Efficiency.

Taxanes including paclitaxel and docetaxel are effective anticancer agents preferably sufficient for liposomal drug delivery. However, the encapsulation of these drugs with effective amounts into conventional liposomes is difficult due to their high hydrophobicity. Therefore, an effective encapsulation strategy for liposomal taxanes has been eagerly anticipated. In this study, the mixture of polyethoxylated castor oil (Cremophor EL) and ethanol containing phosphate buffered saline termed as CEP was employed as a solvent of the inner hydrophilic core of liposomes where taxanes should be incorporated. Docetaxel-, paclitaxel-, or 7-oxacetylglycosylated paclitaxel-encapsulating liposomes were successfully prepared with almost 100% of encapsulation efficiency and 29.9, 15.4, or 29.1 mol% of loading efficiency, respectively. We then applied the docetaxel-encapsulating liposomes for targeted drug delivery. Docetaxel-encapsulating liposomes were successfully developed HER2-targeted drug delivery by coupling HER2-specific binding peptide on liposome surface. The HER2-targeting liposomes exhibited HER2-specific internalization and enhanced anticancer activity in vitro. Therefore, we propose the sophisticated preparation of liposomal taxanes using CEP as a promising formulation for effective cancer therapies.

Tumor growth limited to subcutaneous site vs tumor growth in pulmonary site exhibit differential effects on systemic immunities.

To evaluate systemic immunity associated with tumor growth limited to a subcutaneous site versus growth proceeding at multiple tumor sites, we established syngeneic mouse subcutaneous and pulmonary tumor models by local subcutaneous and intravenous injection of colon carcinoma CT26 cells. We found that splenic myeloid-derived suppressor cell (MDSC) levels were significantly increased in the subcutaneous tumor model but not in the pulmonary tumor model. Furthermore, both CD4+ and CD8+ T cells as well as CD4+ Foxp3+ T cells were significantly decreased in the subcutaneous tumor model and were largely unchanged in the pulmonary tumor model. In addition, the subcutaneous model, but not the pulmonary model, displayed a Th1 polarization bias. This bias was characterized by decreased IL-4, IL-9, and IL-10 production, whereas the pulmonary model displayed increased production of IL-10. These results suggest that the mode of tumor development has differential effects on systemic immunity that may, in turn, influence approaches to treatment of cancer patients.

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Characterization of Gene Expression Patterns among Artificially Developed Cancer Stem Cells Using Spherical Self-Organizing Map.

We performed gene expression microarray analysis coupled with spherical self-organizing map (sSOM) for artificially developed cancer stem cells (CSCs). The CSCs were developed from human induced pluripotent stem cells (hiPSCs) with the conditioned media of cancer cell lines, whereas the CSCs were induced from primary cell culture of human cancer tissues with defined factors (OCT3/4, SOX2, and KLF4). These cells commonly expressed human embryonic stem cell (hESC)/hiPSC-specific genes (POU5F1, SOX2, NANOG, LIN28, and SALL4) at a level equivalent to those of control hiPSC 201B7. The sSOM with unsupervised method demonstrated that the CSCs could be divided into three groups based on their culture conditions and original cancer tissues. Furthermore, with supervised method, sSOM nominated TMED9, RNASE1, NGFR, ST3GAL1, TNS4, BTG2, SLC16A3, CD177, CES1, GDF15, STMN2, FAM20A, NPPB, CD99, MYL7, PRSS23, AHNAK, and LOC152573 genes commonly upregulating among the CSCs compared to hiPSC, suggesting the gene signature of the CSCs.

iPSC-derived cancer stem cells provide a model of tumor vasculature.

To grow beyond a size of approximately 1-2 mm3, tumor cells activate many processes to develop blood vasculature. Growing evidences indicate that the formation of the tumor vascular network is very complex, and is not restricted to angiogenesis. Cancer cell-derived tumor vasculatures have been recently described. Among them, endothelial differentiation of tumor cells have been directly related to cancer stem cells, which are cells within a tumor that possess the capacity to self-renew, and to exhibit multipotential heterogeneous lineages of cancer cells. Vasculogenic mimicry has been described to be formed by cancer cells expressing stemness markers. Thus, cancer stem cells have been proposed to contribute to vasculogenic mimicry, though its relation is yet to be clarified. Here, we analyzed the tumor vasculature by using a model of mouse cancer stem cells, miPS-LLCcm cells, which we have previously established from mouse induced pluripotent stem cells and we introduced the DsRed gene in miPS-LLCcm to trace them in vivo. Various features of vasculature were evaluated in ovo, in vitro, and in vivo. The tumors formed in allograft nude mice exhibited angiogenesis in chick chorioallantoic membrane assay. In those tumors, along with penetrated host endothelial vessels, we detected endothelial differentiation from cancer stem cells and formation of vasculogenic mimicry. The angiogenic factors such as VEGF-A and FGF2 were expressed predominantly in the cancer stem cells subpopulation of miPS-LLCcm cells. Our results suggested that cancer stem cells play key roles in not only the recruitment of host endothelial vessels into tumor, but also in maturation of endothelial linage of cancer stem cell's progenies. Furthermore, the undifferentiated subpopulation of the miPS-LLCcm participates directly in the vasculogenic mimicry formation. Collectively, we show that miPS-LLCcm cells have advantages to further study tumor vasculature and to develop novel targeting strategies in the future.

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

Analyses of chicken sialyltransferases related to N-glycosylation.

Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-ß1,4-N-acetylglucosamine (Galß1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.

Defining membrane spanning domains and crucial membrane-localized acidic amino acid residues for K⁺ transport of a Kup/HAK/KT-type Escherichia coli potassium transporter.

Potassium (K(+))-uptake transport proteins present in prokaryote and eukaryote cells are categorized into two classes; Trk/Ktr/HKT, K(+) channel, and Kdp belong to the same superfamily, whereas the remaining K(+)-uptake family, Kup/HAK/KT has no homology to the others, and neither its membrane topology nor crucial residues for K(+) uptake have been identified. We examined the topology of Kup from Escherichia coli. Results from the reporter fusion and cysteine labeling assays support a model with 12 membrane-spanning domains. A model for proton-coupled K(+) uptake mediated by Kup has been proposed. However, this study did not show any stimulation of Kup activity at low pH and any evidence of involvement of the three His in Kup-mediated K(+) uptake. Moreover, replacement of all four cysteines of Kup with serine did not abolish K(+) transport activity. To gain insight on crucial residues of Kup-mediated K(+) uptake activity, we focused on acidic residues in the predicted external and transmembrane regions, and identified four residues in the membrane regions required for K(+) uptake activity. This is different from no membrane-localized acidic residues essential for Trk/Ktr/HKTs, K(+) channels and Kdp. Taken together, these results demonstrate that Kup belongs to a distinct type of K(+) transport system.