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Sample records for polyomavirus bk large

  1. The oncogenic potential of BK-polyomavirus is linked to viral integration into the human genome.

    PubMed

    Kenan, Daniel J; Mieczkowski, Piotr A; Burger-Calderon, Raquel; Singh, Harsharan K; Nickeleit, Volker

    2015-11-01

    It has been suggested that BK-polyomavirus is linked to oncogenesis via high expression levels of large T-antigen in some urothelial neoplasms arising following kidney transplantation. However, a causal association between BK-polyomavirus, large T-antigen expression and oncogenesis has never been demonstrated in humans. Here we describe an investigation using high-throughput sequencing of tumour DNA obtained from an urothelial carcinoma arising in a renal allograft. We show that a novel BK-polyomavirus strain, named CH-1, is integrated into exon 26 of the myosin-binding protein C1 gene (MYBPC1) on chromosome 12 in tumour cells but not in normal renal cells. Integration of the BK-polyomavirus results in a number of discrete alterations in viral gene expression, including: (a) disruption of VP1 protein expression and robust expression of large T-antigen; (b) preclusion of viral replication; and (c) deletions in the non-coding control region (NCCR), with presumed alterations in promoter feedback loops. Viral integration disrupts one MYBPC1 gene copy and likely alters its expression. Circular episomal BK-polyomavirus gene sequences are not found, and the renal allograft shows no productive polyomavirus infection or polyomavirus nephropathy. These findings support the hypothesis that integration of polyomaviruses is essential to tumourigenesis. It is likely that dysregulation of large T-antigen, with persistent over-expression in non-lytic cells, promotes cell growth, genetic instability and neoplastic transformation.

  2. Genome Sequence of a Central Chimpanzee-Associated Polyomavirus Related to BK and JC Polyomaviruses, Pan troglodytes troglodytes Polyomavirus 1.

    PubMed

    Madinda, Nadège F; Robbins, Martha M; Boesch, Christophe; Leendertz, Fabian H; Ehlers, Bernhard; Calvignac-Spencer, Sébastien

    2015-09-03

    We amplified and sequenced the genome of a polyomavirus infecting a central chimpanzee (Pan troglodytes troglodytes). This virus, which is closely related to BK and JC polyomaviruses, may help shed a new light on these human pathogens' evolutionary history. Copyright © 2015 Madinda et al.

  3. BK Polyomavirus and the Transplanted Kidney: Immunopathology and Therapeutic Approaches

    PubMed Central

    Lamarche, Caroline; Orio, Julie; Collette, Suzon; Senécal, Lynne; Hébert, Marie-Josée; Renoult, Édith; Tibbles, Lee Anne; Delisle, Jean-Sébastien

    2016-01-01

    Abstract BK polyomavirus is ubiquitous, with a seropositivity rate of over 75% in the adult population. Primary infection is thought to occur in the respiratory tract, but asymptomatic BK virus latency is established in the urothelium. In immunocompromised host, the virus can reactivate but rarely compromises kidney function except in renal grafts, where it causes a tubulointerstitial inflammatory response similar to acute rejection. Restoring host immunity against the virus is the cornerstone of treatment. This review covers the virus-intrinsic features, the posttransplant microenvironment as well as the host immune factors that underlie the pathophysiology of polyomavirus-associated nephropathy. Current and promising therapeutic approaches to treat or prevent this complication are discussed in relation to the complex immunopathology of this condition. PMID:27391196

  4. Evolution of the BK polyomavirus: epidemiological, anthropological and clinical implications.

    PubMed

    Yogo, Yoshiaki; Sugimoto, Chie; Zhong, Shan; Homma, Yukio

    2009-07-01

    BK polyomavirus (BKV) is essentially ubiquitous in all human populations worldwide. Asymptomatic infection with this virus occurs during early childhood, leading to life-long persistence in the kidney. BKV has four subtypes that can be identified using serological and genotyping methods. The evolutionary aspects of BKV have remained poorly understood due to the limited availability of BKV genomes, since urinary excretion of BKV DNA is detected primarily in immunocompromised individuals. However, we have found that BKV DNA sequences can often be amplified from non-immunocompromised elderly individuals, using a highly sensitive polymerase chain reaction (PCR) with highly concentrated urinary DNA as the source of viral DNA. Using this approach, we have PCR-amplified and sequenced a large number of partial and complete BKV genomes from various human populations worldwide and conducted a series of evolutionary studies using these sequences. We have shown that subtypes I and IV evolved into four and six subgroups, respectively, with each having a close relationship with a particular human population. In addition, we have provided evidence supporting the hypothesis that BKV strains with the archetypal transcriptional control region (TCR) circulate in the human population. In this review, we describe these findings and discuss their epidemiological, anthropological and clinical implications.

  5. Polyomavirus BK Neutralizing Activity in Human Immunoglobulin Preparations

    PubMed Central

    Randhawa, Parmjeet S; Schonder, Kristine; Shapiro, Ron; Farasati, Nousha; Huang, Yuchen

    2011-01-01

    Background Polyomavirus BK (BKV) infection can cause nephropathy in the allograft kidney. No well-established drug treatment is available at this time. Human intravenous immunoglobulins (IVIG) have been used as an empiric therapy without proof of effectiveness. Methods We tested five lots of commercially available IVIG preparations from two different suppliers for polyomavirus neutralizing activity. BKV and mouse polyomavirus were used to infect human and murine host cells, respectively, with or without prior treatment with IVIG. Neutralization activity was measured by quantitation of viral DNA after 7 days in culture. Results Coincubation of BKV but not mouse polyomavirus with clinically relevant concentrations of IVIG derived from healthy and hepatitis B vaccinated subjects caused more than 90% inhibition of viral DNA yield after 7 days in culture. Consistent with a direct neutralizing mechanism, this effect was significantly diminished if viral infection was performed in immunoglobulin pretreated cells or if immunoglobulin treatment was delayed 2 hr after addition of infectious virus. Conclusion Human IVIG preparations contain BKV neutralizing antibodies. Data on neutralizing capacity of these antibodies are presented to aid dose exploration in clinical trials seeking to validate the use of IVIG in patients with BKV infection. PMID:20568674

  6. BK and JC polyomavirus infections in Tunisian renal transplant recipients.

    PubMed

    Boukoum, Hanen; Nahdi, Imen; Sahtout, Wissal; Skiri, Habib; Aloui, Sabra; Achour, Abdelatif; Segondy, Michel; Aouni, Mahjoub

    2015-10-01

    The aim of this prospective study was to investigate the rate of BK (BKPyV) and JC (JCPyV) polyomavirus infections and their influence on allograft function in Tunisian renal transplant recipients. A total of 72 renal transplant recipients were studied. BKPyV and JCPyV were detected and quantified by real-time PCR in urine and plasma. Demographic and laboratory characteristics were collected for each patient. Polyomavirus DNAuria was detected in 54 (75%) of renal transplant recipients: 26 (36%) had BKPyV DNAuria, 20 (28%) had JCPyV DNAuria, and 8 (11%) had a dual BKPyV/JCPyV DNAuria. BKPyV DNAemia was detected in four (5.5%) patients, whereas no patient had JCPyV viremia. More than 70% of BKPyV and JCPyV infections started within the first 3 months post-transplant. The risk for positive DNAemia was observed in patients with DNAuria level >10(7) copies/ml. BK Polyomavirus-associated nephropathy (BKPyVAN) was observed in two patients. This study highlights the high frequency of BKPyV and JCPyV viruria during the first year post-transplant with the highest incidence observed in the third month. We identified several risk factors that were associated with BKV DNAuria including age, sex of patients, and the use of tacrolimus instead of cyclosporine A at month 3. The use of cyclosporine A instead of tacrolimus was identified as risk factor for JCV viruria in month 3. No statistical difference in the allograft function was found between BKPyV and/or JCPyV infected and uninfected patients.

  7. Polyomavirus (BK)-associated pleomorphic giant cell carcinoma of the urinary bladder: a case report.

    PubMed

    Alexiev, Borislav A; Papadimitriou, John C; Chai, Toby C; Ramos, Emilio; Staats, Paul N; Drachenberg, Cinthia B

    2013-04-01

    This report describes the morphological features of a pleomorphic giant cell carcinoma with focal trophoblastic differentiation of the urinary bladder in a male, 12 years post living related donor renal transplant. The voided urine cytology demonstrated rare decoy cells admixed with markedly atypical urothelial cell clusters, papillae and giant cells. Cystoprostatectomy demonstrated a nodular mass involving the trigone and right lateral-posterior wall, adjacent to the ureteral orifice. Hematoxylin-eosin stained sections showed two synchronous malignancies: (a) pleomorphic giant cell carcinoma with focal trophoblastic differentiation of the urinary bladder, metastatic to the omentum and (b) prostatic adenocarcinoma, Gleason score 3+4=7, involving the right prostate lobe. Strong diffuse expression of polyomavirus large T antigen was demonstrated in the primary and metastatic pleomorphic giant cell carcinoma, supporting a possible role for polyomavirus (BK) in the oncogenetic pathway. The prostatic adenocarcinoma was negative for polyomavirus large T antigen. Our findings of p63, CK7 and CK903 expression in pleomorphic giant cell carcinoma suggest that the tumor is of urothelial derivation. This is the first report describing the morphological features of urinary bladder pleomorphic giant cell carcinoma with trophoblastic differentiation, positive for polyomavirus large T antigen, arising in the background of BKV reactivation.

  8. BK Polyomavirus: Clinical Aspects, Immune Regulation, and Emerging Therapies.

    PubMed

    Ambalathingal, George R; Francis, Ross S; Smyth, Mark J; Smith, Corey; Khanna, Rajiv

    2017-04-01

    BK polyomavirus (BKV) causes frequent infections during childhood and establishes persistent infections within renal tubular cells and the uroepithelium, with minimal clinical implications. However, reactivation of BKV in immunocompromised individuals following renal or hematopoietic stem cell transplantation may cause serious complications, including BKV-associated nephropathy (BKVAN), ureteric stenosis, or hemorrhagic cystitis. Implementation of more potent immunosuppression and increased posttransplant surveillance has resulted in a higher incidence of BKVAN. Antiviral immunity plays a crucial role in controlling BKV replication, and our increasing knowledge about host-virus interactions has led to the development of improved diagnostic tools and clinical management strategies. Currently, there are no effective antiviral agents for BKV infection, and the mainstay of managing reactivation is reduction of immunosuppression. Development of immune-based therapies to combat BKV may provide new and exciting opportunities for the successful treatment of BKV-associated complications.

  9. The polyomavirus BK agnoprotein co-localizes with lipid droplets

    SciTech Connect

    Unterstab, Gunhild; Gosert, Rainer; Leuenberger, David; Lorentz, Pascal; Rinaldo, Christine H.; Hirsch, Hans H.

    2010-04-10

    Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66 were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function.

  10. Polyomavirus BK Induces Inflammation via Up-regulation of CXCL10 at Translation Levels in Renal Transplant Patients with Nephropathy.

    PubMed

    Kariminik, Ashraf; Dabiri, Shahriar; Yaghobi, Ramin

    2016-08-01

    Polyomavirus BK-associated nephropathy (BKAN) is an important mechanism for renal losing after kidney transplantation. It seems that Polyomavirus BK can induce nephropathy by direct cell lysis and stimulation of the immune system and induction of inflammation. CXCL10 is a pro-inflammatory cytokine which stimulates the migration and activation of immune cells to the infected sites. Therefore, the aim of the current study was to evaluate the messenger RNA (mRNA) and protein levels of CXCL10 in the Polyomavirus BK-infected and Polyomavirus BK-non-infected post renal transplanted nephropathic patients in comparison to healthy controls. In this cross-sectional study, Polyomavirus BK-infected post renal transplanted nephropathic patients, Polyomavirus BK-non-infected post renal transplanted nephropathic patients, and healthy controls were enrolled to evaluate mRNA and protein levels of CXCL10 by real-time PCR and ELISA techniques, respectively. mRNA levels of CXCL10 were not significantly different among participants, while serum levels of CXCL10 were significantly elevated in the Polyomavirus BK-infected patients when compared to non-infected patients as well as controls and in non-infected patients when compared to healthy controls. Due to the results, it seems that Polyomavirus BK may potentially induce renal losing through stimulation of inflammation via increasing translation of CXCL10, as a pro-inflammatory chemokine.

  11. Meaning of early polyomavirus-BK replication post kidney transplant.

    PubMed

    Mitterhofer, A P; Pietropaolo, V; Barile, M; Tinti, F; Fioriti, D; Mischitelli, M; Limonta, A; Meçule, A; Ferretti, G; Poli, L; Chiarini, F; Berloco, P B; Taliani, G

    2010-05-01

    Polyomavirus BK (BKV) infection is ubiquitous in the human population. Under immunosuppression, BKV can undergo reactivation resulting in viral replication. What really happens in the early hours posttransplantation is not clearly defined; the meaning of early viremia and viruria is not clear. BKV viremia is considered a marker of infection. The aim of our study was to investigate the prevalence of early BKV infection in kidney transplant patients, to evaluate the relationship to infections at 3 and 6 months and the association with recipient, donor, and graft features. We enrolled 36 kidney transplanted patients from May 2006 to April 2007. BKV load was measured on plasma and urine samples by Q-PCR at 12 hours (T(0)/early) as well as 3 (T(3)) and 6 (T(6)) months thereafter. A high percentage of BKV infections were detectable in the first hours after transplantation (33.3%), which remained unchanged to month 6 post transplantation. Moreover, patients who were positive at T(0) had a high probability of remaining positive thereafter. The number of copies in plasma samples tended to increase at 3 months and to decrease thereafter, whereas the urine viral load tended to steadily increase. Among BKV-positive patients, we identified 2 groups according to viremic state at T(0): 9 patients (group A); who were already positive and remained so to T(6) 5 and 3 patients who turned positive at 3 or at 6 months, respectively (group B). Group A included 75% of positive patients at T(0) and 90% of positive patients at either T(3) or T(6) (P = .007). The most important contribution of our study was to highlight the presence of BKV infection in renal transplant recipients from the first hours posttransplantation. This condition seemed to be the most important risk factor for persistent infection in the first 6 months. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  12. BK Polyomavirus Genotypes Represent Distinct Serotypes with Distinct Entry Tropism

    PubMed Central

    Pastrana, Diana V.; Ray, Upasana; Magaldi, Thomas G.; Schowalter, Rachel M.; Çuburu, Nicolas

    2013-01-01

    BK polyomavirus (BKV) causes significant urinary tract pathogenesis in immunosuppressed individuals, including kidney and bone marrow transplant recipients. It is currently unclear whether BKV-neutralizing antibodies can moderate or prevent BKV disease. We developed reporter pseudoviruses based on seven divergent BKV isolates and performed neutralization assays on sera from healthy human subjects. The results demonstrate that BKV genotypes I, II, III, and IV are fully distinct serotypes. While nearly all healthy subjects had BKV genotype I-neutralizing antibodies, a majority of subjects did not detectably neutralize genotype III or IV. Surprisingly, BKV subgenotypes Ib1 and Ib2 can behave as fully distinct serotypes. This difference is governed by as few as two residues adjacent to the cellular glycan receptor-binding site on the virion surface. Serological analysis of mice given virus-like particle (VLP)-based BKV vaccines confirmed these findings. Mice administered a multivalent VLP vaccine showed high-titer serum antibody responses that potently cross-neutralized all tested BKV genotypes. Interestingly, each of the neutralization serotypes bound a distinct spectrum of cell surface receptors, suggesting a possible connection between escape from recognition by neutralizing antibodies and cellular attachment mechanisms. The finding implies that different BKV genotypes have different cellular tropisms and pathogenic potentials in vivo. Individuals who are infected with one BKV serotype may remain humorally vulnerable to other BKV serotypes after implementation of T cell immunosuppression. Thus, prevaccinating organ transplant recipients with a multivalent BKV VLP vaccine might reduce the risk of developing posttransplant BKV disease. PMID:23843634

  13. Neutralization Serotyping of BK Polyomavirus Infection in Kidney Transplant Recipients

    PubMed Central

    Pastrana, Diana V.; Brennan, Daniel C.; Çuburu, Nicolas; Storch, Gregory A.; Viscidi, Raphael P.; Randhawa, Parmjeet S.; Buck, Christopher B.

    2012-01-01

    BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy. PMID:22511874

  14. Viral DNA Replication-Dependent DNA Damage Response Activation during BK Polyomavirus Infection

    PubMed Central

    Verhalen, Brandy; Justice, Joshua L.; Imperiale, Michael J.

    2015-01-01

    ABSTRACT BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. IMPORTANCE Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR

  15. Efficient propagation of archetype BK and JC polyomaviruses.

    PubMed

    Broekema, Nicole M; Imperiale, Michael J

    2012-01-20

    BKPyV and JCPyV are closely related, ubiquitous human pathogens that cause disease in immunocompromised patients. The DNA sequence of the regulatory regions distinguishes two forms of these viruses, designated archetype and rearranged. Although cell culture systems exist for rearranged BKPyV and JCPyV, currently there is no robust cell culture system to study the archetype viruses. Large T antigen (TAg) is a virally encoded protein required to initiate viral DNA synthesis. Because archetype virus produces undetectable levels of TAg, we hypothesized that TAg overexpression would stimulate archetype virus replication. Efficient propagation of the archetype forms of BKPyV and JCPyV was observed in 293TT cells, human embryonic kidney cells overexpressing SV40 TAg. Importantly, the archetypal structure of the regulatory region was maintained during viral growth. Significant replication was not observed for Merkel cell, KI, or WU polyomaviruses. 293TT cells provide a means of propagating archetype BKPyV and JCPyV for detailed study.

  16. Simultaneous BK Polyomavirus (BKPyV)-associated nephropathy and hemorrhagic cystitis after living donor kidney transplantation.

    PubMed

    Helanterä, Ilkka; Hirsch, Hans H; Wernli, Marion; Ortiz, Fernanda; Lempinen, Marko; Räisänen-Sokolowski, Anne; Auvinen, Eeva; Mannonen, Laura; Lautenschlager, Irmeli

    2016-03-01

    BK polyomavirus (BKPyV) commonly reactivates after kidney transplantation, and can cause polyomavirus-associated nephropathy (PyVAN), whereas after allogeneic stem cell transplantation the most frequent manifestation of BKPyV is polyomavirus-associated hemorrhagic cystitis (PyVHC). Despite high-level BKPyV replication in both, the pathogenesis and manifestation of both BKPyV entities appears to differ substantially. We describe an unusual case of simultaneous PyVAN and PyVHC presenting with acute symptoms in a BKPyV-IgG positive recipient eight months after kidney transplantation from a haploidentical living donor, who was BKPyV-IgG negative. Symptoms of cystitis and viremia subsided rapidly after reduction of immunosuppression.

  17. Antibody response to BK polyomavirus as a prognostic biomarker and potential therapeutic target in prostate cancer

    PubMed Central

    Keller, Xavier Etienne; Kardas, Piotr; Acevedo, Claudio; Sais, Giovanni; Poyet, Cédric; Banzola, Irina; Mortezavi, Ashkan; Seifert, Burkhardt; Sulser, Tullio

    2015-01-01

    Infectious agents, including the BK polyomavirus (BKPyV), have been proposed as important inflammatory pathogens in prostate cancer. Here, we evaluated whether the preoperative antibody response to BKPyV large T antigen (LTag) and viral capsid protein 1 (VP1) was associated with the risk of biochemical recurrence in 226 patients undergoing radical prostatectomy for primary prostate cancer. Essentially, the multivariate Cox regression analysis revealed that preoperative seropositivity to BKPyV LTag significantly reduced the risk of biochemical recurrence, independently of established predictors of biochemical recurrence such as tumor stage, Gleason score and surgical margin status. The predictive accuracy of the regression model was denotatively increased by the inclusion of the BKPyV LTag serostatus. In contrast, the VP1 serostatus was of no prognostic value. Finally, the BKPyV LTag serostatus was associated with a peculiar cytokine gene expression profile upon assessment of the cellular immune response elicited by LTag. Taken together, our findings suggest that the BKPyV LTag serology may serve as a prognostic factor in prostate cancer. If validated in additional studies, this biomarker may allow for better treatment decisions after radical prostatectomy. Finally, the favorable outcome of LTag seropositive patients may provide a potential opportunity for novel therapeutic approaches targeting a viral antigen. PMID:25749042

  18. Biologic diversity of polyomavirus BK genomic sequences: Implications for molecular diagnostic laboratories.

    PubMed

    Luo, C; Bueno, M; Kant, J; Randhawa, P

    2008-10-01

    Data on polyomavirus genomic diversity has greatly expanded in the past few years. The implications of viral DNA sequence variation on the performance of molecular diagnostic assays have not been systematically examined. 716 BK, 1626 JC, and 73 SV40 virus sequences available in GenBank were aligned using Clustal-X. Five different published BKV PCR assays currently in use at major medical centers were evaluated for primer and probe mismatches with available GenBank sequences. Coverage of naturally occurring BKV strains varied amongst different assay methods. Targeted viral sequences showed major mismatch with primer or probe sequence in up to 30.7% of known BKV strains. BKV subtypes IVa, IVb, and IVc were more prone to this problem, reflecting common use of Type I Dun sequence for assay design. Despite the known polymorphism of this gene, 484 VP-1 sequences with conserved areas potentially suitable for PCR assay design are available. Assay targets in the Large T-antigen and agnogene are less subject to genetic variation, but sequence information corresponding to the latter two genes is available only for 164 and 174 published strains, respectively. Cross reactivity of appropriately selected BKV primers with JCV and SV40 sequences available in current databases was not a significant problem.

  19. Early and late promoters of BK polyomavirus, Merkel cell polyomavirus, Trichodysplasia spinulosa-associated polyomavirus and human polyomavirus 12 are among the strongest of all known human polyomaviruses in 10 different cell lines.

    PubMed

    Moens, Ugo; Van Ghelue, Marijke; Ludvigsen, Maria; Korup-Schulz, Sarah; Ehlers, Bernhard

    2015-08-01

    Recently, 11 new human polyomaviruses (HPyVs) have been isolated and named KI, WU, Merkel cell polyomavirus (MCPyV), HPyV6,  -7,  -9,  -10 and  -12, Trichodysplasia spinulosa-associated polyomavirus (TSPyV), STLPyV and NJPyV-2013. Little is known about cell tropism of the novel HPyVs, and cell cultures allowing virus propagation are lacking. Because viral tropism partially depends on the interaction of cellular transcription factors with the viral promoter, we monitored the promoter activity of all known HPyVs. Therefore, we compared the relative early and late promoter activity of the BK polyomavirus (BKPyV) (WW strain) with the corresponding activities of the other HPyVs in 10 different cell lines derived from brain, colon, kidney, liver, lung, the oral cavity and skin. Our results show that the BKPyV, MCPyV, TSPyV and HPyV12 early promoters displayed the strongest activity in most cell lines tested, while the remaining HPyV had relative low early promoter activity. HPyV12 showed the highest late promoter activity of all HPyVs in most cell lines, but also the BKPyV, MCPyV and TSPyV late promoters belonged to the stronger ones among HPyVs. The HPyVs with weak early promoter activity had in general also weak late promoter activity, except for HPyV10 whose late promoter was relatively strong in six of the 10 cell lines. A 20 bp deletion in the promoter of an HPyV12 variant significantly affected both early and late promoter activity in most cell lines. In conclusion, our findings suggest which cell lines may be suitable for virus propagation and may give an indication of the cell tropism of the HPyVs.

  20. BK polyomavirus encephalitis in a patient with thrombotic microangiopathy after an allogeneic hematopoietic stem cell transplant.

    PubMed

    Jun, Jae-Bum; Choi, Yunsuk; Kim, Hawk; Lee, Sun Ho; Jeong, Joseph; Jung, Jiwon

    2016-12-01

    To date, only one case of BK polyomavirus (BKPyV) encephalitis combined with transplant-associated thrombotic microangiopathy has been reported in an hematopoietic stem cell transplantation (HCT) recipient. We report the case of an HCT recipient who developed thrombotic microangiopathy and subsequent BKPyV encephalitis. She died despite treatment with cidofovir, ciprofloxacin, and intravenous immunoglobulin without improvement in mental status. Early suspicion of BKPyV encephalitis in an HCT recipient presenting with altered mental status and hemorrhagic cystitis is important. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Vaccine to Prevent BK Polyomavirus-associated Kidney and Bladder Infections in Organ Transplant Recipients | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute is seeking statements of capability or interest from parties interested in collaborative research to co-develop, evaluate or commercialize therapeutic compositions and methods for pre-vaccination of organ transplant recipients against BK polyomavirus (BKV) as well as methods for producing a BKV vaccine against all four known BKV serotypes.

  2. Ureteral stents: a risk factor for polyomavirus BK viremia in kidney transplant recipients undergoing protocol screening.

    PubMed

    Siparsky, N F; Kushnir, L F; Gallichio, M H; Conti, D J

    2011-09-01

    Polyomavirus BK nephropathy (BKN) remains a common cause of early renal transplant dysfunction and graft loss. To date, little has been reported on the role, if any, of transplant ureteral stents in the development of polyomavirus BK viremia (BVK) and BKN. We performed a single-center, retrospective analysis of renal transplant recipients who underwent renal transplantation followed by monthly BKV screening at Albany Medical Center between January 1, 2006, and December 31, 2009. A transplant ureteral stent was placed at the discretion of the surgeon. The immunosuppression protocol employed for deceased donor and unrelated living -donor recipients was antithymocyte antibody induction with methylprednisolone, mycophenolate mofetil, tacrolimus, and sirolimus. During the study period, 186 recipients were identified; 124 (67%) underwent intraoperative transplant ureteral stent placement, while 62 patients (33%) did not undergo stent placement. With our monthly screening protocol, we detected BKV in 32 of the 186 recipients (17%) following transplantation; 27 of the 32 (84%) viremic patients were stent recipients. In all patients who developed BKV, an immunosuppression dose reduction protocol was employed. Ureteral stent placement conferred a statistically significant elevated risk of developing BKV (odds ratio = 3.17, 95% confidence interval 1.16-8.70). Patient gender, age, ethnicity, diabetes status, and retransplant status were not statistically significant factors in the development of BKV. Our study demonstrated the elevated risk of BKV in recipients who undergo transplant ureteral stenting. Monthly BK polymerase chain reaction screening appears to be a useful tool for the early detection of BKV in this higher-risk group. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Pre-transplant immune factors may be associated with BK polyomavirus reactivation in kidney transplant recipients.

    PubMed

    DeWolfe, David; Gandhi, Jinal; Mackenzie, Matthew R; Broge, Thomas A; Bord, Evelyn; Babwah, Amaara; Mandelbrot, Didier A; Pavlakis, Martha; Cardarelli, Francesca; Viscidi, Raphael; Chandraker, Anil; Tan, Chen S

    2017-01-01

    BK polyomavirus (BKPyV) reactivation in kidney transplant recipients can lead to allograft damage and loss. The elements of the adaptive immune system that are permissive of reactivation and responsible for viral control remain incompletely described. We performed a prospective study evaluating BKPyV-specific T-cell response, humoral response and overall T-cell phenotype beginning pre-transplant through one year post-transplant in 28 patients at two centers. We performed an exploratory analysis of risk factors for the development of viremia and viruria as well as compared the immune response to BKPyV in these groups and those who remained BK negative. 6 patients developed viruria and 3 developed viremia. BKPyV-specific CD8+ T-cells increased post-transplant in viremic and viruric but not BK negative patients. BKPyV-specific CD4+ T-cells increased in viremic, but not viruric or BK negative patients. Anti-BKPyV IgG antibodies increased in viruric and viremic patients but remained unchanged in BK negative patients. Viremic patients had a greater proportion of CD8+ effector cells pre-transplant and at 12 months post-transplant. Viremic patients had fewer CD4+ effector memory cells at 3 months post-transplant. Exploratory analysis demonstrated lower CD4 and higher total CD8 proportions, higher anti-BKPyV antibody titers and the cause of renal failure were associated BKPyV reactivation. In conclusion, low CD4, high CD8 and increased effector CD8 cells were found pre-transplant in patients who became viremic, a phenotype associated with immune senescence. This pre-transplant T-cell senescence phenotype could potentially be used to identify patients at increased risk of BKPyV reactivation.

  4. Pre-transplant immune factors may be associated with BK polyomavirus reactivation in kidney transplant recipients

    PubMed Central

    Gandhi, Jinal; Mackenzie, Matthew R.; Broge, Thomas A.; Bord, Evelyn; Babwah, Amaara; Mandelbrot, Didier A.; Pavlakis, Martha; Cardarelli, Francesca; Viscidi, Raphael; Chandraker, Anil

    2017-01-01

    BK polyomavirus (BKPyV) reactivation in kidney transplant recipients can lead to allograft damage and loss. The elements of the adaptive immune system that are permissive of reactivation and responsible for viral control remain incompletely described. We performed a prospective study evaluating BKPyV-specific T-cell response, humoral response and overall T-cell phenotype beginning pre-transplant through one year post-transplant in 28 patients at two centers. We performed an exploratory analysis of risk factors for the development of viremia and viruria as well as compared the immune response to BKPyV in these groups and those who remained BK negative. 6 patients developed viruria and 3 developed viremia. BKPyV-specific CD8+ T-cells increased post-transplant in viremic and viruric but not BK negative patients. BKPyV-specific CD4+ T-cells increased in viremic, but not viruric or BK negative patients. Anti-BKPyV IgG antibodies increased in viruric and viremic patients but remained unchanged in BK negative patients. Viremic patients had a greater proportion of CD8+ effector cells pre-transplant and at 12 months post-transplant. Viremic patients had fewer CD4+ effector memory cells at 3 months post-transplant. Exploratory analysis demonstrated lower CD4 and higher total CD8 proportions, higher anti-BKPyV antibody titers and the cause of renal failure were associated BKPyV reactivation. In conclusion, low CD4, high CD8 and increased effector CD8 cells were found pre-transplant in patients who became viremic, a phenotype associated with immune senescence. This pre-transplant T-cell senescence phenotype could potentially be used to identify patients at increased risk of BKPyV reactivation. PMID:28562595

  5. Fluoroquinolone prophylaxis in preventing BK polyomavirus infection after renal transplant: A systematic review and meta-analysis.

    PubMed

    Song, Tu-Run; Rao, Zheng-Sheng; Qiu, Yang; Liu, Jin-Peng; Huang, Zhong-Li; Wang, Xian-Ding; Lin, Tao

    2016-03-01

    Previous studies regarding the prevention of BK viremia following renal transplantation with fluoroquinolone have yielded conflicting results. The purpose of this systematic review was to examine the evidence regarding the efficacy of fluoroquinolone in preventing BK polyomavirus infection following renal transplantation. We searched PubMed, Embase, and the Cochrane Central Register of Controlled Trials for research articles published prior to January 2015 using keywords such as "fluoroquinolone," "BK viremia," and "renal transplantation." We extracted all types of study published in English. The primary outcome was BK viremia and viruria at 1 year post-transplantation. Secondary outcomes were BK virus-associated nephropathy (BKVN), graft failure, and fluoroquinolone-resistant infection. We identified eight trials, including a total of 1477 participants with a mean duration of fluoroquinolone prophylaxis of >1 month. At 1 year, fluoroquinolone prophylaxis was not associated with a decreased incidence of BK viremia [risk ratio (RR), 0.84; 95% confidence interval (95% CI), 0.58-1.20). No significant differences in BKVN (RR, 0.88; 95% CI, 0.37-2.11), risk of graft failure due to BKVN (RR, 0.68; 95% CI, 0.29-1.59), or fluoroquinolone-resistant infection (RR, 1.08; 95% CI, 0.64-1.83) were observed between the fluoroquinolone prophylaxis and control groups. The results of this study suggest that fluoroquinolone is ineffective in preventing BK polyomavirus infection following renal transplantation. Copyright © 2016. Published by Elsevier Taiwan.

  6. BK polyomavirus infection and nephropathy: the virus-immune system interplay.

    PubMed

    Babel, Nina; Volk, Hans-Dieter; Reinke, Petra

    2011-05-24

    Reactivation of latent BK polyomavirus (BKV) infection continues to be a major challenge in renal graft recipients. Progression of BKV infection to BKV-associated nephropathy (BKVAN) leads to graft loss in up to 60% of affected patients. Interestingly, although >80% of healthy adults are seropositive for BKV, BKVAN occurs almost exclusively in transplanted kidneys, which raises questions about its underlying pathogenetic mechanisms. Intragraft inflammation and an insufficient antiviral immune response seem to be the most important risk factors. Early studies revealed an association between the rate of recovery of BKV-specific cellular immunity (which shows high interindividual variation) and BK viral clearance, which determines the clinical course of BKV infection. In patients with prompt recovery of BKV-specific T cells, BKV infection can be controlled at the early reactivation stage and does not progress to BKVAN. By contrast, in patients with persistent BKV reactivation caused by insufficient BKV-specific immunity, continued viral replication and inflammation ultimately lead to graft injury and/or BKVAN. As the chronic course of BKV infection can be prevented in most patients by prompt restoration of BKV-specific immunity, frequent monitoring of BK viral load and targeted, timely modification or reduction of immunosuppression is strongly recommended for affected patients.

  7. Polyomavirus (BK) in pediatric renal transplants: evaluation of viremic patients with and without BK associated nephritis.

    PubMed

    Hymes, Leonard C; Warshaw, Barry L

    2006-12-01

    Polyoma BK virus (BKV) is emerging as a significant complication in renal transplantation, which may lead to renal dysfunction and graft loss caused by BK nephritis (BKN). We report the management and outcome of 20 children who developed BK viremia. Serum polymerase chain reaction (PCR) for BKV DNA was measured monthly for the first year in transplant recipients and every six months thereafter, or for unexplained creatinine elevation. With seroconversion to +PCR, patients were managed with reduction of immunosuppression. Renal biopsy was performed if PCR or creatinine did not improve. From June 2003 to January 2006, 20 children seroconverted for BKV at 23 to 1410 days post-transplant (mean 467 days). Sixteen underwent renal biopsy. Eight displayed BKN, three acute rejection and five were normal. Patients with BKN displayed higher PCR and serum creatinine and presented later than children with viremia without BKN. There were no differences between the two groups for age, gender, donor source or immunosuppression. Seven children with BKN received treatment with cidofovir. Thirteen patients (65%) remained PCR+ after reduction of immunosuppression or treatment with cidofovir. Renal function was stable in 16 children (80%) at 13 +/- 6 months after seroconversion. Four patients with BKN demonstrated progressive loss of renal function. BKV infection in children can occur as an early complication or may develop years after transplantation. Patients with BKN presented later and displayed higher viral loads and serum creatinine than viremic patients without BKN. Children with BKN remained PCR+ despite reduction of immunosuppression or treatment with cidofovir and were at greater risk for loss of renal function.

  8. BK polyomavirus in kidney transplant recipients: screening, monitoring and clinical management.

    PubMed

    Varella, Rafael Brandão; Almeida, Jorge Reis; Lopes, Patrícia de Fátima; Matos, Jorge Paulo Strogoff de; Menezes, Paulo; Lugon, Jocemir Ronaldo

    2014-01-01

    BK polyomavirus (BKPyV) is a causal agent of nephropathy, ureteral stenosis and hemorrhagic cystitis in kidney transplant recipients, and is considered an important emerging disease in transplantation. Regular screening for BKPyV reactivation mainly during the first 2 years posttransplant, with subsequent pre-emptive reduction of immunosuppression is considered the best option to avoid disease progression, since successful clearance or reduction of viremia is achieved in the vast majority of patients within 6 months. The use of drugs with antiviral properties for patients with persistent viremia has been attempted despite unclear benefits. Clinical manifestations of BKPyV nephropathy, current strategies for diagnosis and monitoring of BKPyV infection, management of immunosuppressive regimen after detection of BKPyV reactivation and the use of antiviral drugs are discussed in this review.

  9. Rapid detection of urinary polyomavirus BK by heterodyne-based surface plasmon resonance biosensor

    NASA Astrophysics Data System (ADS)

    Su, Li-Chen; Tian, Ya-Chung; Chang, Ying-Feng; Chou, Chien; Lai, Chao-Sung

    2014-01-01

    In renal transplant patients, immunosuppressive therapy may result in the reactivation of polyomavirus BK (BKV), leading to polyomavirus-associated nephropathy (PVAN), which inevitably causes allograft failure. Since the treatment outcomes of PVAN remain unsatisfactory, early identification and continuous monitoring of BKV reactivation and reduction of immunosuppressants are essential to prevent PVAN development. The present study demonstrated that the developed dual-channel heterodyne-based surface plasmon resonance (SPR) biosensor is applicable for the rapid detection of urinary BKV. The use of a symmetrical reference channel integrated with the poly(ethylene glycol)-based low-fouling self-assembled monolayer to reduce the environmental variations and the nonspecific noise was proven to enhance the sensitivity in urinary BKV detection. Experimentally, the detection limit of the biosensor for BKV detection was estimated to be around 8500 copies/mL. In addition, urine samples from five renal transplant patients were tested to rapidly distinguish PVAN-positive and PVAN-negative renal transplant patients. By virtue of its simplicity, rapidity, and applicability, the SPR biosensor is a remarkable potential to be used for continuous clinical monitoring of BKV reactivation.

  10. Quantitative Proteomic Analysis of Enriched Nuclear Fractions from BK Polyomavirus-infected Primary Renal Proximal Tubule Epithelial Cells

    PubMed Central

    Justice, Joshua L.; Verhalen, Brandy; Kumar, Ranjit; Lefkowitz, Elliot J.; Imperiale, Michael J.; Jiang, Mengxi

    2016-01-01

    Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with LC-MS/MS. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection. PMID:26354146

  11. The association between polyomavirus BK strains and BKV viruria in liver transplant recipients

    PubMed Central

    Wang, Robert Y. L.; Li, Yi-Jung; Lee, Wei-Chen; Wu, Hsin-Hsu; Lin, Chan-Yu; Lee, Cheng-Chia; Chen, Yung-Chang; Hung, Cheng-Chieh; Yang, Chih-Wei; Tian, Ya-Chung

    2016-01-01

    BK virus (BKV) is a polyomavirus that cause of allograft dysfunction among kidney transplant recipients. The role of BKV infection in non-renal solid organ transplant recipients is not well understood neither for the relationship between various BKV strains with occurrence of BKV viral viruria. This study aimed to understand the prevalence of BKV infection and identified of BKV various strains in the urine of liver transplant recipients. There was not significant difference of renal outcome between high BKV viruria and low BKV viruria in the liver transplant recipients. The WW-non-coding control region (NCCR) BKV detected in urine was associated with higher urinary BKV load, whereas the Dunlop-NCCR BKV was detected in the urine of low urinary BKV load. An in vitro cultivation system demonstrated that WW-BKV strain exhibiting the higher viral DNA replication efficiency and higher BKV load. Altogether, this is the first study to demonstrate the impact of BKV strains on the occurrence of BK viruria in the liver transplant recipients. PMID:27338010

  12. New Structural Insights into the Genome and Minor Capsid Proteins of BK Polyomavirus using Cryo-Electron Microscopy

    PubMed Central

    Hurdiss, Daniel L.; Morgan, Ethan L.; Thompson, Rebecca F.; Prescott, Emma L.; Panou, Margarita M.; Macdonald, Andrew; Ranson, Neil A.

    2016-01-01

    Summary BK polyomavirus is the causative agent of several diseases in transplant patients and the immunosuppressed. In order to better understand the structure and life cycle of BK, we produced infectious virions and VP1-only virus-like particles in cell culture, and determined their three-dimensional structures using cryo-electron microscopy (EM) and single-particle image processing. The resulting 7.6-Å resolution structure of BK and 9.1-Å resolution of the virus-like particles are the highest-resolution cryo-EM structures of any polyomavirus. These structures confirm that the architecture of the major structural protein components of these human polyomaviruses are similar to previous structures from other hosts, but give new insight into the location and role of the enigmatic minor structural proteins, VP2 and VP3. We also observe two shells of electron density, which we attribute to a structurally ordered part of the viral genome, and discrete contacts between this density and both VP1 and the minor capsid proteins. PMID:26996963

  13. Immunity to Polyomavirus BK Infection: Immune Monitoring to Regulate the Balance between Risk of BKV Nephropathy and Induction of Alloimmunity

    PubMed Central

    Cioni, Michela; Basso, Sabrina; Gagliardone, Chiara; Potenza, Leonardo; Verrina, Enrico; Luppi, Mario; Zecca, Marco; Ghiggeri, Gian Marco; Ginevri, Fabrizio

    2013-01-01

    Polyomavirus BK-associated nephropathy (PyVAN) is the main infectious cause of allograft damage after kidney transplantation. A number of studies revealed an association between the presence of BKV-specific cellular immunity and BK viral clearance, with patients failing to recover specific T cells progressing to PyVAN. Evolution to allograft dysfunction can be prevented by restoration of BKV-specific immunity through a stepwise reduction of maintenance immunosuppressive drugs. Prospective monitoring of BK viral load and specific immunity, together with B-cell alloimmune surveillance, may allow a targeted modification/reduction of immunosuppression, with the aim of obtaining viral clearance while preventing graft injury due to deposition of de novo donor-specific HLA antibodies and late/chronic antibody-mediated allograft injury. Innovative, immune-based therapies may further contribute to BKV infection prevention and control. PMID:24000288

  14. BK virus-specific immunity kinetics: a predictor of recovery from polyomavirus BK-associated nephropathy.

    PubMed

    Schachtner, T; Müller, K; Stein, M; Diezemann, C; Sefrin, A; Babel, N; Reinke, P

    2011-11-01

    Impaired BKV-specific immunity is associated with development of BKV-associated nephropathy. Suitable immunological parameters to identify patients at risk, however, are still debated. We monitored 18 kidney-transplant recipients through the course of self-limited BKV-reactivation (n = 11) and BKV-associated nephropathy (n = 7). BKV-specific cellular immunity directed to nonstructural small and Large T-antigen, and structural VP1-3 was analyzed in an interferon-γ Elispot assay. BKV-specific IgM and IgG were measured using an enzyme-linked immunosorbent assay simultaneously. BKV-specific cellular immunity directed to five BKV-proteins increased significantly from diagnosis to resolution of BKV-reactivation (p < 0.001). Patients with self-limited BKV-reactivation developed BKV-specific T cells without therapeutic interventions, and cleared BKV-reactivation within a median period of 1 month. Patients with BKV-associated nephropathy, however, showed BKV-specific T cells after a median period of 5 months after therapeutic interventions only, and cleared BKV-reactivation after a median period of 8 months. Anti-structural T cells were detected earlier than anti-nonstructural T cells, which coincided with BKV-clearance. Patients with BKV-associated nephropathy showed the highest frequencies of BKV-specific T cells at recovery, the highest increase in BKV-specific IgG and persistence of increased IgM levels (p < 0.05). Our results suggest prognostic values of BKV-specific immune monitoring to identify those patients at risk of BKV-associated nephropathy and to aid in the management of therapeutic interventions.

  15. Commercially available immunoglobulins contain virus neutralizing antibodies against all major genotypes of polyomavirus BK.

    PubMed

    Randhawa, P; Pastrana, D V; Zeng, G; Huang, Y; Shapiro, R; Sood, P; Puttarajappa, C; Berger, M; Hariharan, S; Buck, C B

    2015-04-01

    Neutralizing antibodies (NAbs) form the basis of immunotherapeutic strategies against many important human viral infections. Accordingly, we studied the prevalence, titer, genotype-specificity, and mechanism of action of anti-polyomavirus BK (BKV) NAbs in commercially available human immune globulin (IG) preparations designed for intravenous (IV) use. Pseudovirions (PsV) of genotypes Ia, Ib2, Ic, II, III, and IV were generated by co-transfecting a reporter plasmid encoding luciferase and expression plasmids containing synthetic codon-modified VP1, VP2, and VP3 capsid protein genes into 293TT cells. NAbs were measured using luminometry. All IG preparations neutralized all BKV genotypes, with mean EC50 titers as high as 254 899 for genotype Ia and 6,666 for genotype IV. Neutralizing titers against genotypes II and III were higher than expected, adding to growing evidence that infections with these genotypes are more common than currently appreciated. Batch to batch variation in different lots of IG was within the limits of experimental error. Antibody mediated virus neutralizing was dose dependent, modestly enhanced by complement, genotype-specific, and achieved without effect on viral aggregation, capsid morphology, elution, or host cell release. IG contains potent NAbs capable of neutralizing all major BKV genotypes. Clinical trials based on sound pharmacokinetic principles are needed to explore prophylactic and therapeutic applications of these anti-viral effects, until effective small molecule inhibitors of BKV replication can be developed.

  16. Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

    SciTech Connect

    Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer; Hirsch, Hans H.; Rinaldo, Christine Hanssen

    2010-03-01

    High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.

  17. Ureteral stent duration and the risk of BK polyomavirus viremia or bacteriuria after kidney transplantation.

    PubMed

    Wingate, Jonathan T; Brandenberger, Jared; Weiss, Andrew; Scovel, Lauren G; Kuhr, Christian S

    2017-02-01

    Ureteral stents are used in kidney transplantation (KTX) to decrease post-operative complications, but they are associated with BK polyomavirus viremia (BKV). Our primary outcome was to determine the association between ureteral stent duration and BKV. Secondary outcome measures were the association between bacteriuria and stent duration or use of ureteral stent strings. Between January 2010 and January 2015, 403 patients underwent KTX at the Virginia Mason Medical Center and met inclusion criteria. Stent duration was classified as short (<3 weeks) or long (>3 weeks). Multivariate logistic regression models were created to assess for factors associated with BKV. The covariates in the BKV model were chosen a priori based on stent duration and risk factors previously described in the literature. Ureteral stents were placed in 304 (75.4%) transplants. Stent strings were left attached in 166 (54.6%) patients. On multivariate analyses, long stent duration was significantly associated with increased risk of BKV compared with no stent (odds ratio [OR] 1.92, P=.044, 95% confidence interval [CI] 1.04-3.74). Short stent duration was not associated with BKV. Sixty-two (15.4%) patients had bacteriuria. Bacteriuria was associated with female gender (OR 2.77, P<.001, 95% CI 1.58-4.95), and there was a dose-dependent effect with stent duration compared with no stent-short duration (OR 2.46, P=.049, 95% CI 1.05-6.49) and long duration (OR 3.58, P=.004, 95% CI 1.58-9.25). Stent strings were not associated with either complication. The association between ureteral stents and BKV may be dose dependent. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Conserved archetypal configuration of the transcriptional control region during the course of BK polyomavirus evolution.

    PubMed

    Yogo, Yoshiaki; Zhong, Shan; Xu, Yawei; Zhu, Mengyun; Chao, Yuegen; Sugimoto, Chie; Ikegaya, Hiroshi; Shibuya, Ayako; Kitamura, Tadaichi

    2008-08-01

    BK polyomavirus (BKV) is widespread among humans, asymptomatically infecting children and then persisting in renal tissue. The transcriptional control region (TCR) of the BKV genome is variable among clinical isolates. Thus, archetypal TCRs with a common basic configuration generally occur in BKV isolates from the urine of immunocompromised patients, but rearranged TCRs that possibly arise from the archetypal configuration have also been detected in clinical specimens. To examine the hypothesis that archetypal strains represent wild-type strains circulating in the human population (the archetype hypothesis), we analysed 145 complete viral genomes amplified directly from the urine of non-immunocompromised individuals worldwide. These genomes included 82, three, two and 58 sequences classified as belonging to subtypes I, II, III and IV, respectively. Rearranged TCRs with long duplications or deletions were detected from two subtype I and two subtype IV genomes, but not from the other 141 genomes (thus, the TCRs of these genomes were judged to be archetypal). The variations in the archetypal TCRs were nucleotide substitutions and single-nucleotide deletions, most of which were unique to particular subtypes or subgroups. We confirmed that the four complete BKV genomes with rearranged TCRs did not form a unique lineage on a phylogenetic tree. Collectively, the findings demonstrate that the archetypal TCR configuration has been conserved during the evolution of BKV, providing support for the archetype hypothesis. Additionally, we suggest that 'archetype' should be used as a conceptual term that denotes a prototypical structure that can generate various rearranged TCRs during viral growth in vivo and in vitro.

  19. BK viremia and polyomavirus nephropathy in 352 kidney transplants; risk factors and potential role of mTOR inhibition.

    PubMed

    Jacobi, Johannes; Prignitz, Antonina; Büttner, Maike; Korn, Klaus; Weidemann, Alexander; Hilgers, Karl F; Heller, Katharina; Velden, Joachim; Knöll, Antje; Wullich, Bernd; May, Christoph; Eckardt, Kai-Uwe; Amann, Kerstin U

    2013-10-02

    Polyomavirus BK nephropathy (PyVAN) remains an important cause of early graft dysfunction and graft loss in kidney transplantation. In this retrospective, single centre cohort study we studied the incidence and outcome of BK viral infection in 352 patients transplanted in 2008-2011. During follow-up viral replication was detected in 48 patients (13.6%); 22 patients (6.2%) had biopsy proven PyVAN.In multivariate logistic regression analyses risk factors for BK-viremia were lack of enrolment into randomized controlled trials (RCTs), biopsy proven acute rejections, cytomegaly virus (CMV) serostatus of both donor and recipient and previous transplantation.In patients without PyVAN reduction or switch of immunosuppression was associated with rapid viral clearance and stable graft function. In contrast, in most patients with PyVAN graft function deteriorated and 5 patients prematurely lost their allograft. Switch of immunosuppression to a low dose cyclosporine plus mTOR inhibitor based regimen in patients with PyVAN was safe, well tolerated and tended to be associated with a better short-term outcome in terms of graft function compared to reduction of existing immunosuppression alone. With the lack of licensed anti-polyoma viral drugs reduction or conversion of immunosuppression remains the mainstay of therapy in patients with PyVAN. The combination of low dose cyclosporine plus mTOR inhibition appears to be safe and warrants further investigation.

  20. Specific IgG Antibodies React to Mimotopes of BK Polyomavirus, a Small DNA Tumor Virus, in Healthy Adult Sera.

    PubMed

    Pietrobon, Silvia; Bononi, Ilaria; Mazzoni, Elisa; Lotito, Francesca; Manfrini, Marco; Puozzo, Andrea; Destro, Federica; Guerra, Giovanni; Nocini, Pier Francesco; Martini, Fernanda; Tognon, Mauro G

    2017-01-01

    BK polyomavirus (BKPyV) was isolated in 1971 from the urine of a kidney transplant patient. Soon after its identification, BKPyV was characterized as a kidney-tropic virus, which is responsible of a significant fraction of the rejection of transplant kidney in the host. Moreover, in experimental conditions, BKPyV is able to transform different types of animal and human cells and to induce tumors of different histotypes in experimental animals. BKPyV DNA sequences have been detected in healthy individuals and cancer patients using polymerase chain reaction/Shouthern blot hybridization methods. Serum antibodies against this polyomavirus were revealed using immunological techniques, which, however, cross-react with other polyomaviruses such as JC (JCPyV) and Simian Virus 40. These non-specific data indicate the need of novel immunological methods and new investigations to check in a specific manner, BKPyV spread in humans. To this aim, mimotopes from BKPyV structural capsid protein 1 (VP1) were employed for specific immunological reactions to IgG antibodies of human serum samples. An indirect enzyme-linked immunosorbent assay with synthetic peptides mimicking immunogenic epitopes of BKPyV VP1 was set up and employed to test sera of healthy adult subjects. Data from this innovative immunological assay indicate that serum antibodies against BKPyV VP1 mimotopes are detectable in healthy subjects ranging from 18 to 90 years old. The overall prevalence of serum samples that reacted to BKPyV VP1 mimotopes was 72%. The strong points from this investigation are the novelty of the immunological method, its simplicity of the approach, and the specificity of BKPyV antibody reaction to VP1 mimotopes.

  1. Specific IgG Antibodies React to Mimotopes of BK Polyomavirus, a Small DNA Tumor Virus, in Healthy Adult Sera

    PubMed Central

    Pietrobon, Silvia; Bononi, Ilaria; Mazzoni, Elisa; Lotito, Francesca; Manfrini, Marco; Puozzo, Andrea; Destro, Federica; Guerra, Giovanni; Nocini, Pier Francesco; Martini, Fernanda; Tognon, Mauro G.

    2017-01-01

    BK polyomavirus (BKPyV) was isolated in 1971 from the urine of a kidney transplant patient. Soon after its identification, BKPyV was characterized as a kidney-tropic virus, which is responsible of a significant fraction of the rejection of transplant kidney in the host. Moreover, in experimental conditions, BKPyV is able to transform different types of animal and human cells and to induce tumors of different histotypes in experimental animals. BKPyV DNA sequences have been detected in healthy individuals and cancer patients using polymerase chain reaction/Shouthern blot hybridization methods. Serum antibodies against this polyomavirus were revealed using immunological techniques, which, however, cross-react with other polyomaviruses such as JC (JCPyV) and Simian Virus 40. These non-specific data indicate the need of novel immunological methods and new investigations to check in a specific manner, BKPyV spread in humans. To this aim, mimotopes from BKPyV structural capsid protein 1 (VP1) were employed for specific immunological reactions to IgG antibodies of human serum samples. An indirect enzyme-linked immunosorbent assay with synthetic peptides mimicking immunogenic epitopes of BKPyV VP1 was set up and employed to test sera of healthy adult subjects. Data from this innovative immunological assay indicate that serum antibodies against BKPyV VP1 mimotopes are detectable in healthy subjects ranging from 18 to 90 years old. The overall prevalence of serum samples that reacted to BKPyV VP1 mimotopes was 72%. The strong points from this investigation are the novelty of the immunological method, its simplicity of the approach, and the specificity of BKPyV antibody reaction to VP1 mimotopes. PMID:28321224

  2. BK polyomavirus with archetypal and rearranged non-coding control regions is present in cerebrospinal fluids from patients with neurological complications.

    PubMed

    Bárcena-Panero, Ana; Echevarría, Juan E; Van Ghelue, Marijke; Fedele, Giovanni; Royuela, Enrique; Gerits, Nancy; Moens, Ugo

    2012-08-01

    BK polyomavirus (BKPyV) has recently been postulated as an emerging opportunistic pathogen of the human central nervous system (CNS), but it is not known whether specific strains are associated with the neurotropic character of BKPyV. The presence of BKPyV large T-antigen DNA was examined in 2406 cerebrospinal fluid (CSF) samples from neurological patients with suspected JC polyomavirus infection. Twenty patients had a large T-antigen DNA-positive specimen. The non-coding control region (NCCR) of the BKPyV strains amplified from CSF from these 20 patients, strains circulating in renal and bone marrow transplant recipients and from healthy pregnant women was sequenced. The archetypal conformation was the most prevalent in all groups and 14 of the neurological patients harboured archetypal strains, while the remaining six patients possessed BKPyV with rearranged NCCR similar to previously reported variants from non-neurological patients. Transfection studies in Vero cells revealed that five of six early and four of six late rearranged promoters of these CSF isolates showed significantly higher activity than the corresponding archetypal promoter. From seven of the neurological patients with BKPyV DNA-positive CSF, paired serum samples were available. Five of them were negative for BKPyV DNA, while serum from the remaining two patients harboured BKPyV strains with archetypal NCCR that differed from those present in their CSF. Our results suggest that NCCR rearrangements are not a hallmark for BKPyV neurotropism and the dissemination of a rearranged NCCR from the blood may not be the origin of BKPyV CNS infection.

  3. Human polyomaviruses JC and BK in the urine of Brazilian children and adolescents vertically infected by HIV.

    PubMed

    Machado, Daisy Maria; Fink, Maria Cristina; Pannuti, Cláudio Sérgio; Succi, Regina Célia de Menezes; Machado, Alessandra Aparecida; Carmo, Fabiana Bononi do; Gouvêa, Aída de Fátima Barbosa; Urbano, Paulo Roberto; Beltrão, Suenia Vasconcelos; Santos, Isabel Cristina Lopes dos; Machado, Clarisse Martins

    2011-12-01

    The aim of this study was to characterize the urinary excretion of the BK (BKV) and JC (JCV) human polyomaviruses in a cohort of human immunodeficiency virus (HIV)-infected children and adolescents. One hundred and fifty-six patients were enrolled: Group I included 116 HIV-infected children and adolescents [median age = 11.4 years (y); range 1-22 y]; Group II included 40 non-HIV-infected healthy controls (median age = 11.37 y; range 7-16 y). Single urine samples from both groups were screened for the presence of JCV and BKV DNA by polymerase chain reaction at enrolment. The overall rate of JCV and BKV urinary excretion was found to be 24.4% and 40.4%, respectively (n = 156). Group I had urinary excretion of JCV and BKV in 27.6% and 54.3% of subjects, respectively. In contrast, Group II showed positive results for JCV in 17.5% of subjects and for BKV in 12.5% of subjects (p Pearson JCV = 0.20; p Pearson BKV < 0.0001). In Group I, there was no association between JCV/BKV shedding and age, gender or CD4 values. Patients with an HIV viral load < 50 copies/mL had a lower excretion of BKV (p < 0.001) and a trend of lower JCV excretion (p = 0.07). One patient in Group I (1/116, 0.9%) showed clinical and radiological features consistent with progressive multifocal leukoencephalopathy, suggesting that children with HIV/polyomavirus coinfection should be kept under surveillance.

  4. Comparative evaluation of laboratory developed real-time PCR assays and RealStar(®) BKV PCR Kit for quantitative detection of BK polyomavirus.

    PubMed

    Hasan, Mohammad R; Tan, Rusung; Al-Rawahi, Ghada; Thomas, Eva; Tilley, Peter

    2016-08-01

    Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency

  5. Global profiling of histone modifications in the polyomavirus BK virion minichromosome.

    PubMed

    Fang, Chiung-Yao; Shen, Cheng-Huang; Wang, Meilin; Chen, Pei-Lain; Chan, Michael W Y; Hsu, Pang-Hung; Chang, Deching

    2015-09-01

    During polyomavirus infection, the viral DNA adopts histones from host cells and forms minichromosomes as an important part of the viral life cycle. However, the detailed mechanisms of this histone incorporation remain unclear. Here, we profiled the histone posttranslational modifications (PTMs) in BKPyV minichromosomes and in the chromatin of BKPyV host cells. Through Triton-acetic acid-urea (TAU)-PAGE separation followed by nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis, we identified different kinds of PTMs on histones from BKPyV minichromosomes and from host cells. We observed not only the common PTMs on histones such as acetylation, methylation, phosphorylation, ubiquitination, and formylation but also several novel PTM sites. Our results also confirmed that the BKPyV minichromosome is hyperacetylated. Our detailed histone PTM profiles for the BKPyV minichromosome provide insights for future exploration of the underlying mechanisms and biological relevance of these histone PTMs. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Mapping of phosphorylation sites in polyomavirus large T antigen

    SciTech Connect

    Hassauer, M.; Scheidtmann, K.H.; Walter, G.

    1986-06-01

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, /sup 32/P/sub i/-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.

  7. Polyomavirus large T antigen is prevalent in urothelial carcinoma post-kidney transplant.

    PubMed

    Yan, Ling; Salama, Mohamed E; Lanciault, Christian; Matsumura, Linh; Troxell, Megan L

    2016-02-01

    Viral pathogens have been associated with both infectious disease and neoplasia in transplant recipients. Polyomavirus is emerging as a potential causative agent for genitourinary tract cancer in post-kidney transplant patients. Human papillomavirus (HPV) has a proven role in squamous cancers, but has not been studied in genitourinary malignancies in transplantation. Of 2345 kidney transplants performed at our center over the past 20 years, we identified 16 patients with 20 genitourinary cancers (0.7%), including 13 bladder/ureter carcinomas, 5 renal cell carcinomas (RCCs), and 2 prostate carcinomas. We performed immunohistochemical staining for polyomavirus large T antigen and p16, followed by in situ hybridization for HPV in p16+ cases. Four cases of high-grade invasive urothelial bladder carcinomas were positive for large T. Large T+ urothelial carcinomas developed at least 8 years posttransplant in young men, 3 with history of BK polyoma viremia, 2 of whom had native kidney failure due to reflux/obstruction. In situ hybridization for high-risk HPV was negative in all tested cases. Overall, 3 patients died of carcinoma. All 5 RCCs were negative for both large T and p16; 2 prostate cancers were p16 negative and p16+/HPV negative, respectively. Thus, our study shows a relatively high prevalence of large T antigen in urothelial carcinoma in kidney transplant patients (31%), but not in RCC. Although sample size is small, young patients with obstructive disease may be at particular risk for developing large T-positive urothelial carcinoma. Overall, our data further support the necessities of long-term cancer surveillance for renal transplant patients. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Polyomavirus BK Replication in De Novo Kidney Transplant Patients Receiving Tacrolimus or Cyclosporine: A Prospective, Randomized, Multicenter Study

    PubMed Central

    Hirsch, H H; Vincenti, F; Friman, S; Tuncer, M; Citterio, F; Wiecek, A; Scheuermann, E H; Klinger, M; Russ, G; Pescovitz, M D; Prestele, H

    2013-01-01

    Polyomavirus BK (BKV)-associated nephropathy causes premature kidney transplant (KT) failure. BKV viruria and viremia are biomarkers of disease progression, but associated risk factors are controversial. A total of 682 KT patients receiving basiliximab, mycophenolic acid (MPA), corticosteroids were randomized 1:1 to cyclosporine (CsA) or tacrolimus (Tac). Risk factors were analyzed in 629 (92.2%) patients having at least 2 BKV measurements until month 12 posttransplant. Univariate analysis associated CsA-MPA with lower rates of viremia than Tac-MPA at month 6 (10.6% vs. 16.3%, p = 0.048) and 12 (4.8% vs. 12.1%, p = 0.004) and lower plasma BKV loads at month 12 (3.9 vs. 5.1 log10 copies/mL; p = 0.028). In multivariate models, CsA-MPA remained associated with less viremia than Tac-MPA at month 6 (OR 0.60; 95% CI 0.36–0.99) and month 12 (OR 0.33; 95% CI 0.16–0.68). Viremia at month 6 was also independently associated with higher steroid exposure until month 3 (OR 1.19 per 1 g), and with male gender (OR 2.49) and recipient age (OR 1.14 per 10 years) at month 12. The data suggest a dynamic risk factor evolution of BKV viremia consisting of higher corticosteroids until month 3, Tac-MPA compared to CsA-MPA at month 6 and Tac-MPA, older age, male gender at month 12 posttransplant. PMID:23137180

  9. Perception, diagnosis and management of BK polyomavirus replication and disease in paediatric kidney transplant recipients in Europe.

    PubMed

    Pape, Lars; Tönshoff, Burkhard; Hirsch, Hans H

    2016-05-01

    BK polyomavirus (BKPyV)-associated nephropathy remains a challenge to the success of kidney transplantation, but its impact varies in different transplant programmes. We investigated current practice through a web-based questionnaire made available by the European Society for Paediatric Nephrology (ESPN). A total of 90 physicians (23% of 391 active members) from 27 countries participated in the study. BKPyV-associated nephropathy is seen in 1-5% of patients annually with treatment success in 30-60%, and graft loss in 10%. Quantitative BKPyV load testing is available to >90% of physicians. Screening is performed in urine alone in 26%, in urine and blood in 37% and in blood alone in 37%. Most physicians (47%) screen at month 1, 2, 3, 6, 9 and 12 post-transplant. For patients with baseline renal function and plasma BKPyV loads of 10 000-1 000 000 copies/mL, 50% report performing renal biopsies prior to intervention. Intervention consists of reducing immunosuppression first with mycophenolate (Myc) in 40%, first with calcineurin inhibitors (CNI) in 29% or with both in 31%. Changing immunosuppressive drugs is considered mainly for biopsy-proven nephropathy consisting of discontinuation of Myc in 75%, and switching from CNI to mTOR inhibitors (52%). Cidofovir, intravenous immunoglobulin G, leflunomide and fluoroquinolones are used in less than one-third of this group. Furthermore, 66% of participants see a need for new antiviral drugs and new immmunosuppressive strategies, and almost 90% are willing to participate in future observational and interventional trials. This ESPN survey suggests that prompt translation of a positive screening test into reducing immunosuppression could improve outcomes. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  10. Polyomavirus BK--a potential new therapeutic target for painful bladder syndrome/interstitial cystitis?

    PubMed

    Van der Aa, Frank; Beckley, Ian; de Ridder, Dirk

    2014-09-01

    To investigate the role of urinary BK polyoma virus (BKPyV) in the pathophysiology and prognosis of patients with painful bladder syndrome/interstitial cystitis (PBS/IC). Urine samples were collected from 15 patients with PBS/IC and 8 control patients (with urolithiasis, overactive bladder and benign prostatic hyperplasia). BKPyV titres were quantitatively determined using real time PCR. Fisher's exact test was used to compare virus titre levels between the two groups. The PBS/IC patients subsequently underwent cystoscopy, hydrodistension and bladder biopsy. Finally, a chart review was performed in order to correlate PBS/IC subtype and treatment outcomes with BKPyV status. Positive BKPyV titres were found in 11 out of 15 PBS/IC patients but none of the controls. Cystoscopy was performed in 13 of the 15 PBS/IC patients (in 2 BKPyV positive patients, cystoscopy was not performed). Bladder ulceration and glomerulations were observed in all 9 BKPyV positive PBS/IC patients but only 1 out of 4 BKPyV negative patients. None of the non-ulcerative PBS/IC patients had BKPyV positive urine. Viral titres were not predictive of the clinical course however, 3 patients with the highest viral titres eventually underwent cystectomy. We identified BKPyV in the urine of virtually all our patients with ulcerative PBS/IC. This finding suggests there may be a pathophysiological association between the virus and the haemorrhagic manifestations of PBS/IC. Classifying PBS/IC patients into BKPyV positive or negative groups may prove useful in future research on markers of disease prognosis and the subtypes of PBS/IC. We believe that BKPyV may therefore have a role as a potential therapeutic target in PBS/IC. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Distribution and genetic diversity of the human polyomaviruses JC and BK in surface water and sewage treatment plant during 2009 in Porto Alegre, Southern Brazil.

    PubMed

    Comerlato, J; Souza-Campos, F; Souza-Arantes, T; Roos-Kulmann, M I; Trindade-Oliveira, M; Rosado-Spilki, F; Guedes-Frazzon, A P; Roehe, P M; Franco, A C

    2017-01-01

    The human polyomaviruses JC and BK (JCPyV and BKPyV) are ubiquitous, species-specific viruses that belong to the family Polyomaviridae. These viruses are known to be excreted in human urine, and they are potential indicators of human wastewater contamination. In order to assess the distribution of both JCPyV and BKPyV in urban water samples collected from a sewage treatment plant (STP) and from a canalized water stream of Porto Alegre, Brazil, two nested-PCR assays were optimized and applied to the samples collected. The amplicons obtained were submitted to sequencing, and the sequences were analyzed with sequences of human polyomaviruses previously deposited in GenBank. Twelve out of 30 water samples (40%) were JCPyV positive, whereas six samples (20%) were BKPyV positive. The sequencing results confirmed the presence of JCPyV subtypes 1 and 3, whereas only BKPyV Ia and Ib were found. This study shows for the first time the presence of human polyomaviruses in surface water and in samples collected in a sewage treatment plant in southern Brazil.

  12. Polyomavirus-associated nephritis in 2 horses.

    PubMed

    Jennings, S H; Wise, A G; Nickeleit, V; Maes, R K; Cianciolo, R E; Del Piero, F; Law, J M; Kim, Y; McCalla, A C; Breuhaus, B A; Roberts, M C; Linder, K E

    2013-09-01

    Polyomaviruses produce latent and asymptomatic infections in many species, but productive and lytic infections are rare. In immunocompromised humans, polyomaviruses can cause tubulointerstitial nephritis, demyelination, or meningoencephalitis in the central nervous system and interstitial pneumonia. This report describes 2 Standardbred horses with tubular necrosis and tubulointerstitial nephritis associated with productive equine polyomavirus infection that resembles BK polyomavirus nephropathy in immunocompromised humans.

  13. High diversity of human polyomaviruses in environmental and clinical samples in Argentina: Detection of JC, BK, Merkel-cell, Malawi, and human 6 and 7 polyomaviruses.

    PubMed

    Torres, Carolina; Barrios, Melina Elizabeth; Cammarata, Robertina Viviana; Cisterna, Daniel Marcelo; Estrada, Tatiana; Martini Novas, Sergio; Cahn, Pedro; Blanco Fernández, María Dolores; Mbayed, Viviana Andrea

    2016-01-15

    New human polyomaviruses have been recently described. The aim of this work was to detect and characterize human polyomaviruses circulating in Argentina by recovering viruses from environmental and sewage samples and evaluating their potential role as viral indicators of human waste contamination. Analysis was performed in a wider context including viruses from clinical samples from an immunocompromised population. River water and sewage samples were analyzed as a strategy to study the molecular epidemiology of viruses excreted by millions of people. Samples belonged to the Matanza-Riachuelo River (2005-2006: n=25 and 2012: n=20) and sewage from Buenos Aires city and suburbs (2011 and 2013: n=24). Viral detection was performed by PCR and the amplified viral genomes were characterized by phylogenetic analysis. Polyomaviruses were detected in 95.8% of sewage samples, identifying BKPyV (87.5%), JCPyV (83.3%), MCPyV (8.3%) and HPyV6 (8.3%). Besides, one sample collected in 2009 resulted positive for HPyV7. In 2005-2006, polyomaviruses were detected in 84.0% of river water samples, with the highest detection for MCPyV (52.0%), followed by BKPyV (44.0%), JCPyV (20.0%) and MWPyV (4.0%). In 2012, polyomaviruses were detected in 85.0% of river samples, finding JCPyV (85.0%), BKPyV (75.0%), MCPyV (25.0%) and HPyV6 (25.0%). Also, polyomaviruses, including JCPyV, BKPyV and MCPyV, were detected in 63.2% of urine samples from patients infected with HIV (n=19). Characterization indicated the coexistence of different genotypes and variants for each virus, particularly in sewage. MCPyV sequences (the only sequences from Argentina) formed a monophyletic group with the single sequence available for South America (French Guiana). The high level of detection and viral diversity found by environmental surveillance, which involved the characterization of viruses not previously described in South America, reinforces the usefulness of this approach to monitor viral contamination and

  14. BK virus in solid organ transplant recipients: an emerging syndrome.

    PubMed

    Mylonakis, E; Goes, N; Rubin, R H; Cosimi, A B; Colvin, R B; Fishman, J A

    2001-11-27

    BK virus is a human polyomavirus associated with a range of clinical presentations from asymptomatic viruria with pyuria to ureteral ulceration with ureteral stenosis in renal transplant patients or hemorrhagic cystitis in bone marrow transplant recipients. Infection of renal allografts has been associated with diminished graft function in some individuals. Fortunately, however, the majority of patients with BK virus infections are asymptomatic. The type, duration, and intensity of immunosuppression are major contributors to susceptibility to the activation of BK virus infection. Histopathology is required for the demonstration of renal parenchymal involvement; urine cytology and viral polymerase chain reaction methods are useful adjunctive diagnostic tools. Current, treatment of immunosuppressed patients with polyomavirus viruria is largely supportive and directed toward minimizing immunosuppression. Improved diagnostic tools and antiviral therapies are needed for polyomavirus infections.

  15. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    SciTech Connect

    Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

    2010-03-15

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  16. The conserved core enzymatic activities and the distinct dynamics of polyomavirus large T antigens

    PubMed Central

    An, Ping; Brodsky, Jeffrey L.; Pipas, James M.

    2016-01-01

    Several human polyomaviruses including JCV, BKV and TSV are associated with diseases, particularly in immunosuppressed patients. While the large T antigen (LT) encoded by the monkey polyomavirus SV40 is well studied, and possesses intrinsic ATPase and DNA helicase activities, the LTs of the human polyomaviruses are relatively uncharacterized. In order to evaluate whether these enzymatic activities, which are required for viral DNA replication, are conserved between polyomaviruses, we performed a comparative study using the LTs from JCV, TSV and SV40. The ATPase and DNA helicase activities and the interaction with the cellular tumor suppressor p53 were assayed for the purified Zn-ATPase domains of the three LTs. We found that all Zn-ATPases were active ATPases. The Zn-ATPase domains also functioned as DNA helicases, although the measured kinetic constants differed among the three proteins. In addition, when tested against four small molecule ATPase inhibitors, the Zn-ATPase domains of TSV was more resistant than that of SV40 and JCV. Our results show that, while LTs from JCV and TSV share the core ATPase and DNA helicase activities, they possess important functional differences that might translate into their respective abilities to infect and replicate in hosts. PMID:25752954

  17. BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP‐12

    PubMed Central

    Yakhontova, K.; Lu, M.; Manzetti, J.

    2015-01-01

    BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV‐specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)‐ and calcineurin‐inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR–SP6‐kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC‐1 kinase inhibitor torin‐1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP‐12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP‐12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation. PMID:26639422

  18. BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12.

    PubMed

    Hirsch, H H; Yakhontova, K; Lu, M; Manzetti, J

    2016-03-01

    BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV-specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)- and calcineurin-inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR-SP6-kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC-1 kinase inhibitor torin-1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP-12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP-12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation.

  19. Antigen-Specificity of T Cell Infiltrates in Biopsies With T Cell-Mediated Rejection and BK Polyomavirus Viremia: Analysis by Next Generation Sequencing.

    PubMed

    Zeng, G; Huang, Y; Huang, Y; Lyu, Z; Lesniak, D; Randhawa, P

    2016-11-01

    This study interrogates the antigen-specificity of inflammatory infiltrates in renal biopsies with BK polyomavirus (BKPyV) viremia (BKPyVM) with or without allograft nephropathy (BKPyVN). Peripheral blood mononuclear cells (PBMC) from five healthy HLA-A0101 subjects were stimulated by peptides derived from the BKPYV proteome or polymorphic regions of HLA. Next generation sequencing of the T cell-receptor complementary DNA was performed on peptide-stimulated PBMC and 23 biopsies with T cell-mediated rejection (TCMR) or BKPyVN. Biopsies from patients with BKPyVM or BKVPyVN contained 7.7732 times more alloreactive than virus-reactive clones. Biopsies with TCMR also contained BKPyV-specific clones, presumably a manifestation of heterologous immunity. The mean cumulative T cell clonal frequency was 0.1378 for alloreactive clones and 0.0375 for BKPyV-reactive clones. Samples with BKPyVN and TCMR clustered separately in dendrograms of V-family and J-gene utilization patterns. Dendrograms also revealed that V-gene, J-gene, and D-gene usage patterns were a function of HLA type. In conclusion, biopsies with BKPyVN contain abundant allospecific clones that exceed the number of virus-reactive clones. The T cell component of tissue injury in viral nephropathy appears to be mediated primarily by an "innocent bystander" mechanism in which the principal element is secondary T cell influx triggered by both antiviral and anti-HLA immunity. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  20. Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples▿

    PubMed Central

    McQuaig, Shannon M.; Scott, Troy M.; Lukasik, Jerzy O.; Paul, John H.; Harwood, Valerie J.

    2009-01-01

    In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens. PMID:19346361

  1. Large conductance, calcium- and voltage-gated potassium (BK) channels: regulation by cholesterol

    PubMed Central

    Dopico, Alejandro M.; Bukiya, Anna N.; Singh, Aditya K.

    2012-01-01

    Cholesterol (CLR) is an essential component of eukaryotic plasma membranes. CLR regulates the membrane physical state, microdomain formation and the activity of membrane-spanning proteins, including ion channels. Large conductance, voltage- and Ca2+-gated K+ (BK) channels link membrane potential to cell Ca2+ homeostasis. Thus, they control many physiological processes and participate in pathophysiological mechanisms leading to human disease. Because plasmalemma BK channels cluster in CLR-rich membrane microdomains, a major driving force for studying BK channel-CLR interactions is determining how membrane CLR controls the BK current phenotype, including its pharmacology, channel sorting, distribution, and role in cell physiology. Since both BK channels and CLR tissue levels play a pathophysiological role in human disease, identifying functional and structural aspects of the CLR-BK channel interaction may open new avenues for therapeutic intervention. Here, we review the studies documenting membrane CLR-BK channel interactions, dissecting out the many factors that determine the final BK current response to changes in membrane CLR content. We also summarize work in reductionist systems where recombinant BK protein is studied in artificial lipid bilayers, which documents a direct inhibition of BK channel activity by CLR and builds a strong case for a direct interaction between CLR and the BK channel-forming protein. Bilayer lipid-mediated mechanisms in CLR action are also discussed. Finally, we review studies of BK channel function during hypercholesterolemia, and underscore the many consequences that the CLR-BK channel interaction brings to cell physiology and human disease. PMID:22584144

  2. Biology, evolution, and medical importance of polyomaviruses: An update.

    PubMed

    Moens, Ugo; Krumbholz, Andi; Ehlers, Bernhard; Zell, Roland; Johne, Reimar; Calvignac-Spencer, Sébastien; Lauber, Chris

    2017-10-01

    The family Polyomaviridae encompasses non-enveloped viruses with a circular dsDNA genome that is typically approximately 5000bp in length. Originally isolated from mammals, polyomavirus sequences have now been detected in invertebrates, fish, amphibians, reptiles and birds, although it remains to be determined whether all these animals are genuine hosts. The genomes of all polyomaviruses encode at least two regulatory proteins (large and small tumour antigen) and two structural proteins (capsid proteins VP1 and VP2) whose functions have been defined. The large and small tumour antigens have domains conserved among the polyomaviruses, which are responsible for specific interactions with cellular proteins and may result in alteration of the cell cycle. Additional open reading frames (ORFs) are present in the genomes of the different polyomavirus species. Some of these ORFs are transcribed and translated in viral proteins, but their functions remain poorly understood. Polyomaviruses have a restricted host specificity. This may indicate that co-divergence with their hosts, which has been demonstrated in a few cases, was an important factor during polyomavirus diversification. However, a strict co-divergence scenario fails to explain family-wide patterns of diversity, suggesting an important contribution of lineage duplication and, possibly to a lesser extent, recombination and cross-species transmission. Polyomaviruses are pathogens that can cause various malignant and non-malignant diseases in birds and mammals, including humans, but so far they have not been linked to disease in lower vertebrates. In immunosuppressed individuals, reactivation of polyomavirus BK or JC can cause serious disease of the urogenital tract and brain, respectively, while Merkel cell polyomavirus is most probably associated with the development of a highly aggressive neuroendocrine skin tumour in elderly or patients with pre-existing conditions. This review provides an update on the life

  3. Nationwide survey of BK polyomavirus associated hemorrhagic cystitis in adult allogeneic stem cell transplantation among haematologists and urologists.

    PubMed

    Schneidewind, Laila; Neumann, Thomas; Kranz, Jennifer; Knoll, Florian; Pelzer, Alexandre Egon; Schmidt, Christian; Krüger, William

    2017-02-03

    There are no epidemiological data on BK virus associated hemorrhagic cystitis (BKHC) in adult allogeneic stem cell transplantation in Germany available and associations with clinical conditions like GvHD are controversially discussed. Therefore, we conducted a nationwide survey among haematologists and urologists about this disease. We developed two questionnaires, one for haematologists (26 items) and one for urologists (20 items) concerning BKHC in adult allogeneic stem cell transplantation with epidemiological data and clinical implications. The survey was sent out at least three times to EBMT registered centres performing at least five transplantations a year, leading to 39 centres. The recruiting time was between January and June 2016. Total response rates were 76.9% among haematologists and 74.4% among urologists. BKHC seems to appear less frequent in this survey than it is described in the literature. Six deaths in the last 5 years due to this disease have been reported. Interestingly, haematologists as well as urologists mostly think that local therapy is most effective while 50.0% stated that there is no real effective oral or intravenous medication. Associations with other clinical conditions mentioned were heterogeneous, e.g. transplantation type, CMV reactivation, acute GvHD, nephropathy and worse clinical outcome. There was a significant discrepancy between haematologists and urologist concerning the association with acute GvHD (p = 0.004). We need prospective, multicentric clinical studies to evaluate local therapy and for developing a risk stratification model since this disease can be severe with morbidity and rarely mortality. In our opinion, this should be an interdisciplinary approach.

  4. Clinically Relevant Reactivation of Polyomavirus BK (BKPyV) in HLA-A02-Positive Renal Transplant Recipients Is Associated with Impaired Effector-Memory Differentiation of BKPyV-Specific CD8+ T Cells

    PubMed Central

    Remmerswaal, Ester B. M.; Heutinck, Kirstin M.; ten Brinke, Anja; Feltkamp, Mariet C. W.; van der Weerd, Neelke C.; van der Pant, Karlijn A. M. I.; Bemelman, Frederike J.; van Lier, René A. W.; ten Berge, Ineke J. M.

    2016-01-01

    Polyomavirus BK (BKPyV) frequently reactivates in immunosuppressed renal transplant recipients (RTRs) and may lead to graft loss due to BKPyV-induced interstitial nephritis (BKVN). Little is known on the differentiation of CD8+ T cells targeting BKPyV in RTRs. Here we investigated whether BKPyV-specific CD8+ T cell differentiation differs in RTRs with varying degrees of BKPyV reactivation and/or BKVN. Using combinatorial encoding with tetramers carrying BKPyV major capsid protein (VP1) and large T antigen protein (LTAG) epitopes, we investigated CD8+ T cell responses to BKPyV in longitudinally obtained PBMC samples from 46 HLA-A02-positive RTRs and 20 healthy adults. We were also able to isolate BKPyV-specific CD8+ T cells from five renal allografts, two of which were affected by BKVN. Before transplantation, BKPyV-specific CD8+ T cells targeting VP1 and LTAG epitopes appeared predominantly as central-memory and CD27+/CD28+ effector-memory (TEM), and naïve-like PD-1-expressing cells, respectively. After viral reactivation, BKPyV-specific CD8+ T cells assumed CD28− TEM and TEMRA states in patients who were able to control BKPyV, whereas differentiation lagged behind in patients with severe viral reactivation or BKVN. Furthermore, VP1-specific CD69+/CD103+ tissue-resident memory (TRM) cells accumulated in BKVN-affected allografts but lacked signs of effector differentiation. In contrast, granzyme B-expressing effector cells were detected in allografts not affected by BKVN. In conclusion, effector-memory differentiation of BKPyV-specific CD8+ T cells in patients with high viral load or BKVN is impaired. Further characterization of the specific mechanisms behind this altered cellular differentiation is necessary to develop therapies that can prevent the emergence of BKVN. PMID:27723787

  5. Merkel cell polyomavirus infection in both components of a combined Merkel cell carcinoma and basal cell carcinoma with ductal differentiation; each component had a similar but different novel Merkel cell polyomavirus large T antigen truncating mutation.

    PubMed

    Iwasaki, Takeshi; Kodama, Hajime; Matsushita, Michiko; Kuroda, Naoto; Yamasaki, Yoshikazu; Murakami, Ichiro; Yamamoto, Osamu; Hayashi, Kazuhiko

    2013-03-01

    Merkel cell polyomavirus infects up to 80% of patients with Merkel cell carcinoma. Combined Merkel cell carcinoma and cutaneous tumors occur occasionally. Previous reports have suggested that Merkel cell polyomavirus is absent from combined Merkel cell carcinoma and squamous cell carcinomas. This is the first report that Merkel cell polyomavirus infected in both lesions of a combined Merkel cell carcinoma and basal cell carcinoma. A 92-year-old Japanese man presented with a right thigh small subcutaneous mass. Histologic examination revealed a combined tumor with Merkel cell carcinoma and basal cell carcinoma with ductal differentiation. Both tumors and intermingled Merkel cells in basal cell carcinoma expressed Merkel cell polyomavirus large T antigen, and 17 and 240 copies of Merkel cell polyomavirus/cell were detected in the microdissected Merkel cell carcinoma and basal cell carcinoma specimens, respectively. Mutation analysis of Merkel cell polyomavirus large T antigen revealed a novel truncating mutation in Merkel cell carcinoma and a similar but different mutation in the basal cell carcinoma. These results suggest that each was infected by a different Merkel cell polyomavirus subclone derived from a single Merkel cell polyomavirus.

  6. Polyomavirus Large T Antigen Binds Symmetrical Repeats at the Viral Origin in an Asymmetrical Manner

    PubMed Central

    Harrison, Celia; Jiang, Tao; Banerjee, Pubali; Meinke, Gretchen; D'Abramo, Claudia M.; Schaffhausen, Brian

    2013-01-01

    Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication. PMID:24109229

  7. Polyomavirus large T antigen binds symmetrical repeats at the viral origin in an asymmetrical manner.

    PubMed

    Harrison, Celia; Jiang, Tao; Banerjee, Pubali; Meinke, Gretchen; D'Abramo, Claudia M; Schaffhausen, Brian; Bohm, Andrew

    2013-12-01

    Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.

  8. Phylogenetics, evolution, and medical importance of polyomaviruses.

    PubMed

    Krumbholz, Andi; Bininda-Emonds, Olaf R P; Wutzler, Peter; Zell, Roland

    2009-09-01

    The increasing frequency of tissue transplantation, recent progress in the development and application of immunomodulators, and the depressingly high number of AIDS patients worldwide have placed human polyomaviruses, a group of pathogens that can become reactivated under the status of immunosuppression, suddenly in the spotlight. Since the first description of a polyomavirus a half-century ago in 1953, a multiplicity of human and animal polyomaviruses have been discovered. After reviewing the history of research into this group, with a special focus is made on the clinical importance of human polyomaviruses, we conclude by elucidating the phylogenetic relationships and thus evolutionary history of these viruses. Our phylogenetic analyses are based on all available putative polyomavirus species as well as including all subtypes, subgroups, and (sub)lineages of the human BK and JC polyomaviruses. Finally, we reveal that the hypothesis of a strict codivergence of polyomaviruses with their respective hosts does not represent a realistic assumption in light of phylogenetic findings presented here.

  9. The Large Conductance, Calcium-activated K+ (BK) Channel is regulated by Cysteine String Protein

    PubMed Central

    Kyle, Barry D.; Ahrendt, Eva; Braun, Andrew P.; Braun, Janice E. A.

    2013-01-01

    Large-conductance, calcium-activated-K+ (BK) channels are widely distributed throughout the nervous system, where they regulate action potential duration and firing frequency, along with presynaptic neurotransmitter release. Our recent efforts to identify chaperones that target neuronal ion channels have revealed cysteine string protein (CSPα) as a key regulator of BK channel expression and current density. CSPα is a vesicle-associated protein and mutations in CSPα cause the hereditary neurodegenerative disorder, adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL). CSPα null mice show 2.5 fold higher BK channel expression compared to wild type mice, which is not seen with other neuronal channels (i.e. Cav2.2, Kv1.1 and Kv1.2). Furthermore, mutations in either CSPα's J domain or cysteine string region markedly increase BK expression and current amplitude. We conclude that CSPα acts to regulate BK channel expression, and consequently CSPα-associated changes in BK activity may contribute to the pathogenesis of neurodegenerative disorders, such as ANCL. PMID:23945775

  10. Glucocorticoids facilitate the stable transformation of embryonal rat fibroblasts by a polyomavirus large tumor antigen-deficient mutant.

    PubMed Central

    Martens, I; Nilsson, M; Magnusson, G; Linder, S

    1988-01-01

    The addition of glucocorticoids to the growth medium could substitute for the expression of the polyomavirus large tumor antigen in the transformation of rat fibroblasts in vitro. After transfection with a large tumor antigen-deficient mutant of polyomavirus, pbc1051, high-frequency permanent transformation was observed, if the cells were grown in medium containing dexamethasone. Growth of pbc1051-transfected rat fibroblasts was strictly dependent on the presence of glucocorticoids during the initial phase of transformation. In the second phase, the growth of pbc1051-transfected cells was stimulated by dexamethasone, but the hormone was not essential for growth. After approximately 10 weeks in culture, pbc1051-transfected cells had progressed to hormone independent growth. Rat embryo cells transfected with wild-type polyomavirus DNA had the second phase in which growth was stimulated by glucocorticoid, and after this phase growth was steroid independent. Addition of glucocorticoids to rat fibroblasts transfected with a plasmid encoding only the middle-sized tumor antigen resulted in only a weak stimulation of growth. In contrast, embryo cells transfected with a plasmid containing the human homologue of the cellular T24 Ha-ras gene linked to murine sarcoma virus and simian virus 40 enhancers could be efficiently established as cell lines in medium supplemented with glucocorticoids. The data suggest that, in the transformation of primary rodent cells by polyomavirus, the activity of large tumor antigen can be substituted for by stimulating normal cellular functions with dexamethasone. Images PMID:2840668

  11. Erectile dysfunction in mice lacking the large-conductance calcium-activated potassium (BK) channel

    PubMed Central

    Werner, Matthias E; Zvara, Peter; Meredith, Andrea L; Aldrich, Richard W; Nelson, Mark T

    2005-01-01

    Penile erection is dependent on the nitric oxide (NO)/cGMP-dependent protein kinase I (PKGI) pathway. One important target of PKGI in smooth muscle is the large-conductance, calcium-activated potassium (BK) channel, which upon activation hyperpolarizes the smooth muscle cell membrane, causing relaxation. Relaxation of arterial and corpus cavernosum smooth muscle (CCSM) is necessary to increase blood flow into the corpora cavernosa that leads to penile tumescence. We investigated the functional role of BK channels in the corpus cavernosum utilizing a knock-out mouse lacking the Slo gene (Slo−/−) responsible for the pore-forming subunit of the BK channel. Whole-cell currents were recorded from isolated CCSM cells of Slo+/+ and Slo−/− mice. Iberiotoxin-sensitive voltage- and [Ca2+]-activated K+ currents, the latter activated by local transient calcium releases (calcium sparks), were present in Slo+/+ CCSM cells, but absent in Slo−/− cells. CCSM strips from Slo−/− mice demonstrated a four-fold increase in phasic contractions, in the presence of phenylephrine. Nerve-evoked relaxations of precontracted strips were reduced by 50%, both in strips from Slo−/− mice and by blocking BK channels with iberiotoxin in the Slo+/+ strips. Consistent with the in vitro results, in vivo intracavernous pressure exhibited pronounced oscillations in Slo−/− mice, but not in Slo+/+ mice. Furthermore, intracavernous pressure increases to nerve stimulation, in vivo, were reduced by 22% in Slo−/−mice. These results indicate that the BK channel has an important role in erectile function, and loss of the BK channel leads to erectile dysfunction. PMID:16020453

  12. Optogenetic Control of Ca(2+) and Voltage-Dependent Large Conductance (BK) Potassium Channels.

    PubMed

    Mager, Thomas; Wood, Phillip G; Bamberg, Ernst

    2017-03-24

    Ca(2+) concentration jumps for the activation of Ca(2+)-dependent ion channels or transporters can be obtained either by fast solution exchange or by the use of caged Ca(2+). Here, we report on an alternate optogenetic method for the activation of Ca(2+) and voltage-dependent large conductance (BK) potassium channels. This was achieved through the use of the light-gated channelrhodopsin 2 variant, CatCh(Calcium translocating Channelrhodopsin) with enhanced Ca, which produces locally [Ca(2+)] in the μM range on the inner side of the membrane, without significant [Ca(2+)] increase in the cytosol. BK channel subunits α and β1 were expressed together with CatCh in HEK293 cells, and voltage and Ca(2+) dependence were analyzed. Light activation of endogenous BK channels under native conditions in astrocytes and glioma cells was also investigated. Additionally, BK channels were used as sensors for the calibration of the [Ca(2+)] on the inner surface of the cell membrane. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding

    SciTech Connect

    Freund, R.; Bauer, P.H.; Benjamin, T.L.; Crissman, H.A.; Bradbury, E.M. |

    1994-11-01

    The authors have examined the growth properties of polyomavirus large T-antigen mutants that ar unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts for mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G{sub 0} or G{sub 1} through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle. 48 refs., 6 figs., 5 tabs.

  14. J Domain-Independent Regulation of the Rb Family by Polyomavirus Large T Antigen

    PubMed Central

    Sheng, Qing; Love, Tara M.; Schaffhausen, Brian

    2000-01-01

    The ability of polyomavirus large T antigen (LT) to promote cell cycling, to immortalize primary cells, and to block differentiation has been linked to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family. Our previous studies have shown that LT requires an intact N-terminal DnaJ domain, in addition to an Rb binding site, for activation of simple E2F-containing promoters and stimulation of cell cycle progression. Here we show that some LT effects dependent on interaction with the Rb family are largely DnaJ independent. In differentiating C2C12 myoblasts, overexpression of LT caused apoptosis. Although this activity of LT completely depended on Rb binding, LTs with mutations in the J domain remained able to kill. Comparisons of Rb− and J− LTs revealed additional differences. Wild-type but not Rb− LT activated the cyclin A promoter under serum starvation conditions. Genetic analysis of the promoter linked the Rb requirement to an E2F site in the promoter. LTs with mutations in the J domain were still able to activate the promoter. Finally, J mutant LTs caused changes in phosphorylation of both pRb and p130. In the case of p130, Thr-986 was shown to be a site that is regulated by J mutant LT. Taken together, these observations reveal that LT regulation of Rb function can be separated into both DnaJ-dependent and DnaJ-independent pathways. PMID:10799605

  15. Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

    SciTech Connect

    C Harrison; G Meinke; H Kwun; H Rogalin; P Phelan; P Bullock; Y Chang; P Moore; A Bohm

    2011-12-31

    The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 {angstrom} crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be {approx} 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.

  16. Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin.

    PubMed

    Harrison, Celia J; Meinke, Gretchen; Kwun, Hyun Jin; Rogalin, Henry; Phelan, Paul J; Bullock, Peter A; Chang, Yuan; Moore, Patrick S; Bohm, Andrew

    2011-06-17

    The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ~740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.

  17. Merkel Cell Polyomavirus Large T Antigen Disrupts Host Genomic Integrity and Inhibits Cellular Proliferation

    PubMed Central

    Li, Jing; Wang, Xin; Diaz, Jason; Tsang, Sabrina H.; Buck, Christopher B.

    2013-01-01

    Clonal integration of Merkel cell polyomavirus (MCV) DNA into the host genome has been observed in at least 80% of Merkel cell carcinoma (MCC). The integrated viral genome typically carries mutations that truncate the C-terminal DNA binding and helicase domains of the MCV large T antigen (LT), suggesting a selective pressure to remove this MCV LT region during tumor development. In this study, we show that MCV infection leads to the activation of host DNA damage responses (DDR). This activity was mapped to the C-terminal helicase-containing region of the MCV LT. The MCV LT-activated DNA damage kinases, in turn, led to enhanced p53 phosphorylation, upregulation of p53 downstream target genes, and cell cycle arrest. Compared to the N-terminal MCV LT fragment that is usually preserved in mutants isolated from MCC tumors, full-length MCV LT shows a decreased potential to support cellular proliferation, focus formation, and anchorage-independent cell growth. These apparently antitumorigenic effects can be reversed by a dominant-negative p53 inhibitor. Our results demonstrate that MCV LT-induced DDR activates p53 pathway, leading to the inhibition of cellular proliferation. This study reveals a key difference between MCV LT and simian vacuolating virus 40 LT, which activates a DDR but inhibits p53 function. This study also explains, in part, why truncation mutations that remove the MCV LT C-terminal region are necessary for the oncogenic progression of MCV-associated cancers. PMID:23760247

  18. The large tumor antigen: a "Swiss Army knife" protein possessing the functions required for the polyomavirus life cycle.

    PubMed

    Topalis, D; Andrei, G; Snoeck, R

    2013-02-01

    The SV40 large tumor antigen (L-Tag) is involved in the replication and cell transformation processes that take place during the polyomavirus life cycle. The ability of the L-Tag to interact with and to inactivate the tumor suppressor proteins p53 and pRb, makes this polyfunctional protein an interesting target in the search for compounds with antiviral and/or antiproliferative activities designed for the management of polyomavirus-associated diseases. The severe diseases caused by polyomaviruses, mainly in immunocompromised hosts, and the absence of licensed treatments, make the discovery of new antipolyomavirus drugs urgent. Parallels can be made between the SV40 L-Tag and the human papillomavirus (HPV) oncoproteins (E6 and E7) as they are also able to deregulate the cell cycle in order to promote cell transformation and its maintenance. In this review, a presentation of the SV40 L-Tag characteristics, regarding viral replication and cellular transformation, will show how similar these two processes are between the polyoma- and papillomavirus families. Insights at the molecular level will highlight similarities in the binding of polyoma- and papillomavirus replicative helicases to the viral DNA and in their disruptions of the p53 and pRb tumor suppressor proteins.

  19. Prospective study of polyomavirus BK replication and nephropathy in renal transplant recipients in China: a single-center analysis of incidence, reduction in immunosuppression and clinical course.

    PubMed

    Huang, Gang; Chen, Li-Zhong; Qiu, Jiang; Wang, Chang-Xi; Fei, Ji-Guang; Deng, Su-Xiong; Li, Jun; Chen, Guo-Dong; Zhang, Lei; Fu, Qian; Zeng, Wen-Tao; Zhao, Da-Qiang

    2010-01-01

    BK virus (BKV)-associated nephropathy (BKVAN) in renal transplant recipients is an important cause of renal transplant dysfunction. Our aim was to determine the kinetics of BKV load within one yr after kidney transplantation under the impact of intensive monitoring and reduction in maintenance immunosuppression, the incidence of BKVAN, and the outcome of BKVAN treatment. Urine and peripheral blood (PB) were taken from 90 renal transplant recipients for BKV cytological testing and real-time PCR for BKV DNA at one, three, six, nine, and 12 months after transplantation and treatment. Graft biopsies and urinary sediments of recipients with BKVAN were taken to monitor viral particles by conventional transmission electron microscopy (TEM). By one post-transplant year, urinary decoy cells (median, 8/10 HPF), BKV viruria (median, 2.60 × 10(5) copies/mL), viremia (median, 9.65 × 10(3) copies/mL), and BKVAN occurred in 42.2%, 45.6%, 22.2%, and 5.6% of patients, respectively. The incidence of BK infection was lower in patients who received cyclosporine A (CsA) (28.9%) compared to tacrolimus (FK506) (57.7%) (p = 0.007). An increased hazard of BK infection was associated with the use of FK506 (HR 2.6, p = 0.009) relative to CsA. After reduction in immunosuppression, viremia resolved in 95%, without increased acute rejection, allograft dysfunction, or graft loss. BKVAN was diagnosed in five patients (5.6%). The treatment of immunosuppression reduction was effective (i.e., decreased the viral load and number of decoy cells, and improved graft function) in our five patients with BKVAN. Quantitative count of decoy cells (e.g., >10 per 10 HPF) as a marker of viremia and BKVAN had increased positive predictive values of 85.7% and 57.1%, respectively. Choice of FK506 as immunosuppressive agent is an independent risk factor affecting BKV infection. Monitoring and pre-emptive of immunosuppression reduction were associated with resolution of viremia and showed effective in BKVAN

  20. Polyomavirus T Antigens Activate an Antiviral State

    PubMed Central

    Giacobbi, Nicholas S.; Gupta, Tushar; Coxon, Andrew; Pipas, James M.

    2014-01-01

    Ectopic expression of Simian Virus 40 (SV40) large T antigen (LT) in mouse embryonic fibroblasts (MEFs) increased levels of mRNAs encoding interferon stimulated genes (ISGs). The mechanism by which T antigen increases levels of ISGs in MEFs remains unclear. We present evidence that expression of T antigen from SV40, Human Polyomaviruses BK (BKV) or JC (JCV) upregulate production of ISGs in MEFs, and subsequently result in an antiviral state, as determined by inhibition of VSV or EMCV growth. The first 136 amino acids of LT are sufficient for these activities. Furthermore, increased ISG expression and induction of the antiviral state requires STAT1. Finally, the RB binding motif of LT is necessary for activation of STAT1. We conclude that the induction of the STAT1 mediated innate immune response in MEFs is a common feature shared by SV40, BKV and JCV. PMID:25589241

  1. Discovery of African bat polyomaviruses and infrequent recombination in the large T antigen in the Polyomaviridae.

    PubMed

    Carr, Michael; Gonzalez, Gabriel; Sasaki, Michihito; Ito, Kimihito; Ishii, Akihiro; Hang'ombe, Bernard M; Mweene, Aaron S; Orba, Yasuko; Sawa, Hirofumi

    2017-04-01

    Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.

  2. Variability in assessing for BK viremia: whole blood is not reliable and plasma is not above reproach - a retrospective analysis.

    PubMed

    Agrawal, Neerja; Echenique, Ignacio A; Meehan, Shane M; Limaye, Ajit P; Cook, Linda; Chang, Anthony; Harland, Robert C; Javaid, Basit; Kadambi, Pradeep V; Matushek, Scott; Williams, James; Josephson, Michelle A

    2017-07-01

    Polyomavirus nephropathy (PVN) is a major complication of kidney transplantation. Most reports describe polyomavirus viremia either precedes or is detectable at the time of diagnosis of PVN. This association is the basis of current screening recommendations. We retrospectively reviewed the PCR results of blood and urine samples from 29 kidney transplant recipients with biopsy-proven PVN. Biopsies were performed for a rise in serum creatinine or persistent high-level BK viruria. All biopsies showed polyoma virus large T-antigen expression in tubular epithelium using immunohistochemistry. All had viruria preceding or at the time of biopsy (range, 5.2 × 10(4) to >25 × 10(6) BKV DNA copies/ml). Twenty (69%) had viremia ranging from 2.5 × 10(3) to 4.3 × 10(6) copies/ml at the time of the biopsy. Via blood BK PCR assay, nine (31%) had no BK viremia detected either preceding or at the time of the biopsy. In five recipients where sufficient specimen permitted, additional plasma BK assessment revealed positive detection of viremia. A comparative analysis of assays from two centres was performed with spiked samples. BK DNA may not be detected in the blood of some kidney transplant recipients with histologically confirmed PVN. This may reflect limitation of whole blood as opposed to plasma-based BK DNA assessment. © 2017 Steunstichting ESOT.

  3. The risk of polyomavirus BK-associated hemorrhagic cystitis after allogeneic hematopoietic SCT is associated with myeloablative conditioning, CMV viremia and severe acute GVHD.

    PubMed

    Uhm, J; Hamad, N; Michelis, F V; Shanavas, M; Kuruvilla, J; Gupta, V; Lipton, J H; Messner, H A; Seftel, M; Kim, D D

    2014-12-01

    Hemorrhagic cystitis (HC) is a common complication after allogeneic hematopoietic SCT (allo-HSCT). Several risk factors have been suggested including BU-containing myeloablative conditioning, unrelated donors and GVHD, but these have not been consistently reported. We conducted a retrospective study including 339 allo-HSCT recipients between 2009 and 2012. Of 339 patients, 79 (23.3%) developed HC with 2-year cumulative incidence of 24.0% (95% confidence interval, 19.4-28.9). The median onset time was 45 days (range, 16-430) after allo-HSCT. Sixty-two patients (84%) out of 74 evaluated for urine BK virus PCR testing showed a positive result (mean 2.0 × 10(10) copies of DNA per mL). In univariate analysis, myeloablative conditioning, HLA-mismatched donor, CMV viremia and acute GVHD (aGVHD) grade 3-4 were significantly associated with the risk of HC. Multivariate analysis confirmed all associating factors identified in univariate analysis except for HLA-mismatched donor: myeloablative conditioning (hazard ratio (HR) 2.63, P=0.003), CMV viremia (HR 1.88, P=0.014) and aGVHD grade 3-4 (HR 1.71, P=0.029). HC did not affect OS or non-relapse mortality. Symptomatic HC is a frequent complication following allo-HSCT, with a 2-year cumulative incidence of 24.0%. Three clinical factors associated with HC were identified including myeloablative conditioning, CMV viremia and severe aGVHD.

  4. Early monitoring of the human polyomavirus BK replication and sequencing analysis in a cohort of adult kidney transplant patients treated with basiliximab

    PubMed Central

    2011-01-01

    Background Nowadays, better immunosuppressors have decreased the rates of acute rejection in kidney transplantation, but have also led to the emergence of BKV-associated nephropathy (BKVAN). Therefore, we prospectively investigated BKV load in plasma and urine samples in a cohort of kidney transplants, receiving basiliximab combined with a mycophenolate mofetil-based triple immunotherapy, to evaluate the difference between BKV replication during the first 3 months post-transplantation, characterized by the non-depleting action of basiliximab, versus the second 3 months, in which the maintenance therapy acts alone. We also performed sequencing analysis to assess whether a particular BKV subtype/subgroup or transcriptional control region (TCR) variants were present. Methods We monitored BK viruria and viremia by quantitative polymerase chain reaction (Q-PCR) at 12 hours (Tx), 1 (T1), 3 (T2) and 6 (T3) months post-transplantation among 60 kidney transplant patients. Sequencing analysis was performed by nested-PCR with specific primers for TCR and VP1 regions. Data were statistically analyzed using χ2 test and Student's t-test. Results BKV was detected at Tx in 4/60 urine and in 16/60 plasma, with median viral loads of 3,70 log GEq/mL and 3,79 log GEq/mL, respectively, followed by a significant increase of both BKV-positive transplants (32/60) and median values of viruria (5,78 log GEq/mL) and viremia (4,52 log GEq/mL) at T2. Conversely, a significantly decrease of patients with viruria and viremia (17/60) was observed at T3, together with a reduction of the median urinary and plasma viral loads (4,09 log GEq/mL and 4,00 log GEq/mL, respectively). BKV TCR sequence analysis always showed the presence of archetypal sequences, with a few single-nucleotide substitutions and one nucleotide insertion that, interestingly, were all representative of the particular subtypes/subgroups we identified by VP1 sequencing analysis: I/b-2 and IV/c-2. Conclusions Our results confirm

  5. The Role of Merkel Cell Polyomavirus and Other Human Polyomaviruses in Emerging Hallmarks of Cancer

    PubMed Central

    Moens, Ugo; Rasheed, Kashif; Abdulsalam, Ibrahim; Sveinbjørnsson, Baldur

    2015-01-01

    Polyomaviruses are non-enveloped, dsDNA viruses that are common in mammals, including humans. All polyomaviruses encode the large T-antigen and small t-antigen proteins that share conserved functional domains, comprising binding motifs for the tumor suppressors pRb and p53, and for protein phosphatase 2A, respectively. At present, 13 different human polyomaviruses are known, and for some of them their large T-antigen and small t-antigen have been shown to possess oncogenic properties in cell culture and animal models, while similar functions are assumed for the large T- and small t-antigen of other human polyomaviruses. However, so far the Merkel cell polyomavirus seems to be the only human polyomavirus associated with cancer. The large T- and small t-antigen exert their tumorigenic effects through classical hallmarks of cancer: inhibiting tumor suppressors, activating tumor promoters, preventing apoptosis, inducing angiogenesis and stimulating metastasis. This review elaborates on the putative roles of human polyomaviruses in some of the emerging hallmarks of cancer. The reciprocal interactions between human polyomaviruses and the immune system response are discussed, a plausible role of polyomavirus-encoded and polyomavirus-induced microRNA in cancer is described, and the effect of polyomaviruses on energy homeostasis and exosomes is explored. Therapeutic strategies against these emerging hallmarks of cancer are also suggested. PMID:25866902

  6. The role of Merkel cell polyomavirus and other human polyomaviruses in emerging hallmarks of cancer.

    PubMed

    Moens, Ugo; Rasheed, Kashif; Abdulsalam, Ibrahim; Sveinbjørnsson, Baldur

    2015-04-10

    Polyomaviruses are non-enveloped, dsDNA viruses that are common in mammals, including humans. All polyomaviruses encode the large T-antigen and small t-antigen proteins that share conserved functional domains, comprising binding motifs for the tumor suppressors pRb and p53, and for protein phosphatase 2A, respectively. At present, 13 different human polyomaviruses are known, and for some of them their large T-antigen and small t-antigen have been shown to possess oncogenic properties in cell culture and animal models, while similar functions are assumed for the large T- and small t-antigen of other human polyomaviruses. However, so far the Merkel cell polyomavirus seems to be the only human polyomavirus associated with cancer. The large T- and small t-antigen exert their tumorigenic effects through classical hallmarks of cancer: inhibiting tumor suppressors, activating tumor promoters, preventing apoptosis, inducing angiogenesis and stimulating metastasis. This review elaborates on the putative roles of human polyomaviruses in some of the emerging hallmarks of cancer. The reciprocal interactions between human polyomaviruses and the immune system response are discussed, a plausible role of polyomavirus-encoded and polyomavirus-induced microRNA in cancer is described, and the effect of polyomaviruses on energy homeostasis and exosomes is explored. Therapeutic strategies against these emerging hallmarks of cancer are also suggested.

  7. Polyomavirus Large T Antigen Binds Cooperatively to Its Multiple Binding Sites in the Viral Origin of DNA Replication

    PubMed Central

    Peng, Yu-Cai; Acheson, Nicholas H.

    1998-01-01

    Polyomavirus large T antigen binds to multiple 5′-G(A/G)GGC-3′ pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a “handover” mechanism mediated by these protein-protein contacts. PMID:9696829

  8. Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA.

    PubMed Central

    Bertin, J; Sunstrom, N A; Acheson, N H

    1993-01-01

    During transcription of the late strand of polyomavirus DNA, RNA polymerase II stalls and accumulates nearby the binding sites on viral DNA recognized by polyomavirus large T antigen. Stalling by RNA polymerases is eliminated when thermolabile large T antigen is inactivated by using a temperature-sensitive virus mutant (J. Bertin, N.-A. Sunstrom, P. Jain, and N. H. Acheson, Virology 189:715-724, 1992). To determine whether stalling by RNA polymerases is mediated through the interaction of large T antigen with one or more of its binding sites, viable polyomavirus mutants that contain altered large-T-antigen-binding sites were constructed. Point mutations were introduced by site-directed mutagenesis into the multiple, clustered G(A/G)GGC pentanucleotides known to be the target sequence for large T-antigen binding. Mutation of the G(A/G)GGC pentanucleotides in the first two binding sites encountered by RNA polymerases in the intergenic region (sites C and B) had no detectable effect on stalling as measured by transcriptional run-on analysis. However, mutation of the two GAGGC pentanucleotides in binding site A, which lies adjacent to the origin of viral DNA replication, eliminated stalling by RNA polymerases. We conclude that binding of large T antigen to site A blocks elongation by RNA polymerase II. Further characterization of virus containing mutated site A did not reveal any effects on early transcription levels or on virus DNA replication. However, the mutant virus gave rise to small plaques, suggesting impairment in some stage of virus growth. Stalling of RNA polymerases by large T antigen bound to the intergenic region of viral DNA may function to prevent transcription from displacing proteins whose binding is required for the normal growth of polyomavirus. Images PMID:8396655

  9. Large Conductance Voltage- and Ca2+-gated Potassium (BK) Channel β4 Subunit Influences Sensitivity and Tolerance to Alcohol by Altering Its Response to Kinases*

    PubMed Central

    Velázquez-Marrero, Cristina; Seale, Garrett E.; Treistman, Steven N.; Martin, Gilles E.

    2014-01-01

    Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca2+-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the β4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αβ4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the β4 subunit on tolerance may result from a striking effect of β4 on kinase modulation of the BK channel. We examine the influence of the β4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the β4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the β4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol. PMID:25190810

  10. Activation of galanin receptor 2 stimulates large conductance Ca(2+)-dependent K(+) (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells.

    PubMed

    Pan, Na Clara; Bai, Yun-Fei; Yang, Yutao; Hökfelt, Tomas; Xu, Zhi-Qing David

    2014-03-28

    The large conductance Ca(2+)-activated K(+) (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca(2+) flux and Ca(2+)-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8±16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca(2+)-free solution, suggesting that extracellular Ca(2+) is not essential. Chelating intracellular Ca(2+) by either the slow Ca(2+) buffer EGTA or the fast Ca(2+) buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca(2+). The role of Ca(2+) efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca(2+) from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca(2+) activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca(2+) is due to Ca(2+) efflux from ER through IP3R sequentially opening BK channels.

  11. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens

    SciTech Connect

    Bradley, M.K.; Smith, T.F.; Lathrop, R.H.; Livingston, D.M.; Webster, T.A.

    1987-06-01

    The location and sequence composition of a consensus element of the nucleotide binding site in both simian virus 40 (SV40) and polyomavirus (PyV) large tumor antigens (T antigens) can be predicted with the assistance of a computer-based pattern-matching system, ARIADNE. The latter was used to optimally align elements of T antigen primary sequence and predicted secondary structure with a descriptor for a mononucleotide binding fold. Additional consensus elements of the nucleotide binding site in these two proteins were derived from comparisons of T antigen primary and predicted secondary structures with x-ray structures of the nucleotide binding sites in four otherwise unrelated proteins. Each of these elements was predicted to be encompassed within a 110-residue segment that is highly conserved between the two T antigens residues 418-528 in SV 40 T antigen and residues 565-675 in PyV. Results of biochemical and immunologic experiments on the nucleotide binding behavior of these proteins using (/sup 32/P)-Amp-labeled SV40 T antigen, were found to be consistent with these predictions. Taken together, the latter have resulted in a topological model of the ATP binding site in these two oncogene products.

  12. High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

    PubMed Central

    Borchert, Sophie; Czech-Sioli, Manja; Neumann, Friederike; Schmidt, Claudia; Wimmer, Peter; Dobner, Thomas

    2014-01-01

    ABSTRACT Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare

  13. Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

    PubMed

    Stimers, Joseph R; Song, Li; Rusch, Nancy J; Rhee, Sung W

    2015-01-01

    Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

  14. Ca2+-dependent gating mechanisms for dSlo, a large-conductance Ca2+-activated K+ (BK) channel.

    PubMed Central

    Moss, B L; Silberberg, S D; Nimigean, C M; Magleby, K L

    1999-01-01

    The Ca2+-dependent gating mechanism of cloned BK channels from Drosophila (dSlo) was studied. Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane. Stability plots were used to define stable segments of data. Unlike native BK channels from rat skeletal muscle in which increasing internal Ca2+ concentration (Cai2+) in the range of 5 to 30 microM increases mean open time, increasing Cai2+ in this range for dSlo had little effect on mean open time. However, further increases in Cai2+ to 300 or 3000 microM then typically increased dSlo mean open time. Kinetic schemes for the observed Ca2+-dependent gating kinetics of dSlo were evaluated by fitting two-dimensional dwell-time distributions using maximum likelihood techniques and by comparing observed dependency plots with those predicted by the models. Previously described kinetic schemes that largely account for the Ca2+-dependent kinetics of native BK channels from rat skeletal muscle did not adequately describe the Ca2+ dependence of dSlo. An expanded version of these schemes which, in addition to the Ca2+-activation steps, permitted a Ca2+-facilitated transition from each open state to a closed state, could approximate the Ca2+-dependent kinetics of dSlo, suggesting that Ca2+ may exert dual effects on gating. PMID:10354435

  15. Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume

    PubMed Central

    Niu, Xiaowei

    2011-01-01

    Large-conductance Ca2+- and voltage-activated K+ (BK) channels have the largest conductance (250–300 pS) of all K+-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA− increased currents and MTSET+ decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K+]i. Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger. PMID:21576375

  16. [New, newer, newest human polyomaviruses: how far?].

    PubMed

    Us, Dürdal

    2013-04-01

    Polyomaviruses, classified in Polyomaviridae family, are non-enveloped small (40-45 nm) viruses with icosahedral symmetry and circular double-stranded DNA genome. Polyomaviruses can infect a variety of vertebrates including birds, rodents, cattle, monkeys and humans. The characteristics such as establishment of latent infections, reactivations during immunosuppression and oncogenic potencies render the human polyomaviruses (HPyVs) of considerable importance for public health. The first polyomavirus (Mouse polyomavirus) has been identified in 1953 as filterable tumor-causing agents in mice, followed by Simian vacuolating virus (SV40) isolated from rhesus monkey kidney cells that had been used for poliovirus vaccine preparation in 1960. Due to the known transforming capacity of SV40, it was initially thought that the incidence of cancer could increase following the administration of SV40-contaminated polio vaccines, however advanced studies yielded inconsistent results, without any evidence to conclude whether or not the contaminated polio vaccine caused cancer. Several studies have reported the detection of SV40 genome in some of the human tumors, as well as in the clinical samples of healthy subjects. In addition SV40 seropositivity was reported in human populations although in low rates (2-10%). These data have raised the possibility that SV40 infects humans and circulates in human populations unrelated to being exposed to the vaccine. The discovery of the first human polyomaviruses was in 1971 independently from each other, one was BK virus (BKPyV) isolated from the urine sample of a renal transplant patient, and the other was JC virus (JCPyV) isolated from the brain tissue of a patient with progressive multifocal leukoencephalopathy, and both were named after the patients' initials. BK and JC viruses were the only well-known human polyomaviruses throughout 36 years, however drammatical increase in number of newly identified human polyomaviruses was recorded in

  17. The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

    PubMed Central

    Fimia, Gian Maria; Gottifredi, Vanesa; Bellei, Barbara; Ricciardi, Maria Rosaria; Tafuri, Agostino; Amati, Paolo; Maione, Rossella

    1998-01-01

    It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis. PMID:9614186

  18. Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

    PubMed

    Koide, Masayo; Syed, Arsalan U; Braas, Karen M; May, Victor; Wellman, George C

    2014-11-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

  19. Human Polyomavirus Reactivation: Disease Pathogenesis and Treatment Approaches

    PubMed Central

    De Gascun, Cillian F.; Carr, Michael J.

    2013-01-01

    JC and BK polyomaviruses were discovered over 40 years ago and have become increasingly prevalent causes of morbidity and mortality in a variety of distinct, immunocompromised patient cohorts. The recent discoveries of eight new members of the Polyomaviridae family that are capable of infecting humans suggest that there are more to be discovered and raise the possibility that they may play a more significant role in human disease than previously understood. In spite of this, there remains a dearth of specific therapeutic options for human polyomavirus infections and an incomplete understanding of the relationship between the virus and the host immune system. This review summarises the human polyomaviruses with particular emphasis on pathogenesis in those directly implicated in disease aetiology and the therapeutic options available for treatment in the immunocompromised host. PMID:23737811

  20. Multiple Skin Cancers in a Renal Transplant Recipient: A Patient Report with Analyses of Human Papillomavirus and Human Polyomavirus Infection

    PubMed Central

    Kaneda, Tokinobu; Matsushita, Michiko; Iwasaki, Takeshi; Ishiguro, Naoko; Koide, Takashi; Hayashi, Kazuhiko; Kitamura, Yukisato

    2015-01-01

    Skin cancer is an important complication in renal transplant recipients. Associations of transplant-related skin tumor with ultraviolet radiation, age at transplantation, type of immunosuppressant drug administered, and viral infection have been reported; however, the details remain unclear. We report a 61-year-old man who had underwent renal transplantation at 38 years of age and developed multiple skin tumors or squamous cell carcinomas (SCCs). Polymerase chain reaction (PCR) analyses of the patient’s 12 tumors for viral DNAs of cutaneous or mucosal human papillomavirus (HPV) and 6 human polyomaviruses (MCPyV, trichodysplasia spinulosa-associated, BK, JC, KI and WU polyomaviruses) only detected cutaneous HPV-DNA in only 5 of the tumors; no other viruses were detected. Real-time PCR showed high loads of cutaneous HPV in 3 SCCs and very low loads of MCPyV in 9. Immunohistochemistry revealed no tumor cell expression for MCPyV-large T-antigen or mucosal HPV. Our report not only reconfirmed the association of cutaneous HPV5 with skin cancer in renal transplant recipients in previous studies but also showed no relevant association of 6 human polyomaviruses and mucosal HPV with skin tumors. PMID:26538801

  1. Increased Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel in Adult-Onset Neuronal Ceroid Lipofuscinosis

    PubMed Central

    Donnelier, Julien; Braun, Samuel T.; Dolzhanskaya, Natalia; Ahrendt, Eva; Braun, Andrew P.; Velinov, Milen; Braun, Janice E. A.

    2015-01-01

    Cysteine string protein (CSPα) is a presynaptic J protein co-chaperone that opposes neurodegeneration. Mutations in CSPα (i.e., Leu115 to Arg substitution or deletion (Δ) of Leu116) cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly inherited neurodegenerative disease. We have previously demonstrated that CSPα limits the expression of large conductance, calcium-activated K+ (BK) channels in neurons, which may impact synaptic excitability and neurotransmission. Here we show by western blot analysis that expression of the pore-forming BKα subunit is elevated ~2.5 fold in the post-mortem cortex of a 36-year-old patient with the Leu116∆ CSPα mutation. Moreover, we find that the increase in BKα subunit level is selective for ANCL and not a general feature of neurodegenerative conditions. While reduced levels of CSPα are found in some postmortem cortex specimens from Alzheimer’s disease patients, we find no concomitant increase in BKα subunit expression in Alzheimer’s specimens. Both CSPα monomer and oligomer expression are reduced in synaptosomes prepared from ANCL cortex compared with control. In a cultured neuronal cell model, CSPα oligomers are short lived. The results of this study indicate that the Leu116∆ mutation leads to elevated BKα subunit levels in human cortex and extend our initial work in rodent models demonstrating the modulation of BKα subunit levels by the same CSPα mutation. While the precise sequence of pathogenic events still remains to be elucidated, our findings suggest that dysregulation of BK channels may contribute to neurodegeneration in ANCL. PMID:25905915

  2. Polyomavirus inactivation - a review.

    PubMed

    Nims, Raymond W; Plavsic, Mark

    2013-03-01

    Polyomavirus inactivation has been studied since the 1950s when it became apparent that certain polio vaccines were contaminated with SV40. Relatively high temperatures (≥70 °C) are required to effect thermal inactivation of the polyomaviruses. The chemical inactivants that are effective (β-propiolactone, ethanol, sodium hydroxide, and formaldehyde) are those that have displayed efficacy for other small, non-enveloped viruses, such as the circoviruses. Low pH inactivation can be effective, especially at pH at or below 3 and at higher temperatures. Polyomaviruses are more resistant to UV-C irradiation than are other small non-enveloped viruses such as the parvoviruses and caliciviruses. The efficacy of photodynamic inactivation of polyomaviruses is very much dye-dependent, with toluidine blue, acridine orange, and methylene blue dyes being effective photosensitizers. Ionizing radiation can be effective, depending on the conditions employed and the inactivation matrix. Inactivation of the oncogenic properties of the polyomaviruses may require higher doses of inactivant than those required to inactivate infectivity. While the polyomaviruses are considered to be highly resistant to inactivation, the degree of resistance is dependent upon the specific approach under consideration. For certain approaches, such as UV-C and gamma-irradiation, the polyomaviruses appear to be more resistant than other small non-enveloped viruses. Copyright © 2012 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  3. Natural History of Polyomaviruses in Men: The HPV Infection in Men (HIM) Study

    PubMed Central

    Hampras, Shalaka S.; Giuliano, Anna R.; Lin, Hui-Yi; Fisher, Kate J.; Abrahamsen, Martha E.; McKay-Chopin, Sandrine; Gheit, Tarik; Tommasino, Massimo; Rollison, Dana E.

    2015-01-01

    Background. Several new polyomaviruses have been discovered in the last decade, including Merkel cell polyomavirus (MCPyV). Little is known about the natural history of the more recently discovered polyomaviruses. We estimated the incidence, prevalence, and persistence of 9 polyomaviruses (MCPyV, BK polyomavirus, KI polyomavirus, JC polyomavirus, WU polyomavirus, Human polyomavirus 6 [HPyV6], HPyV7, HPyV9, and Trichodysplasia spinulosa–associated polyomavirus) and examined factors associated with MCPyV infection in a prospective cohort of 209 men initially enrolled in the HPV Infection in Men (HIM) study. Methods. Participants enrolled at the US site of the HIM study were recruited into a substudy of cutaneous viral infections and followed for a median of 12.6 months. Eyebrow hair and normal skin swab specimens were obtained at each study visit, and the viral DNA load was measured using multiplex polymerase chain reaction. Results. MCPyV infection showed the highest prevalence (65.1% of normal skin swab specimens and 30.6% of eyebrow hair specimens), incidence (81.7 cases per 1000 person-months among normal skin swab specimens, and 24.1 cases per 1000 person-months among eyebrow hair specimens), and persistence (85.8% of normal skin swab specimens and 58.9% of eyebrow hair specimens) among all polyomaviruses examined. Age of >44 years (odds ratio [OR], 2.11; 95% confidence interval [CI], 1.03–4.33) and Hispanic race (OR, 2.64; 95% CI, 1.01–6.88) were associated with an increased prevalence of MCPyV infection in eyebrow hair and normal skin swab specimens, respectively. Conclusion. MCPyV infection is highly prevalent in adults, with age and race being predisposing factors. PMID:25387582

  4. Development of GoSlo-SR-5-69, a potent activator of large conductance Ca2+-activated K+ (BK) channels.

    PubMed

    Roy, Subhrangsu; Large, Roddy J; Akande, Adebola Morayo; Kshatri, Aravind; Webb, Tim I; Domene, Carmen; Sergeant, Gerard P; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

    2014-03-21

    We have designed, synthesised and characterised the effects of a number of novel anthraquinone derivatives and assessed their effects on large conductance, Ca(2+) activated K(+) (BK) channels recorded from rabbit bladder smooth muscle cells using the excised, inside/out configuration of the patch clamp technique. These compounds are members of the GoSlo-SR family of compounds, which potently open BK channels and shift the voltage required for half maximal activation (V1/2) negatively. The efficacy of the anilinoanthraquinone derivatives was enhanced when the size of ring D was increased, since the cyclopentane and cyclohexane derivatives shifted the V1/2, by -24 ± 6 mV and -54 ± 8 mV, respectively, whereas the cycloheptane and cyclooctane derivatives shifted the V1/2 by -61 ± 6 mV and -106 ± 6 mV. To examine if a combination of hydrophobicity and steric bulking of this region further enhanced their ability to open BK channels, we synthesised a number of naphthalene and tetrahydro-naphthalene derivatives. The tetrahydro-2-naphthalene derivative GoSlo-SR-5-69 was the most potent and efficacious of the series since it was able to shift the activation V1/2 by greater than -100 mV when applied at a concentration of 1 μM and had an EC50 of 251 nM, making it one of the most potent and efficacious BK channel openers synthesised to date.

  5. Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing.

    PubMed

    Chen, Lie; Tian, Lijun; MacDonald, Stephen H-F; McClafferty, Heather; Hammond, Martin S L; Huibant, Jean-Marc; Ruth, Peter; Knaus, Hans-Guenther; Shipston, Michael J

    2005-09-30

    The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.

  6. First epidemiologic study in Argentina of the prevalence of BK viruria in kidney transplant patients.

    PubMed

    Schiavelli, R; Bonaventura, R; Rial, M C; Petrone, H; Pujol, G Soler; Gaite, L J; Acosta, M; Gutierrez, A; Acosta, F; Valdez, G; Raffaele, P; Chanta, G; Perez, M; Potes, L; Suso, E; Cremades, G; Ibañez, J; Imperiali, N; Luxardo, R; Castellanos, M; Maggiora, E; Carreño, C Agost; Cobos, M; Marinic, K; Sinchi, J L; Otero, A B; Freire, M C

    2014-11-01

    The worldwide seroprevalence of human BK polyomavirus (BKV) in adults is 80%. About 10%-60% of renal transplant recipients experience BKV infection, nephropathy of the graft may occur in 5% of the cases, and up to 45% lose the graft. The aim of this work was to describe the prevalence of BK viruria during the 1st year after transplantation. An epidemiologic multicenter cross-sectional study was carried out in consecutive patients at each site with kidney transplantation from August 2011 to July 2012. Clinically significant viruria was defined as >10(7) copies/mL. Viral DNA was extracted with the use of silica columns. Quantification was performed with the use of real-time polymerase chain reaction with primers that amplify a fragment of the large T-antigen gene and with a specific Taqman-MGB probe for BKV. For each assay, a standard curve with a quantified plasmid was included. Of 402 renal transplant recipients at 18 renal transplant sites, we analyzed 382; median age was 46.33 years, and 46.40% were female. The median of the temporal distribution for urine samples was 153 days. BK virus was detected in 50/382 samples (13%), 18 with values >10(7) copies/mL (4.7%). The median of the distribution of positive values was 123 days and the highest frequency of positive values was in months 3-7. The conditions of recipient older than 34 years and donor older than 41 years were the only ones that showed statistically significant association with BK viruria. No association with any specific immunosuppressive drug was observed. This is the first multicenter study conducted in Argentina to determine the prevalence of BK viruria in renal transplant recipients. Because of the growing number of the population susceptible to this infection, it is important to register and describe data about its epidemiology and associated risk factors.

  7. BK Virus in Recipients of Kidney Transplants.

    PubMed

    Hendrix, Kelly M

    2014-01-01

    Since its discovery in 1971, the BK virus, a human polyomavirus, has emerged as a significant cause of renal dysfunction and transplant graft loss in kidney transplant recipients. Improved screening methods have been effective in assisting in the early identification of the virus, and thus, prompt intervention to prevent the progression of the disease. Treatment options for the virus are limited; therefore, lowering immunosuppressive medications should be considered the first line of treatment. Current adjunctive therapies are not guaranteed to control the viral activity and may have limited therapeutic value.

  8. Review on the Relationship between Human Polyomaviruses-Associated Tumors and Host Immune System

    PubMed Central

    Delbue, Serena; Comar, Manola; Ferrante, Pasquale

    2012-01-01

    The polyomaviruses are small DNA viruses that can establish latency in the human host. The name polyomavirus is derived from the Greek roots poly-, which means “many,” and -oma, which means “tumours.” These viruses were originally isolated in mouse (mPyV) and in monkey (SV40). In 1971, the first human polyomaviruses BK and JC were isolated and subsequently demonstrated to be ubiquitous in the human population. To date, at least nine members of the Polyomaviridae family have been identified, some of them playing an etiological role in malignancies in immunosuppressed patients. Here, we describe the biology of human polyomaviruses, their nonmalignant and malignant potentials ability, and their relationship with the host immune response. PMID:22489251

  9. Detection and characterization of a novel polyomavirus in wild rodents.

    PubMed

    Orba, Yasuko; Kobayashi, Shintaro; Nakamura, Ichiro; Ishii, Akihiro; Hang'ombe, Bernard M; Mweene, Aaron S; Thomas, Yuka; Kimura, Takashi; Sawa, Hirofumi

    2011-04-01

    To investigate polyomavirus infection in wild rodents, we analysed DNA samples from the spleens of 100 wild rodents from Zambia using a broad-spectrum PCR-based assay. A previously unknown polyomavirus genome was identified in a sample from a multimammate mouse (Mastomys species) and the entire viral genome of 4899 bp was subsequently sequenced. This viral genome contained potential ORFs for the capsid proteins, VP1, VP2 and VP3, and early proteins, small t antigen and large T antigen. Phylogenetic analysis showed that it was a novel member of the family Polyomaviridae, and thus the virus was tentatively named mastomys polyomavirus. After transfection of the viral genome into several mammalian cell lines, transient expression of the VP1 and large T antigen proteins was confirmed by immunoblotting and immunocytochemical analyses. Comparison of large T antigen function in mastomys polyomavirus with that in rhesus monkey polyomavirus SV40 and human polyomavirus JC virus revealed that the large T antigen from mastomys polyomavirus interacted with the tumour suppressor protein pRb, but not with p53.

  10. BK Virus Nephropathy in Heart Transplant Recipients.

    PubMed

    Joseph, Alin; Pilichowska, Monika; Boucher, Helen; Kiernan, Michael; DeNofrio, David; Inker, Lesley A

    2015-06-01

    Polyomavirus-associated nephropathy (PVAN) has become an important cause of kidney failure in kidney transplant recipients. PVAN is reported to affect 1% to 7% of kidney transplant recipients, leading to premature transplant loss in approximately 30% to 50% of diagnosed cases. PVAN occurring in the native kidneys of solid-organ transplant recipients other than kidney only recently has been noted. We report 2 cases of PVAN in heart transplant recipients, which brings the total of reported cases to 7. We briefly review the literature on the hypothesized causes of PVAN in kidney transplant recipients and comment on whether these same mechanisms also may cause PVAN in other solid-organ transplant recipients. PVAN should be considered in the differential diagnosis when evaluating worsening kidney function. BK viremia surveillance studies of nonkidney solid-organ recipients should be conducted to provide data to assist the transplantation community in deciding whether regular monitoring of nonkidney transplant recipients for BK viremia is indicated.

  11. Inhibitory Interactions between BK and JC Virus among Kidney Transplant Recipients

    PubMed Central

    Cheng, Xingxing S.; Bohl, Daniel L.; Storch, Gregory A.; Ryschkewitsch, Caroline; Gaudreault-Keener, Monique; Major, Eugene O.; Randhawa, Parmjeet; Hardinger, Karen L.

    2011-01-01

    BK and JC polyomaviruses can reactivate after transplantation, causing renal dysfunction and graft loss. The incidence of JC reactivation after renal transplant is not well understood. Here, we characterized JC reactivation using samples collected during the first year after transplantation from 200 kidney recipients. We detected BK and JC viruses in the urine of 35 and 16% of transplant recipients, respectively. The median viral load in the urine was 400 times higher for BK virus than JC virus. The presence of BK viruria made concurrent JC viruria less likely: JC viruria was detected in 22% of non-BK viruric recipients compared with 4% of BK viruric recipients (P = 0.001). The co-detection rate was 1.5%, which is less than the expected 5.6% if reactivation of each virus was independent (P = 0.001). We did not observe JC viremia, JC nephropathy, or progressive multifocal leukoencephalopathy. The onset of JC viruria was associated with donor, but not recipient, JC-specific antibody in a titer-dependent fashion and inversely associated with donor and recipient BK-specific antibody. Donor and recipient JC seropositivity did not predict BK viruria or viremia. In conclusion, among renal transplant recipients, infection with one polyomavirus inversely associates with infection with the other. PMID:21511831

  12. An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel

    PubMed Central

    Bukiya, Anna N.; Kuntamallappanavar, Guruprasad; Edwards, Justin; Singh, Aditya K.; Shivakumar, Bangalore; Dopico, Alex M.

    2014-01-01

    Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol–BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel’s physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function. PMID:24927535

  13. An alcohol-sensing site in the calcium- and voltage-gated, large conductance potassium (BK) channel.

    PubMed

    Bukiya, Anna N; Kuntamallappanavar, Guruprasad; Edwards, Justin; Singh, Aditya K; Shivakumar, Bangalore; Dopico, Alex M

    2014-06-24

    Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol-BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel's physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.

  14. A Retrograde Trafficking Inhibitor of Ricin and Shiga-Like Toxins Inhibits Infection of Cells by Human and Monkey Polyomaviruses

    PubMed Central

    Nelson, Christian D. S.; Carney, Dan W.; Derdowski, Aaron; Lipovsky, Alex; Gee, Gretchen V.; O’Hara, Bethany; Williard, Paul; DiMaio, Daniel; Sello, Jason K.; Atwood, Walter J.

    2013-01-01

    ABSTRACT Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2cycl, an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2cycl inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2cycl likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2cycl as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. PMID:24222489

  15. A prospective study of human polyomavirus infection in pregnancy.

    PubMed

    Coleman, D V; Wolfendale, M R; Daniel, R A; Dhanjal, N K; Gardner, S D; Gibson, P E; Field, A M

    1980-07-01

    Urine samples from 1,235 pregnant women were examined by light microscopy for cytologic evidence of virus infection. Smears of urine sediment from 40 women (3.2%) were observed to contain inclusion-bearing cells; polyomavirus infection was confirmed by virologic methods in 24 (60%). A polyomavirus was isolated from 12 women. Five isolates were identified as JC virus and one as BK virus. Another isolate designated AS virus appeared to be unique. Serologic studies on the 40 women were consistent with a high frequency of reactivation of JC virus, and virus excretion was related to gestation. The evidence suggests that selective excretion of JC virus may occur in pregnancy. Among 390 pregnant women without inclusion-bearing cells in their urine, 78 (20%) had a high or rising titer of serum antibody to JC or BK virus or both, a result suggesting virus reactivation, but virus excretion was not detected. In contrast to other reports, no evidence was found for transmission of BK virus to the fetus.

  16. Gamma interferon controls mouse polyomavirus infection in vivo.

    PubMed

    Wilson, Jarad J; Lin, Eugene; Pack, Christopher D; Frost, Elizabeth L; Hadley, Annette; Swimm, Alyson I; Wang, Jun; Dong, Ying; Breeden, Cynthia P; Kalman, Daniel; Newell, Kenneth A; Lukacher, Aron E

    2011-10-01

    Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.

  17. Complete Sequence of the Smallest Polyomavirus Genome, Giant Guitarfish (Rhynchobatus djiddensis) Polyomavirus 1

    PubMed Central

    Dill, Jennifer A.

    2016-01-01

    Polyomaviruses are known to infect mammals and birds. Deep sequencing and metagenomic analysis identified the first polyomavirus from a cartilaginous fish, the giant guitarfish (Rhynchobatus djiddensis). Giant guitarfish polyomavirus 1 (GfPyV1) has typical polyomavirus genome organization, but is the smallest polyomavirus genome (3.96 kb) described to date. PMID:27198025

  18. Complete Sequence of the Smallest Polyomavirus Genome, Giant Guitarfish (Rhynchobatus djiddensis) Polyomavirus 1.

    PubMed

    Dill, Jennifer A; Ng, Terry F F; Camus, Alvin C

    2016-05-19

    Polyomaviruses are known to infect mammals and birds. Deep sequencing and metagenomic analysis identified the first polyomavirus from a cartilaginous fish, the giant guitarfish (Rhynchobatus djiddensis). Giant guitarfish polyomavirus 1 (GfPyV1) has typical polyomavirus genome organization, but is the smallest polyomavirus genome (3.96 kb) described to date. Copyright © 2016 Dill et al.

  19. BK-VP3 as a new target of cellular immunity in BK virus infection.

    PubMed

    Mueller, Karin; Schachtner, Thomas; Sattler, Arne; Meier, Sarah; Friedrich, Peter; Trydzenskaya, Hanna; Hinrichs, Carl; Trappe, Ralf; Thiel, Andreas; Reinke, Petra; Babel, Nina

    2011-01-15

    Polyomavirus BK virus (BKV) infection represents a serious complication leading to BKV-associated nephropathy (BKVAN) and subsequent kidney graft loss in up to 10% of transplant patients. Cellular immunity is known to play a crucial role in the control of BKV replication. However, the knowledge on the BKV-T-cell response is limited: only two (VP1 and large T antigen) of six known BKV proteins were evaluated for their antigenicity so far. By using 10-color flow cytometry and newly created overlapping peptide pools of five BKV antigens (VP1, VP2, VP3, large T antigen, and small t antigen), we performed cross-sectional phenotypic and functional analysis of BKV-specific T cells in kidney transplant patients with a history of BKVAN. Patients with clinically unapparent BKV infection (history of transient/no BKV reactivation) were used as control group. Our data demonstrate for the first time the antigenic properties of all five evaluated proteins with VP3 as a new important target of cellular immunity. Further, we found a correlation between the severity of the previous BKV infection and the magnitude of memory CD4+ T-cell response. Thus, compared with the control group, patients with a history of BKVAN demonstrated significantly higher frequencies of interferon-γ- and interleukin-2-producing effector memory CD4+ T cells. In the control group, more patients with detectable interferon-γ+/interleukin-2+/tumor necrosis factor+ triple producers were found, suggesting possibly a protective function of these multifunctional T cells. In conclusion, our study results suggest an implementation of new targets for monitoring of BKV immunity. Further studies are required to evaluate the protective function of the found BKV-specific T-cell subsets.

  20. Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

    NASA Technical Reports Server (NTRS)

    Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

    2002-01-01

    Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

  1. Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

    NASA Technical Reports Server (NTRS)

    Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

    2002-01-01

    Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

  2. Successful pregnancy in renal transplant recipient with previous known polyomavirus nephropathy.

    PubMed

    Midtvedt, Karsten; Bjorang, Ola; Letting, Anne-Sofie

    2007-01-01

    Pregnancy after renal transplantation has become increasingly common. Studies in non-immunocompromised patients have shown that pregnant women have increased susceptibility to infection or reactivation of latent virus such as BK virus. To what extent a renal transplant recipient is at risk for reactivation of polyoma virus during pregnancy remains unknown. We hereby report successful pregnancy outcome in a renal transplant recipient with a known history of BK virus nephropathy treated with cidofovir i.v. To our knowledge, this is the first published experience with a successful pregnancy in renal transplant recipients with known history of polyomavirus-associated nephropathy.

  3. BK Channels in the Central Nervous System

    PubMed Central

    Contet, C.; Goulding, S. P.; Kuljis, D. A.; Barth, A. L.

    2016-01-01

    Large conductance Ca2+- and voltage-activated K+ (BK) channels are widely distributed in the postnatal central nervous system (CNS). BK channels play a pleiotropic role in regulating the activity of brain and spinal cord neural circuits by providing a negative feedback mechanism for local increases in intracellular Ca2+ concentrations. In neurons, they regulate the timing and duration of K+ influx such that they can either increase or decrease firing depending on the cellular context, and they can suppress neurotransmitter release from presynaptic terminals. In addition, BK channels located in astrocytes and arterial myocytes modulate cerebral blood flow. Not surprisingly, both loss and gain of BK channel function have been associated with CNS disorders such as epilepsy, ataxia, mental retardation, and chronic pain. On the other hand, the neuroprotective role played by BK channels in a number of pathological situations could potentially be leveraged to correct neurological dysfunction. PMID:27238267

  4. BK channel activators and their therapeutic perspectives

    PubMed Central

    Bentzen, Bo H.; Olesen, Søren-Peter; Rønn, Lars C. B.; Grunnet, Morten

    2014-01-01

    The large conductance calcium- and voltage-activated K+ channel (KCa1.1, BK, MaxiK) is ubiquitously expressed in the body, and holds the ability to integrate changes in intracellular calcium and membrane potential. This makes the BK channel an important negative feedback system linking increases in intracellular calcium to outward hyperpolarizing potassium currents. Consequently, the channel has many important physiological roles including regulation of smooth muscle tone, neurotransmitter release and neuronal excitability. Additionally, cardioprotective roles have been revealed in recent years. After a short introduction to the structure, function and regulation of BK channels, we review the small organic molecules activating BK channels and how these tool compounds have helped delineate the roles of BK channels in health and disease. PMID:25346695

  5. Human polyomavirus 9 infection in kidney transplant patients.

    PubMed

    van der Meijden, Els; Wunderink, Herman F; van der Blij-de Brouwer, Caroline S; Zaaijer, Hans L; Rotmans, Joris I; Bavinck, Jan Nico Bouwes; Feltkamp, Mariet C W

    2014-06-01

    Several human polyomaviruses of unknown prevalence and pathogenicity have been identified, including human polyomavirus 9 (HPyV9). To determine rates of HPyV9 infection among immunosuppressed patients, we screened serum samples from 101 kidney transplant patients in the Netherlands for HPyV9 DNA and seroreactivity. A total of 21 patients had positive results for HPyV9 DNA; positivity rates peaked at 3 months after transplantation, but the highest viral loads were measured just after transplantation. During 18 months of follow-up, HPyV9 seroprevalence increased from 33% to 46% among transplant patients; seroprevalence remained stable at ≈30% in a control group of healthy blood donors in whom no HPyV9 DNA was detected. Further analysis revealed an association between detection of HPyV9 and detection of BK polyomavirus but not of cytomegalovirus. Our data indicate that HPyV9 infection is frequent in kidney transplant patients, but the nature of infection-endogenous or donor-derived-and pathogenic potential of this virus remain unknown.

  6. BK virus nephropathy: a pediatric nephrologist's perspective.

    PubMed

    Hymes, Leonard

    2010-01-01

    Polyomavirus (BK)-associated nephropathy (BKVN) is now recognized as significant problem in pediatric renal transplants that may lead to progressive allograft dysfunction. BKVN was first recognized in 1999 in adult renal transplant recipients, and most data have been obtained from this patient population. Today, there is an increasing number of publications pertaining to children with BKVN that allows for a selective analysis of the pediatric population. Most early pediatric publications were predominantly cases reports. However, several retrospective and prospective studies are now available that provide important insights with respect to the incidence of BKVN in the pediatric transplant population, the efficacy of treatment strategies, and the risk factors for developing BKVN. This review analyzes several of the most significant studies that address these issues.

  7. Genotypic diversity of polyomaviruses circulating among kidney transplant recipients in Kuwait.

    PubMed

    Chehadeh, Wassim; Nampoory, Mangalathillam Raman

    2013-09-01

    BK virus (BKV) and JC virus (JCV) are human polyomaviruses that cause asymptomatic latent infections. Under immunosuppression, BKV-associated nephropathy has been documented in Kuwait and elsewhere. Even though different BKV and JCV genotypes with distinct geographical distribution have been described, the genotype of polyomavirus detected in Kuwait is still unknown. The aim of this study was to determine the genotypes of BKV and JCV detected in renal transplant recipients. The detection of polyomavirus DNA was carried out in serum and urine samples of 200 post-transplant recipients during a 1-year follow-up period. Fifty-one (25.5%) post-transplant recipients were tested positive for polyomavirus DNA by semi-nested PCR. JCV DNA could be detected in 29 (57%) patients, and BKV DNA in 22 (43%) patients. In two renal transplant recipients, both BKV and JCV were detected. According to the Bayesian phylogenetic analysis of polyomavirus VP1 sequences, the majority of detected BKV sequences were most closely related to genotypes I and IV, whereas the majority of JCV sequences were most closely related to genotype 3. Polyomavirus VP1 sequences showed strong stability for up to 12 months in most patients; however, in one patient, an amino acid substitution in the BKV VP1 protein was identified over time. The results suggest a close relationship of BKV sequences with the Asian and European strains, and of JCV sequences with the African strains. Long follow-up studies are needed to investigate the association of polyomavirus polymorphism or genotypic shift with the development of nephropathy.

  8. Structure-activity relationships of a novel group of large-conductance Ca(2+)-activated K(+) (BK) channel modulators: the GoSlo-SR family.

    PubMed

    Roy, Subhrangsu; Morayo Akande, Adebola; Large, Roddy J; Webb, Tim I; Camarasu, Costin; Sergeant, Gerard P; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

    2012-10-01

    Opening up ion channels: We synthesised a series of anthraquinone analogues, called the GoSlo-SR family. Their effects on bladder smooth muscle BK channels were examined and, as shown, shifted voltage dependent activation >-100 mV (at 10 μM). They were more efficacious than NS11021 and could provide a new scaffold for the design of efficacious BK openers.

  9. Molecular mechanism of pharmacological activation of BK channels

    PubMed Central

    Gessner, Guido; Cui, Yong-Mei; Otani, Yuko; Ohwada, Tomohiko; Soom, Malle; Hoshi, Toshinori; Heinemann, Stefan H.

    2012-01-01

    Large-conductance voltage- and Ca2+-activated K+ (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain—the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate. PMID:22331907

  10. Butcherbird polyomavirus isolated from a grey butcherbird (Cracticus torquatus) in Queensland, Australia.

    PubMed

    Bennett, Mark D; Gillett, Amber

    2014-01-31

    A novel avian polyomavirus was detected in peri-ocular skin lesions collected from a grey butcherbird (Cracticus torquatus), using a combination of multiply primed rolling circle amplification, nested PCR and long range PCR. The sequence of Butcherbird polyomavirus was determined by combining next generation sequencing and primer walking techniques. The circular double-stranded DNA genome of Butcherbird polyomavirus consisted of 5084 bp, and encoded six open reading frames (ORF-X, VP2, VP3, VP1, small T-antigen and large T-antigen). Phylogenetic analysis placed it amongst other members of the genus Avipolyomavirus, most closely related to Crow polyomavirus. Next generation sequencing enabled the detection of DNA fragments similar to, but distinct from, Canarypox virus within the same lesion from which Butcherbird polyomavirus was amplified, thus confirming an avipolyomavirus-avipoxvirus co-infection in the peri-ocular skin lesions of this grey butcherbird.

  11. Polyomavirus infections and its clinical relevance in cancer patients: A Prospective Study.

    PubMed

    Loutfy, Samah A; Moneer, Manar M; Salem, Salem E; El-Moniem Abada, Emad A; El-Moniem Ahmed, Entsar A; Ibrahim, Lamiaa H; Mohamed, El-Chaimaa B

    BK and JC polyomaviruses (PyV) have been demonstrated to be associated with the pathogenesis of various human cancers. We aimed to investigate the impact of BK and JC polyomavirus infections on several clinical parameters in different human cancers. A total of 150 cancer patients were included in the study (51 patients with solid tumors, 48 patients with lymphomas and 51 patients with leukemias). Amplification of PyV DNA was performed using a semi-nested version of Polymerase chain reaction targeting the T genomic region of PyV. The polyomavirus load was determined using real-time PCR assay. The clinical data were collected. Polyomavirus DNA could be detected in 84 (56%) of 150 of all cancerous patients. The solid tumors had the lowest proportion of JCV (6 (11.8%) of 51), whereas had the highest proportion of JCV (200copies/μl). JCV was more frequent among NHL patients (30%) and absent in HL patients (0%). During follow-up, PyV positivity decreased significantly (p=0.004) in lymphoma patients (n=28). Although PyV positivity decreased significantly from 39% to 7% in 28 of 48 lymphoma patients after treatment, it significantly persisted in leukemic patients after treatment (from 22% to 38%). JC was more frequent among leukemic patients with leukopenia. The presence of JC polyomavirus was more frequent among leukemic patients without any significant impact on their overall survival. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

  12. Characterization of California sea lion polyomavirus 1: expansion of the known host range of the Polyomaviridae to Carnivora.

    PubMed

    Wellehan, James F X; Rivera, Rebecca; Archer, Linda L; Benham, Celeste; Muller, Jennifer K; Colegrove, Kathleen M; Gulland, Frances M D; St Leger, Judy A; Venn-Watson, Stephanie K; Nollens, Hendrik H

    2011-07-01

    The genome of a novel polyomavirus first identified in a proliferative tongue lesion of a California sea lion (Zalophus californianus) is reported. This is only the third described polyomavirus of laurasiatherian mammals, is the first of the three associated with a lesion, and is the first known polyomavirus of a host in the order Carnivora. Predicted large T, small t, VP1, VP2, and VP3 genes were identified based on homology to proteins of known polyomaviruses, and a putative agnoprotein was identified based upon its location in the genome. Phylogenetic analysis of the predicted late region proteins found that the laurasiatherian polyomaviruses, together with Squirrel monkey polyomavirus and Murine pneumotropic virus, form a monophyletic clade. Phylogenetic analysis of the early region was more ambiguous. The noncoding control region of California sea lion polyomavirus 1 is unusual in that only two apparent large T binding sites are present; this is less than any other known polyomavirus. The VP1 of this virus has an unusually long carboxy-terminal region. A quantitative polymerase chain reaction was developed and utilized on various samples from 79 additional animals from either managed or wild stranded California sea lion populations, and California sea lion polyomavirus 1 infection was found in 24% of stranded animals. Sequence of additional samples identified four sites of variation in the t antigens, three of which resulted in predicted coding changes.

  13. Discovery of STL polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique T antigen by alternative splicing.

    PubMed

    Lim, Efrem S; Reyes, Alejandro; Antonio, Martin; Saha, Debasish; Ikumapayi, Usman N; Adeyemi, Mitchell; Stine, O Colin; Skelton, Rebecca; Brennan, Daniel C; Mkakosya, Rajhab S; Manary, Mark J; Gordon, Jeffrey I; Wang, David

    2013-02-20

    The family Polyomaviridae is comprised of circular double-stranded DNA viruses, several of which are associated with diseases, including cancer, in immunocompromised patients. Here we describe a novel polyomavirus recovered from the fecal microbiota of a child in Malawi, provisionally named STL polyomavirus (STLPyV). We detected STLPyV in clinical stool specimens from USA and The Gambia at up to 1% frequency. Complete genome comparisons of two STLPyV strains demonstrated 5.2% nucleotide divergence. Alternative splicing of the STLPyV early region yielded a unique form of T antigen, which we named 229T, in addition to the expected large and small T antigens. STLPyV has a mosaic genome and shares an ancestral recombinant origin with MWPyV. The discovery of STLPyV highlights a novel alternative splicing strategy and advances our understanding of the complex evolutionary history of polyomaviruses.

  14. Large $N$ approach to kaon decays and mixing 28 years later: $$\\Delta I = 1/2$$ rule, $$\\hat B_K$$ and $$\\Delta M_K$$

    DOE PAGES

    Buras, Andrzej J.; Gérard, Jean -Marc; Bardeen, William A.

    2014-05-20

    We review and update our results for K → π π decays and K⁰- K¯⁰ mixing obtained by us in the 1980s within an approach based on the dual representation of QCD as a theory of weakly interacting mesons for large N colours. In our analytic approach the dynamics behind the enhancement of ReA0 and suppression of ReA2, the so-called ΔI = 1/2 rule for K → π π decays, has a simple structure: the usual octet enhancement through quark-gluon renormalization group evolution down to the scales O(1 GeV) is continued as a meson evolution down to zero momentum scalesmore » at which the factorization of hadronic matrix elements is at work. The inclusion of lowest-lying vector meson contributions in addition to the pseudoscalar ones and of Wilson coefficients in a momentum scheme improves significantly the matching between quark-gluon and meson evolutions. In particular, the anomalous dimension matrix governing the meson evolution exhibits the structure of the known anomalous dimension matrix in the quark-gluon evolution. The recent results on ReA2 and ReA0 from the RBC-UKQC collaboration give support for our approach. In particular, the signs of the two main contractions found numerically by these authors follow uniquely from our analytic approach. At NLO in 1/N we obtain R = ReA0/ReA2= 16.0±1.5 which amounts to an order of magnitude enhancement over the strict large N limit value √2. QCD penguins contribute at 15% level to this result. We also find B^K = 0.73± 0.02, with the smallness of 1/N corrections to the large N value B^K = 3/4 resulting within our approach from an approximate cancellation between pseudoscalar and vector meson one-loop contributions. We summarize the status of ΔMK in this approach.« less

  15. Human polyomavirus related to African green monkey lymphotropic polyomavirus.

    PubMed

    Sauvage, Virginie; Foulongne, Vincent; Cheval, Justine; Ar Gouilh, Meriadeg; Pariente, Kevin; Dereure, Olivier; Manuguerra, Jean Claude; Richardson, Jennifer; Lecuit, Marc; Burguière, Ana; Caro, Valérie; Eloit, Marc

    2011-08-01

    While studying the virome of the skin surface of a patient with a Merkel cell carcinoma (MCC) by using unbiased, high-throughput sequencing, we identified a human polyomavirus nearly identical to human polyomavirus 9, a virus recently reported in blood and urine of renal transplantion patients and closely related to the African green monkey lymphotropic polyomavirus. Specific PCR analysis further identified this virus in 2/8 patients with MCC but in only 1/111 controls without MCC. This virus was shed for ≥20 months by the MCC index patient and was on the skin of the spouse of the index patient. These results provide information on the viral ecology of human skin and raise new questions regarding the pathology of virus-associated skin disorders.

  16. Ca2+-dependent inactivation of large conductance Ca2+-activated K+ (BK) channels in rat hippocampal neurones produced by pore block from an associated particle

    PubMed Central

    Hicks, Gareth A; Marrion, Neil V

    1998-01-01

    Recordings of the activity of the large conductance Ca2+-activated K+ (BK) channel from over 90 % of inside-out patches excised from acutely dissociated hippocampal CA1 neurones revealed an inactivation process dependent upon the presence of at least 1 μM intracellular Ca2+. Inactivation was characterized by a sudden switch from sustained high open probability (Po) long open time behaviour to extremely low Po, short open time channel activity. The low Po state (mean Po, 0.001) consisted of very short openings (time constant (τ), ≈0.14 ms) and rare longer duration openings (τ, ≈3.0 ms). Channel inactivation occurred with a highly variable time course being observed either prior to or immediately upon patch excision, or after up to 2 min of inside-out recording. Inactivation persisted whilst recording conditions were constant. Inactivation was reversed by membrane hyperpolarization, the rate of recovery increasing with further hyperpolarization and higher extracellular K+. Inactivation was also reversed when the intracellular Ca2+ concentration was lowered to 100 nM and was permanently removed by application of trypsin to the inner patch surface. In addition, inactivation was perturbed by application of either tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face. During inactivation, channel Po was greater at hyperpolarized rather than depolarized potentials, which was partly the result of a greater number of longer duration openings. Depolarizing voltage steps (−40 to +40 mV) applied during longer duration openings produced only short duration events at the depolarized potential, yielding a transient ensemble average current with a rapid decay (τ, ≈3.8 ms). These data suggest that hippocampal BK channels exhibit a Ca2+-dependent inactivation that is proposed to result from block of the channel by an associated particle. The findings that inactivation was removed by trypsin and prolonged by decreasing extracellular potassium

  17. Polyomavirus detection in multiple sclerosis patients under natalizumab therapy: Profile and frequency of urinary shedding.

    PubMed

    Nali, Luiz Henrique; Fink, Maria Cristina; do Olival, Guilherme S; Moraes, Lenira; Callegaro, Dagoberto; Tilbery, Charles Peter; Vidal, Jose Ernesto; Sumita, Laura Masami; de Oliveira, Augusto C Penalva; Romano, Camila M

    2017-03-01

    Patients undergoing Natalizumab (NTZ) therapy are at risk of progressive multifocal leukoencephalopathy (PML). Besides John Cunningham virus (JCV), BK polyomavirus might represent an additional concern for such patients since it can also infect CNS cells. Currently, data regarding the presence of anti-JCV antibodies added to previous immunosuppressive therapy and prolonged NTZ therapy has been used to classify patients at risk of developing PML. Here, we investigated the profile shedding of JCV and BKV in multiple sclerosis (MS) patients during treatment with NTZ. Serial blood and urine samples from 97 MS patients receiving either NTZ or β-interferon were investigated for polyomavirus shedding. While all blood samples tested negative, 36% of the patients shed polyomavirus in the urine in at least one time point. From these, 21.7%, 9.3%, and 5.1% shed JCV, BKV, and both polyomavirus, respectively. No difference was observed between the rates of urinary shedding of patients treated with NTZ (38.9%) and patients treated with other drugs (34.5%), also no PML event was diagnosed during the follow-up. Therefore, urinary shedding might not be interfered by therapy condition. In our study, we also observed 14/27 (52%) of anti-JCV antibodies prevalence, and nearly half of them (42%) did not present any event of urinary shedding during the follow-up. J. Med. Virol. 89:528-534, 2017. © 2016 Wiley Periodicals, Inc.

  18. First detection of papillomaviruses and polyomaviruses in swimming pool waters: unrecognized recreational water-related pathogens?

    PubMed

    La Rosa, G; Della Libera, S; Petricca, S; Iaconelli, M; Briancesco, R; Paradiso, R; Semproni, M; Di Bonito, P; Bonadonna, L

    2015-12-01

    Viral outbreaks associated with swimming pools have been described worldwide. The objective of this study was to examine the extent of viral contamination in indoor and outdoor swimming pools. Pools were examined for the presence of human enteric viruses (adenovirus, norovirus and enterovirus) and nonenteric viruses (papillomavirus and polyomavirus-BK, JC, KI, WU and Merkel cell). Bacteriological parameters were also evaluated. The analysed pool waters met microbiological quality standards. Enteric viruses were not detected. On the other hand, papillomaviruses (HPV8, 12, 23, 25, 120 and unclassified HPVs) and polyomaviruses (JC and Merkel cell polyomaviruses) were detected in 9/14 samples (64%). The number of HPV DNA copies in pool waters, measured by quantitative Real-time PCR, ranged from 1.27E+04 to 1.13E+05/10L. Results show that a variety of nonenteric viruses may be discharged in pool waters by various secretions and excretions from infected individuals or asymptomatic carriers. To the best of our knowledge, this is the first report on human papillomaviruses and polyomaviruses in swimming pools. The likelihood that these viruses can be transmitted by recreational activities deserves to be explored in future studies. © 2015 The Society for Applied Microbiology.

  19. Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

    PubMed Central

    Pastrana, Diana V.; Tolstov, Yanis L.; Becker, Jürgen C.; Moore, Patrick S.; Chang, Yuan; Buck, Christopher B.

    2009-01-01

    Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection. PMID:19750217

  20. Distribution of human polyomaviruses, adenoviruses, and hepatitis E virus in the environment and in a drinking-water treatment plant.

    PubMed

    Albinana-Gimenez, Nestor; Clemente-Casares, Pilar; Bofill-Mas, Silvia; Hundesa, Ayalkibet; Ribas, Ferran; Girones, Rosina

    2006-12-01

    Large numbers of viruses are excreted in human feces and urine, which even at low concentrations may cause illness when ingested. Some of these viruses have not been traditionally monitored in terms of waterborne diseases and are considered emergent viruses, such as hepatitis E virus (HEV) and JC and BK polyomavirus (JCPyV and BKPyV). The high prevalence of human adenoviruses (HAdV) and polyomaviruses, which both show DNA genomes, in sewage from widely divergent areas has suggested the relevance of evaluating these viruses as possible indicators of viral contamination. The concentration of these viruses was analyzed in sewage and river water and after treatment in a drinking-water treatment plant including chlorination, flocculation, ozonation, and granulate active carbon (GAC) filtration. Samples of GAC-filtered water were collected before a second chlorination treatment. The river used as a source of fresh water presented an average concentration of 2.6 x 10(1) JCPyV and 4 x 10(2) HAdV GC (genome copies)/L. A removal of 2 logarithms (99%) of HAdV and JCPyV was observed in the drinking-water treatment plant. All the GAC-filtered water samples studied contained HAdV, with a mean value of 4.3 HAdV GC/L. HEV strains belonging to genotype 3 were frequently detected in low concentrations in urban sewage and in biosolids or sewage containing swine feces but not in the river water samples studied. The detection of viruses by molecular techniques is useful for genetically describe emergent viruses in community wastewaters and water supplies. Quantification of JCPyV and HAdV using quantitative real-time PCR (QPCR) may be useful for evaluating virus removal efficiency in water treatment plants and as an index of the virological quality of water and of the potential presence of human viruses.

  1. Peptide toxins and small-molecule blockers of BK channels

    PubMed Central

    Yu, Mu; Liu, San-ling; Sun, Pei-bei; Pan, Hao; Tian, Chang-lin; Zhang, Long-hua

    2016-01-01

    Large conductance, Ca2+-activated potassium (BK) channels play important roles in the regulation of neuronal excitability and the control of smooth muscle contractions. BK channels can be activated by changes in both the membrane potential and intracellular Ca2+ concentrations. Here, we provide an overview of the structural and pharmacological properties of BK channel blockers. First, the properties of different venom peptide toxins from scorpions and snakes are described, with a focus on their characteristic structural motifs, including their disulfide bond formation pattern, the binding interface between the toxin and BK channel, and the functional consequence of the blockage of BK channels by these toxins. Then, some representative non-peptide blockers of BK channels are also described, including their molecular formula and pharmacological effects on BK channels. The detailed categorization and descriptions of these BK channel blockers will provide mechanistic insights into the blockade of BK channels. The structures of peptide toxins and non-peptide compounds could provide templates for the design of new channel blockers, and facilitate the optimization of lead compounds for further therapeutic applications in neurological disorders or cardiovascular diseases. PMID:26725735

  2. Serological cross-reactivity between Merkel cell polyomavirus and two closely related chimpanzee polyomaviruses.

    PubMed

    Nicol, Jérôme T J; Liais, Etienne; Potier, Romain; Mazzoni, Elisa; Tognon, Mauro; Coursaget, Pierre; Touzé, Antoine

    2014-01-01

    Phylogenetic analyses based on the major capsid protein sequence indicate that Merkel cell polyomavirus (MCPyV) and chimpanzee polyomaviruses (PtvPyV1, PtvPyV2), and similarly Trichodysplasia spinulosa-associated polyomavirus (TSPyV) and the orangutan polyomavirus (OraPyV1) are closely related. The existence of cross-reactivity between these polyomaviruses was therefore investigated. The findings indicated serological identity between the two chimpanzee polyomaviruses investigated and a high level of cross-reactivity with Merkel cell polyomavirus. In contrast, cross-reactivity was not observed between TSPyV and OraPyV1. Furthermore, specific antibodies to chimpanzee polyomaviruses were detected in chimpanzee sera by pre-incubation of sera with the different antigens, but not in human sera.

  3. Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

    PubMed

    Lu, Tong; Jiang, Bin; Wang, Xiao-Li; Lee, Hon-Chi

    2016-09-01

    The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel β1-subunits (BK-β1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-β1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

  4. Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen.

    PubMed

    Banerjee, Arinjay; Rapin, Noreen; Miller, Megan; Griebel, Philip; Zhou, Yan; Munster, Vincent; Misra, Vikram

    2016-11-01

    It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. The Ancient Evolutionary History of Polyomaviruses

    PubMed Central

    Buck, Christopher B.; Van Doorslaer, Koenraad; Peretti, Alberto; Geoghegan, Eileen M.; Tisza, Michael J.; An, Ping; Katz, Joshua P.; Pipas, James M.; McBride, Alison A.; Camus, Alvin C.; McDermott, Alexa J.; Dill, Jennifer A.; Delwart, Eric; Ng, Terry F. F.; Farkas, Kata; Austin, Charlotte; Kraberger, Simona; Davison, William; Pastrana, Diana V.; Varsani, Arvind

    2016-01-01

    Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae. PMID:27093155

  6. The Ancient Evolutionary History of Polyomaviruses.

    PubMed

    Buck, Christopher B; Van Doorslaer, Koenraad; Peretti, Alberto; Geoghegan, Eileen M; Tisza, Michael J; An, Ping; Katz, Joshua P; Pipas, James M; McBride, Alison A; Camus, Alvin C; McDermott, Alexa J; Dill, Jennifer A; Delwart, Eric; Ng, Terry F F; Farkas, Kata; Austin, Charlotte; Kraberger, Simona; Davison, William; Pastrana, Diana V; Varsani, Arvind

    2016-04-01

    Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

  7. Discovery of a New Human Polyomavirus Associated with Trichodysplasia Spinulosa in an Immunocompromized Patient

    PubMed Central

    van der Meijden, Els; Janssens, René W. A.; Lauber, Chris; Bouwes Bavinck, Jan Nico; Gorbalenya, Alexander E.; Feltkamp, Mariet C. W.

    2010-01-01

    The Polyomaviridae constitute a family of small DNA viruses infecting a variety of hosts. In humans, polyomaviruses can cause infections of the central nervous system, urinary tract, skin, and possibly the respiratory tract. Here we report the identification of a new human polyomavirus in plucked facial spines of a heart transplant patient with trichodysplasia spinulosa, a rare skin disease exclusively seen in immunocompromized patients. The trichodysplasia spinulosa-associated polyomavirus (TSV) genome was amplified through rolling-circle amplification and consists of a 5232-nucleotide circular DNA organized similarly to known polyomaviruses. Two putative “early” (small and large T antigen) and three putative “late” (VP1, VP2, VP3) genes were identified. The TSV large T antigen contains several domains (e.g. J-domain) and motifs (e.g. HPDKGG, pRb family-binding, zinc finger) described for other polyomaviruses and potentially involved in cellular transformation. Phylogenetic analysis revealed a close relationship of TSV with the Bornean orangutan polyomavirus and, more distantly, the Merkel cell polyomavirus that is found integrated in Merkel cell carcinomas of the skin. The presence of TSV in the affected patient's skin was confirmed by newly designed quantitative TSV-specific PCR, indicative of a viral load of 105 copies per cell. After topical cidofovir treatment, the lesions largely resolved coinciding with a reduction in TSV load. PCR screening demonstrated a 4% prevalence of TSV in an unrelated group of immunosuppressed transplant recipients without apparent disease. In conclusion, a new human polyomavirus was discovered and identified as the possible cause of trichodysplasia spinulosa in immunocompromized patients. The presence of TSV also in clinically unaffected individuals suggests frequent virus transmission causing subclinical, probably latent infections. Further studies have to reveal the impact of TSV infection in relation to other populations

  8. JC polyomavirus nephropathy confirmed by using an in-house polymerase chain reaction method.

    PubMed

    Querido, S; Jorge, C; Sousa, H; Birne, R; Matias, P; Weigert, A; Adragão, T; Bruges, M; Ramos, S; Santos, M; Paixão, P; Curran, M D; Machado, D

    2015-10-01

    We report the case of an isolated JC virus (JCV) infection, without co-infection by polyoma BK virus (BKV), associated with nephropathy 4 years after kidney transplantation. Clinical suspicion followed the observation of a decrease in estimated glomerular filtration rate (eGFR) and a renal allograft biopsy revealing polyomavirus-associated tubulointerstitial nephritis and positivity for SV40. An in-house real-time polymerase chain reaction assay, targeting the presence of JCV and the absence of BKV in biopsy tissue, confirmed diagnosis. Thirteen months after diagnosis, and following therapeutic measures, eGFR remains stable. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Introducing the Big Knowledge to Use (BK2U) challenge.

    PubMed

    Perl, Yehoshua; Geller, James; Halper, Michael; Ochs, Christopher; Zheng, Ling; Kapusnik-Uner, Joan

    2017-01-01

    The purpose of the Big Data to Knowledge initiative is to develop methods for discovering new knowledge from large amounts of data. However, if the resulting knowledge is so large that it resists comprehension, referred to here as Big Knowledge (BK), how can it be used properly and creatively? We call this secondary challenge, Big Knowledge to Use. Without a high-level mental representation of the kinds of knowledge in a BK knowledgebase, effective or innovative use of the knowledge may be limited. We describe summarization and visualization techniques that capture the big picture of a BK knowledgebase, possibly created from Big Data. In this research, we distinguish between assertion BK and rule-based BK (rule BK) and demonstrate the usefulness of summarization and visualization techniques of assertion BK for clinical phenotyping. As an example, we illustrate how a summary of many intracranial bleeding concepts can improve phenotyping, compared to the traditional approach. We also demonstrate the usefulness of summarization and visualization techniques of rule BK for drug-drug interaction discovery. © 2016 New York Academy of Sciences.

  10. Efficacy of intravenous immunoglobulin in the treatment of persistent BK viremia and BK virus nephropathy in renal transplant recipients.

    PubMed

    Vu, D; Shah, T; Ansari, J; Naraghi, R; Min, D

    2015-03-01

    BK virus-associated nephropathy (BKVN) can cause clinically significant viral infection in renal transplant recipients, leading to allograft dysfunction and loss. The usual management of BKVN involves the reduction of immunosuppression and the addition of leflunomide, quinolones, and cidofovir, but the rate of graft loss remains high. The aim of this study was to assess the impact of treatment with intravenous human immunoglobulin (IVIG) on the outcome of BKVN in renal transplant recipients. Upon diagnosis of BKVN, patients remained on anti-polyomavirus treatment, consisting of the reduction of immunosuppression and the use of leflunomide therapy. Treatment with IVIG was given only to patients who did not respond to 8 weeks of the adjustment of immunosuppression and leflunomide. All 30 patients had persistent BKV viremia and BKVN with their mean BK viral loads higher than the baseline (range, 15,000-2 million copies/mL). Mean peak BK load was 205,314 copies/mL compared with 697 copies/mL after 1 year of follow-up. Twenty-seven patients (90%) had a positive response in clearing viremia. The actuarial patient and graft survival rates after 12 months were 100% and 96.7%, respectively. IVIG administration appeared to be safe and effective in treating BKV viremia and BKVN and preventing graft loss in patients who had inadequate response to immunosuppression reduction and leflunomide therapy. Copyright © 2015. Published by Elsevier Inc.

  11. Bidirectional control of BK channel open probability by CAMKII and PKC in medial vestibular nucleus neurons

    PubMed Central

    van Welie, Ingrid

    2011-01-01

    Large conductance K+ (BK) channels are a key determinant of neuronal excitability. Medial vestibular nucleus (MVN) neurons regulate eye movements to ensure image stabilization during head movement, and changes in their intrinsic excitability may play a critical role in plasticity of the vestibulo-ocular reflex. Plasticity of intrinsic excitability in MVN neurons is mediated by kinases, and BK channels influence excitability, but whether endogenous BK channels are directly modulated by kinases is unknown. Double somatic patch-clamp recordings from MVN neurons revealed large conductance potassium channel openings during spontaneous action potential firing. These channels displayed Ca2+ and voltage dependence in excised patches, identifying them as BK channels. Recording isolated single channel currents at physiological temperature revealed a novel kinase-mediated bidirectional control in the range of voltages over which BK channels are activated. Application of activated Ca2+/calmodulin-dependent kinase II (CAMKII) increased BK channel open probability by shifting the voltage activation range towards more hyperpolarized potentials. An opposite shift in BK channel open probability was revealed by inhibition of phosphatases and was occluded by blockade of protein kinase C (PKC), suggesting that active PKC associated with BK channel complexes in patches was responsible for this effect. Accordingly, direct activation of endogenous PKC by PMA induced a decrease in BK open probability. BK channel activity affects excitability in MVN neurons and bidirectional control of BK channels by CAMKII, and PKC suggests that cellular signaling cascades engaged during plasticity may dynamically control excitability by regulating BK channel open probability. PMID:21307321

  12. Seroprevalence of trichodysplasia spinulosa-associated polyomavirus.

    PubMed

    van der Meijden, Els; Kazem, Siamaque; Burgers, Manda M; Janssens, Rene; Bouwes Bavinck, Jan Nico; de Melker, Hester; Feltkamp, Mariet C W

    2011-08-01

    We identified a new polyomavirus in skin lesions from a patient with trichodysplasia spinulosa (TS). Apart from TS being an extremely rare disease, little is known of its epidemiology. On the basis of knowledge regarding other polyomaviruses, we anticipated that infections with trichodysplasia spinulosa-associated polyomavirus (TSV) occur frequently and become symptomatic only in immunocompromised patients. To investigate this hypothesis, we developed and used a Luminex-based TSV viral protein 1 immunoassay, excluded cross-reactivity with phylogenetically related Merkel cell polyomavirus, and measured TSV seroreactivity. Highest reactivity was found in a TS patient. In 528 healthy persons in the Netherlands, a wide range of seroreactivities was measured and resulted in an overall TSV seroprevalence of 70% (range 10% in small children to 80% in adults). In 80 renal transplant patients, seroprevalence was 89%. Infection with the new TSV polyomavirus is common and occurs primarily at a young age.

  13. Modulation by the BK accessory β4 subunit of phosphorylation-dependent changes in excitability of dentate gyrus granule neurons

    PubMed Central

    Petrik, David; Wang, Bin; Brenner, Robert

    2011-01-01

    BK channels are large conductance calcium- and voltage-activated potassium channels critical for neuronal excitability. Some neurons express so called fast-gated, type I BK channels. Other neurons express BK channels assembled with the accessory β4 subunit conferring slow-gating of type II BK channels. However, it is not clear how protein phosphorylation modulates these two distinct BK channel types. Using β4 knockout mice, we compared fast- or slow-gated BK channels in response to changes in phosphorylation status of hippocampus dentate gyrus granule neurons. We utilized the selective PP2A/PP4 phosphatase inhibitor, Fostriecin, to study changes in action potential shape and firing properties of the neurons. In β4 knockout neurons, Fostriecin increases BK current, speeds BK channel activation, and reduces action potential amplitudes. Fostriecin increases spiking during early components of an action potential train. In contrast, inhibition of BK channels through β4 in wild type neurons or by BK channel inhibitor Paxilline opposes Fostriecin effects. Voltage clamp recordings of neurons reveal that Fostriecin increases both calcium and BK currents. However, Fostriecin does not activate BK α alone channels in transfected HEK293 cells lacking calcium channels. In summary, these results suggest that the fast-gating, type I BK channels lacking β4 can increase neuronal excitability in response to reduced phosphatase activity and activation of calcium channels. By opposing BK channel activation; the β4 subunit plays an important role in moderating firing frequency regardless of changes in phosphorylation status. PMID:21848922

  14. Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

    PubMed Central

    Bennett, Shauna M.; Jiang, Mengxi

    2013-01-01

    BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells. PMID:23740996

  15. Transient versus Persistent BK Viremia and Long-Term Outcomes after Kidney and Kidney–Pancreas Transplantation

    PubMed Central

    Elfadawy, Nissreen; Schold, Jesse D.; Srinivas, Titte R.; Poggio, Emilio; Fatica, Richard; Avery, Robin; Mossad, Sherif B.

    2014-01-01

    Background and objectives The objective was to study the long-term impact of transient versus persistent BK viremia on kidney transplant outcomes. Design, setting, participants, & measurements In total, 609 recipients who underwent kidney transplant from 2007 to 2011 were screened at months 1–12 for the occurrence of polyomavirus BK viremia; 130 patients (21.7%) developed BK viremia during the first year post-transplant. BK viremia patients were classified according to duration of infection (more or less than 3 months), and BK viral loads (more or less than 10,000 copies/ml) were classified as transient low viremia (n=42), transient high viremia (n=18), persistent low viremia (n=23), and persistent high viremia (n=47). All patients were followed a median of 36 (3–66) months. The rates of BK polyomavirus–associated nephropathy, acute rejection, and 1-year graft function were compared with the polyomavirus BK–negative control group. Results Patient and graft survival were not significantly different among the groups. Graft function (creatinine; milligrams per deciliter) at 1 year was significantly worse in the persistent high viremia (1.75±0.6) and transient high viremia (1.85±0.7) groups compared with aviremic controls (1.47±0.4; P=0.01 and P=0.01, respectively). The incidence of BK polyomavirus–associated nephropathy was limited to the persistent high viremia group (1.3%, P<0.001). The transient high viremia (50%) and persistent high viremia (34%) groups showed significantly (P=0.01) increased incidence of acute rejection versus aviremic controls (21.5%), transient low viremia (19%), or persistent low viremia (17.3%) groups. Conclusion Low viral load BK viremia, either transient or persistent, was not associated with long-term transplant outcomes. Persistent high viremia was associated with a greater risk for BK polyomavirus–associated nephropathy and subsequent graft dysfunction. Although transient high viremia was not associated with BK

  16. Genomic characterization of two novel polyomaviruses in Brazilian insectivorous bats.

    PubMed

    de Sales Lima, Francisco Esmaile; Cibulski, Samuel Paulo; Witt, André Alberto; Franco, Ana Cláudia; Roehe, Paulo Michel

    2015-07-01

    Two novel genomes comprising ≈4.9 kb were identified by next-generation sequencing from pooled organs of Tadarida brasiliensis bats. The overall nucleotide sequence identities between the viral genomes characterized here were less than 80% in comparison to other polyomaviruses (PyVs), members of the family Polyomaviridae. The new genomes display the archetypal organization of PyVs, which includes open reading frames for the regulatory proteins small T antigen (STAg) and large T antigen (LTAg), as well as capsid proteins VP1, VP2 and VP3. In addition, an alternate ORF was identified in the early genome region that is conserved in a large monophyletic group of polyomaviruses. Phylogenetic analysis showed similar clustering with group of PyVs detected in Otomops and Chaerephon bats and some species of monkeys. In this study, the genomes of two novel PyVs were detected in bats of a single species, demonstrating that these mammals can harbor genetically diverse polyomaviruses.

  17. Oncogenic Papillomavirus and Polyomavirus in Water Environments: Is There a Potential for Waterborne Transmission?

    PubMed

    Fratini, M; Di Bonito, P; La Rosa, G

    2014-03-01

    Waterborne exposure to human viruses through contact with sewage-contaminated water environments can result in infections associated with a wide range of illnesses. Gastrointestinal symptoms are the most commonly encountered manifestations of waterborne viral illness. Respiratory diseases, neurological diseases and paralysis can also occur. Whether viral infections resulting in health outcomes like cancer might also be transmitted by the waterborne route is unknown. Recently, viruses belonging to two oncogenic groups-Human Papillomaviruses (HPVs) and Human Polyomaviruses (HPyVs)-have been detected in urban sewages worldwide. The latter have also been identified in other water environments. HPVs are epitheliotropic viruses responsible for several diseases of skin and mucosae, from common warts to squamous intraepithelial lesions that can either heal or progress to invasive carcinoma of the cervix, vulva, vagina, penis, anus or oropharynx. Human PyVs infect different tissues and organs, causing infections that are usually subclinical in immunocompetent individuals but can be serious in immunocompromised hosts. These pathogens belong to a family of DNA tumour viruses. Merkel cell polyomavirus, a HPyV identified in recent years, has attracted much attention due to its link with a rare and aggressive form of human cancer. Merkel cell carcinoma, the incidence of which has tripled over the past two decades. JC polyomavirus and BK polyomavirus are also potentially oncogenic. The observed abundance and wide dissemination of HPVs and HPyVs in water environments strongly suggest the need to shed light on the fate of these viruses in water environments and to elucidate their potential for waterborne transmission. Such information is essential for the improvement of wastewater management programs in terms of both sewage treatment and water quality surveillance.

  18. Polyomavirus Replication and Smoking Are Independent Risk Factors for Bladder Cancer After Renal Transplantation.

    PubMed

    Liu, Sandy; Chaudhry, Muhammad R; Berrebi, Alexander A; Papadimitriou, John C; Drachenberg, Cinthia B; Haririan, Abdolreza; Alexiev, Borislav A

    2017-06-01

    Solid organ transplant recipients are at increased risk for developing malignancies. Polyomaviruses (PV) have been historically associated with experimental tumor development and recently described in association with renourinary malignancies in transplant patients. The aim of this study was to investigate the relationship between PV replication and smoking, and the development of malignant neoplasms in kidney transplant recipients. A retrospective case-control study was conducted for PV replication in all kidney biopsies and urine cytologies performed between 1998 and 2014 from kidney transplant recipients at the University of Maryland Medical Center. Polyomavirus-positive patients (n = 943) were defined as having any of the following: a kidney biopsy with PV associated nephropathy, any urine cytology demonstrating "decoy" cells, and/or significant polyomavirus BK viremia. Polyomavirus-negative matched patients (n = 943) were defined as lacking any evidence of PV replication. The incidence of malignancy (excluding nonmelanoma skin tumors) was determined in these 1886 patients and correlated with demographic data and history of smoking. There was a 7.9% incidence of malignant tumors after a mean posttransplant follow-up of 7.9 ± 5.4 years. Among all cancer subtypes, only bladder carcinoma was significantly associated with PV replication. By multivariate analysis, only PV replication and smoking independently increased the risk of bladder cancer, relative risk, 11.7 (P = 0.0013) and 5.6 (P = 0.0053), respectively. The findings in the current study indicate that kidney transplant recipients with PV replication and smoking are at particular risk to develop bladder carcinomas and support the need for long-term cancer surveillance in these patients.

  19. Age-specific seroprevalences of merkel cell polyomavirus, human polyomaviruses 6, 7, and 9, and trichodysplasia spinulosa-associated polyomavirus.

    PubMed

    Nicol, Jérôme T J; Robinot, Rémy; Carpentier, Audrey; Carandina, Giovanni; Mazzoni, Elisa; Tognon, Mauro; Touzé, Antoine; Coursaget, Pierre

    2013-03-01

    Six new human polyomaviruses have been identified since 2008 (Merkel cell polyomavirus [MCPyV], human polyomavirus 6 [HPyV6], HPyV7, HPyV9, trichodysplasia spinulosa polyomavirus [TSPyV], and Malawi polyomavirus [MWPyV]). The presence of specific antibodies against MCPyV, HPyV6, HPyV7, HPyV9, and TSPyV in 828 Italian subjects aged 1 to 100 years was investigated by virus-like particle-based enzyme-linked immunosorbent assays (ELISAs). The findings indicate that all of these new polyomaviruses circulate widely in humans, with seroprevalences in adulthood ranging from 39.4% for HPyV9 to 87.1% for MCPyV, and that primary exposure is most intense in childhood, with the exception of HPyV7 and HPyV9, for which the seroprevalence increased throughout life. The proportion of subjects with high antibody titers was found to increase with age for MCPyV and to decrease with age for TSPyV.

  20. Seroepidemiology of Human Polyomaviruses in a US Population

    PubMed Central

    Gossai, Anala; Waterboer, Tim; Nelson, Heather H.; Michel, Angelika; Willhauck-Fleckenstein, Martina; Farzan, Shohreh F.; Hoen, Anne G.; Christensen, Brock C.; Kelsey, Karl T.; Marsit, Carmen J.; Pawlita, Michael; Karagas, Margaret R.

    2016-01-01

    Polyomaviruses (PyV) are potentially tumorigenic in humans. However, limited data exist on the population seroprevalence of PyVs and individual characteristics that relate to seropositivity. Using multiplex serology, we determined the seroprevalence of 10 human PyVs (BK, JC, KI, WU, MCV, HPyV6, HPyV7, TSV, HPyV9, and HPyV10) among controls from a population-based skin cancer case-control study (n = 460) conducted in New Hampshire between 1993 and 1995. On a subset of participants (n = 194), methylation at CpG dinucleotides across the genome was measured in peripheral blood using the Illumina Infinium HumanMethylation27 BeadChip array (Illumina Inc., San Diego, California), from which lymphocyte subtype proportions were inferred. All participants were seropositive for at least 1 PyV, with seroprevalences ranging from 17.6% (HPyV9) to 99.1% (HPyV10). Seropositivity to JC, MCV, and HPyV7 increased with age. JC and TSV seropositivity were more common among men than among women. Smokers were more likely to be HPyV9-seropositive but MCV-seronegative, and HPyV7 seropositivity was associated with prolonged glucocorticoid use. Based on DNA methylation profiles, differences were observed in CD8-positive T- and B-cell proportions by BK, JC, and HPyV9 seropositivity. Our findings suggest that PyV seropositivity is common in the United States and varies by sociodemographic and biological characteristics, including those related to immune function. PMID:26667254

  1. Identification of a novel human polyomavirus in organs of the gastrointestinal tract.

    PubMed

    Korup, Sarah; Rietscher, Janita; Calvignac-Spencer, Sébastien; Trusch, Franziska; Hofmann, Jörg; Moens, Ugo; Sauer, Igor; Voigt, Sebastian; Schmuck, Rosa; Ehlers, Bernhard

    2013-01-01

    Polyomaviruses are small, non-enveloped viruses with a circular double-stranded DNA genome. Using a generic polyomavirus PCR targeting the VP1 major structural protein gene, a novel polyomavirus was initially identified in resected human liver tissue and provisionally named Human Polyomavirus 12 (HPyV12). Its 5033 bp genome is predicted to encode large and small T antigens and the 3 structural proteins VP1, VP2 and VP3. Phylogenetic analyses did not reveal a close relationship to any known human or animal polyomavirus. Investigation of organs, body fluids and excretions of diseased individuals and healthy subjects with both HPyV12-specific nested PCR and quantitative real-time PCR revealed additional virus-positive samples of resected liver, cecum and rectum tissues and a positive fecal sample. A capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV12. Seroprevalences of 23% and 17%, respectively, were determined in sera from healthy adults and adolescents and a pediatric group of children. These data indicate that the virus naturally infects humans and that primary infection may already occur in childhood.

  2. Characterization of a Novel Polyomavirus Isolated from a Fibroma on the Trunk of an African Elephant (Loxodonta africana)

    PubMed Central

    Stevens, Hans; Bertelsen, Mads Frost; Sijmons, Steven; Van Ranst, Marc; Maes, Piet

    2013-01-01

    Viruses of the family Polyomaviridae infect a wide variety of avian and mammalian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. In this study a novel polyomavirus, the African elephant polyomavirus 1 (AelPyV-1) found in a protruding hyperplastic fibrous lesion on the trunk of an African elephant (Loxodonta africana) was characterized. The AelPyV-1 genome is 5722 bp in size and is one of the largest polyomaviruses characterized to date. Analysis of the AelPyV-1 genome reveals five putative open-reading frames coding for the classic small and large T antigens in the early region, and the VP1, VP2 and VP3 capsid proteins in the late region. In the area preceding the VP2 start codon three putative open-reading frames, possibly coding for an agnoprotein, could be localized. A regulatory, non-coding region separates the 2 coding regions. Unique for polyomaviruses is the presence of a second 854 bp long non-coding region between the end of the early region and the end of the late region. Based on maximum likelihood phylogenetic analyses of the large T antigen of the AelPyV-1 and 61 other polyomavirus sequences, AelPyV-1 clusters within a heterogeneous group of polyomaviruses that have been isolated from bats, new world primates and rodents. PMID:24205012

  3. Characterization of a novel polyomavirus isolated from a fibroma on the trunk of an African elephant (Loxodonta africana).

    PubMed

    Stevens, Hans; Bertelsen, Mads Frost; Sijmons, Steven; Van Ranst, Marc; Maes, Piet

    2013-01-01

    Viruses of the family Polyomaviridae infect a wide variety of avian and mammalian hosts with a broad spectrum of outcomes including asymptomatic infection, acute systemic disease, and tumor induction. In this study a novel polyomavirus, the African elephant polyomavirus 1 (AelPyV-1) found in a protruding hyperplastic fibrous lesion on the trunk of an African elephant (Loxodonta africana) was characterized. The AelPyV-1 genome is 5722 bp in size and is one of the largest polyomaviruses characterized to date. Analysis of the AelPyV-1 genome reveals five putative open-reading frames coding for the classic small and large T antigens in the early region, and the VP1, VP2 and VP3 capsid proteins in the late region. In the area preceding the VP2 start codon three putative open-reading frames, possibly coding for an agnoprotein, could be localized. A regulatory, non-coding region separates the 2 coding regions. Unique for polyomaviruses is the presence of a second 854 bp long non-coding region between the end of the early region and the end of the late region. Based on maximum likelihood phylogenetic analyses of the large T antigen of the AelPyV-1 and 61 other polyomavirus sequences, AelPyV-1 clusters within a heterogeneous group of polyomaviruses that have been isolated from bats, new world primates and rodents.

  4. Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

    PubMed

    Tian, Lijun; Jeffries, Owen; McClafferty, Heather; Molyvdas, Adam; Rowe, Iain C M; Saleem, Fozia; Chen, Lie; Greaves, Jennifer; Chamberlain, Luke H; Knaus, Hans-Guenther; Ruth, Peter; Shipston, Michael J

    2008-12-30

    Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

  5. Malawi polyomavirus is a prevalent human virus that interacts with known tumor suppressors.

    PubMed

    Berrios, Christian; Jung, Joonil; Primi, Blake; Wang, Michael; Pedamallu, Chandrasekhar; Duke, Fujiko; Marcelus, Christina; Cheng, Jingwei; Garcea, Robert L; Meyerson, Matthew; DeCaprio, James A

    2015-01-01

    Malawi polyomavirus (MWPyV) is a recently identified human polyomavirus. Serology for MWPyV VP1 indicates that infection frequently occurs in childhood and reaches a prevalence of 75% in adults. The MWPyV small T antigen (ST) binds protein phosphatase 2A (PP2A), and the large T antigen (LT) binds pRb, p107, p130, and p53. However, the MWPyV LT was less stable than the simian virus 40 (SV40) LT and was unable to promote the growth of normal cells. This report confirms that MWPyV is a widespread human virus expressing T antigens with low transforming potential.

  6. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

    PubMed Central

    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  7. Outcomes of renal transplant recipients with BK virus infection and BK virus surveillance in the Auckland region from 2006 to 2012

    PubMed Central

    Hsiao, Chun-Yuan; Pilmore, Helen L; Zhou, Lifeng; de Zoysa, Janak R

    2016-01-01

    AIM To evaluate incidence, risk factors and treatment outcome of BK polyomavirus nephropathy (BKVN) in a cohort of renal transplant recipients in the Auckland region without a formal BK polyomavirus (BKV) surveillance programme. METHODS A cohort of 226 patients who received their renal transplants from 2006 to 2012 was retrospectively reviewed. RESULTS Seventy-six recipients (33.6%) had a BK viral load (BKVL) test and 9 patients (3.9%) developed BKVN. Cold ischaemia time (HR = 1.18, 95%CI: 1.04-1.35) was found to be a risk factor for BKVN. Four recipients with BKVN had complete resolution of their BKV infection; 1 recipient had BKVL less than 625 copies/mL; 3 recipients had BKVL more than 1000 copies/mL and 1 had graft failure from BKVN. BKVN has a negative impact on graft function [median estimated glomerular filtration rate (eGFR) 22.5 (IQR 18.5-53.0) mL/min per 1.73 m2, P = 0.015), but no statistically significant difference (P = 0.374) in renal allograft function was found among negative BK viraemia group [median eGFR 60.0 (IQR 48.5-74.2) mL/min per 1.73 m2), positive BK viraemia without BKVN group [median eGFR 55.0 (IQR 47.0-76.0) mL/min per 1.73 m2] and unknown BKV status group [median eGFR 54.0 (IQR 43.8-71.0) mL/min per 1.73 m2]. The incidence and treatment outcomes of BKVN were similar to some centres with BKV surveillance programmes. CONCLUSION Recipients with BVKN have poorer graft function. Although active surveillance for BKV has been shown to be effective in reducing incidence of BKVN, it should be tailored specifically to that transplant centre based on its epidemiology and outcomes of BKVN, particularly in centres with limited resources. PMID:27872831

  8. A novel human polyomavirus closely related to the african green monkey-derived lymphotropic polyomavirus.

    PubMed

    Scuda, Nelly; Hofmann, Jörg; Calvignac-Spencer, Sébastien; Ruprecht, Klemens; Liman, Peter; Kühn, Joachim; Hengel, Hartmut; Ehlers, Bernhard

    2011-05-01

    We identified a novel human polyomavirus from a kidney transplant patient under immunosuppressive treatment, by use of a generic PCR. The genome of the virus was completely amplified and sequenced. In phylogenetic analyses, it appeared as the closest relative to the African green monkey-derived lymphotropic polyomavirus (LPV). Further investigation of clinical samples from immunocompromised patients with specific nested PCR revealed additional positive samples, indicating that the virus naturally infects humans. The virus was tentatively named human polyomavirus 9 (HPyV9). The previously observed seroreactivity to LPV in human populations might find a partial explanation in the circulation of HPyV9.

  9. A novel BK channel-targeted peptide suppresses sound evoked activity in the mouse inferior colliculus

    PubMed Central

    Scott, L. L.; Brecht, E. J.; Philpo, A.; Iyer, S.; Wu, N. S.; Mihic, S. J.; Aldrich, R. W.; Pierce, J.; Walton, J. P.

    2017-01-01

    Large conductance calcium-activated (BK) channels are broadly expressed in neurons and muscle where they modulate cellular activity. Decades of research support an interest in pharmaceutical applications for modulating BK channel function. Here we report a novel BK channel-targeted peptide with functional activity in vitro and in vivo. This 9-amino acid peptide, LS3, has a unique action, suppressing channel gating rather than blocking the pore of heterologously expressed human BK channels. With an IC50 in the high picomolar range, the apparent affinity is higher than known high affinity BK channel toxins. LS3 suppresses locomotor activity via a BK channel-specific mechanism in wild-type or BK channel-humanized Caenorhabditis elegans. Topical application on the dural surface of the auditory midbrain in mouse suppresses sound evoked neural activity, similar to a well-characterized pore blocker of the BK channel. Moreover, this novel ion channel-targeted peptide rapidly crosses the BBB after systemic delivery to modulate auditory processing. Thus, a potent BK channel peptide modulator is open to neurological applications, such as preventing audiogenic seizures that originate in the auditory midbrain. PMID:28195225

  10. Dynamics of pregnancy-associated polyomavirus urinary excretion: a prospective longitudinal study.

    PubMed

    McClure, Gloria B; Gardner, J Suzette; Williams, Jason T; Copeland, Christina M; Sylvester, Sarah K; Garcea, Robert L; Meinerz, Natalie M; Groome, Lynn J; Vanchiere, John A

    2012-08-01

    Asymptomatic polyomaviruria of pregnancy has been documented in point prevalence studies, but little attention has been given to the dynamics of polyomavirus excretion during pregnancy because of its benign course. We tested the hypothesis that the frequency and/or magnitude of polyomavirus excretion would increase as pregnancy progresses. Urine specimens were obtained prospectively from 179 healthy women during uncomplicated pregnancies and 37 healthy non-pregnant women. Real-time polymerase chain reaction was used to determine BK virus (BKV) and JC virus (JCV) viral loads in urine, blood, and rectal and vaginal swabs collected during routine obstetric and gynecologic clinic visits. Asymptomatic urinary shedding of BKV and/or JCV was observed in 384 (48.0%) of 800 specimens from 100 (55.8%) pregnant women. BKV excretion was more common in pregnant than non-pregnant women (41.3% vs. 13.5%, P = 0.0026). The frequency of JCV excretion was no different in pregnant compared to non-pregnant women. The frequency and magnitude of polyomavirus shedding did not vary with gestational age. Post-partum shedding of BKV, but not JCV, rapidly decreased to undetectable levels. Pregnancy-associated BKV excretion begins early in pregnancy and terminates rapidly post-partum. Neither the frequency nor magnitude of BKV or JCV shedding increased with pregnancy progression. Further study into the host factors that regulate pregnancy-associated BKV excretion may allow identification of the host factors that predict susceptibility to BKV-associated diseases in immune compromised patients.

  11. First Detection of Human Papillomaviruses and Human Polyomaviruses in River Waters in Italy.

    PubMed

    Iaconelli, M; Petricca, S; Libera, S Della; Di Bonito, P; La Rosa, G

    2015-12-01

    Waterborne exposure to human viruses is possible through contact with contaminated water environments and can result in infections associated with a wide range of illnesses, including gastrointestinal, respiratory, ear, ocular, and skin infections. Recently, the occurrence in water environments of two groups of human viruses-both known with oncogenic potential, human polyomaviruses (HPyVs) and papillomaviruses (HPVs)-has been reported worldwide. These viruses, responsible for highly prevalent infections worldwide, have recently been proposed as potentially emerging waterborne pathogens. The objective of the present study was to examine the occurrence of HPyVs and HPVs in surface waters, by monitoring two rivers in Northwestern Italy, by nested PCR assays and sequencing. HPyVs (JC, BK, and Merkel cell polyomavirus) were detected in 10/25 (40%) samples. HPVs (HPV8, 17, 21, 25, 32, 80, 99, 105, and putative new HPVs) were identified in 14/25 (56%) river samples. The number of HPV DNA copies in waters was measured by quantitative real-time PCR. To our knowledge, this is the first detection and quantification of HPVs in surface waters. The possibility that HPyVs and HPVs can be transmitted by the waterborne route deserves to be explored in future studies.

  12. EC decay of 244Bk

    NASA Astrophysics Data System (ADS)

    Sodaye, Suparna; Tripathi, R.; Sudarshan, K.; Sharma, S. K.; Pujari, P. K.; Palit, R.; Mukhopadhyay, S.

    2014-12-01

    Berkelium isotopes have been produced in 11B-induced reaction on 238U. The EC decay of 244Bk → 244Cm has been studied by carrying out the single and coincidence measurements of the γ-rays emitted during the de-excitation of the 244Cm levels. Radiochemical separations have been carried out to minimize the contribution from the fission products and target. The new half-life of 244Bk is obtained as 5.02 ± 0.03 h, which is close to the theoretically calculated value. The relative intensities of the decay γ-rays have been re-evaluated. Based on the coincidence measurements, a tentative partial level scheme for 244Bk → 244Cm decay has been proposed.

  13. Detection and quantitation of BK virus DNA by real-time polymerase chain reaction in the LT-ag gene in adult renal transplant recipients.

    PubMed

    Si-Mohamed, Ali; Goff, Jérôme Le; Désiré, Nathalie; Maylin, Sarah; Glotz, Denis; Bélec, Laurent

    2006-01-01

    Determination of polyomavirus BK (BKV) load in urine and plasma has been advocated for monitoring adult renal transplant recipients suffering from BKV-related nephropathy. An "in-house" real-time quantitative PCR assay was developed using the BKV-1/BKV-3 primers set in the large tumor antigen (LT-ag) region to quantitate BK virus loads in plasma and urine in renal transplant patients. This assay was adapted to routine virology laboratory by evaluating two extraction procedures of nucleic acids from urine and plasma, one manual and the other using an automatic extractor, and by evaluating the Light Cycler versus Taqman apparatus. Both the manual and automatic extraction procedures and real-time PCR apparatus were equivalent. The Light Cycler and Taqman instruments allow similarly rapid, accurate, reproducible and specific quantitative detection of the three major BKV subtypes, with a detection limit of 10 BKV DNA copies/ml, and a range from 10(0) to 10(7) copies/ml. Of 855 renal transplant patients, 128 (15%) had BKV DNA in both plasma and urine samples with a mean viral load of 5.1 log/ml in plasma and 6.8 log/ml in urine and in 5 (4%) BKV-associated tubulo-interstitial nephropathy; 332 (39%) BKV DNA was found only in the urine, not in the plasma, without further development of nephropathy and 395 patients had no BKV in plasma and urine. These observations emphasize the usefulness of real-time PCR to assess the BKV load by routine testing, and confirm the need to combine both plasma and urine determinations of the BKV DNA load in order to identify renal transplant patient at high risk for BKV-associated nephropathy.

  14. Role of Nrf2 Signaling in the Regulation of Vascular BK Channel β1 Subunit Expression and BK Channel Function in High-Fat Diet-Induced Diabetic Mice.

    PubMed

    Lu, Tong; Sun, Xiaojing; Li, Yong; Chai, Qiang; Wang, Xiao-Li; Lee, Hon-Chi

    2017-10-01

    The large conductance Ca(2+)-activated K(+) (BK) channel β1-subunit (BK-β1) is a key modulator of BK channel electrophysiology and the downregulation of BK-β1 protein expression in vascular smooth muscle cells (SMCs) underlies diabetic vascular dysfunction. In this study, we hypothesized that the nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway plays a significant role in the regulation of coronary BK channel function and vasodilation in high-fat diet (HFD)-induced obese/diabetic mice. We found that the protein expressions of BK-β1 and Nrf2 were markedly downregulated, whereas those of the nuclear factor-κB (NF-κB) and the muscle ring finger protein 1 (MuRF1 [a ubiquitin E3 ligase for BK-β1]) were significantly upregulated in HFD mouse arteries. Adenoviral expression of Nrf2 suppressed the protein expressions of NF-κB and MuRF1 but enhanced BK-β1 mRNA and protein expressions in cultured coronary SMCs. Knockdown of Nrf2 resulted in reciprocal changes of these proteins. Patch-clamp studies showed that coronary BK-β1-mediated channel activation was diminished in HFD mice. Importantly, the activation of Nrf2 by dimethyl fumarate significantly reduced the body weight and blood glucose levels of HFD mice, enhanced BK-β1 transcription, and attenuated MuRF1-dependent BK-β1 protein degradation, which in turn restored coronary BK channel function and BK channel-mediated coronary vasodilation in HFD mice. Hence, Nrf2 is a novel regulator of BK channel function with therapeutic implications in diabetic vasculopathy. © 2017 by the American Diabetes Association.

  15. Common exposure to STL polyomavirus during childhood.

    PubMed

    Lim, Efrem S; Meinerz, Natalie M; Primi, Blake; Wang, David; Garcea, Robert L

    2014-09-01

    STL polyomavirus (STLPyV) was recently identified in human specimens. To determine seropositivity for STLPyV, we developed an ELISA and screened patient samples from 2 US cities (Denver, Colorado [500]; St. Louis, Missouri [419]). Overall seropositivity was 68%-70%. The age-stratified data suggest that STLPyV infection is widespread and commonly acquired during childhood.

  16. BK Virus Nephropathy in Kidney Transplantation: An Approach Proposal and Update on Risk Factors, Diagnosis, and Treatment.

    PubMed

    Gonzalez, S; Escobar-Serna, D P; Suarez, O; Benavides, X; Escobar-Serna, J F; Lozano, E

    2015-01-01

    BK virus belongs to Polyomaviridae family; it causes 95% of nephropathy cases related to polyomavirus, with the other 5% caused by JC virus. Nephropathy jeopardizes graft function, causing a premature failure of the graft in 1%-10% of patients with kidney transplants. Nowadays, antiviral effective treatment is unknown, which is why blood and urine screening of renal transplantation patients has become the most important recommendation to guide the decrease of immunosuppression, and the only proven method to decrease poor outcomes. Different interventions, such as cidofovir, leflunomide, fluoroquinolones, and intravenous immunoglobulin, have been attempted with no improvement at all. This review aims to summarize the most relevant features of BK virus, historical issues, transmission mechanisms, risk factors, and therapeutic interventions.

  17. BK Channels Reveal Novel Phosphate Sensitivity in SNr Neurons

    PubMed Central

    Ji, Juan Juan; Chen, Lianwan; Duan, Xuezhi; Song, Xueqin; Su, Wenting; Zhang, Peng; Li, Li; Bai, Shuyun; Sun, Yingchun; Inagaki, Nobuya

    2012-01-01

    Whether large conductance Ca2+-activated potassium (BK) channels are present in the substantia nigra pars reticulata (SNr) is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca2+]i) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the KATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with KATP channels under ischemic stresses. PMID:23284908

  18. A Preliminary Study Into the Significance of Intrarenal Reflux in BK Virus Nephropathy After Kidney Transplantation

    PubMed Central

    Kawanishi, Kunio; Honda, Kazuho; Koike, Junki; Hattori, Motoshi; Fuchinoue, Shouhei; Tanabe, Kazunari; Oda, Hideaki; Nagashima, Yoji

    2016-01-01

    Background The BK virus typically colonizes the lower urinary tract and is the causative agent in BK virus nephropathy (BKVN), which can progress to allograft dysfunction and graft loss. Urinary reflux in kidney allografts is induced by vesicoureteral reflux or disturbances in intrarenal reflux (IRR), believed to be associated with BKVN. This study was designed to elucidate the relationship between BKVN and IRR. Methods We examined 30 renal transplant recipients histologically diagnosed with BKVN using anti-Simian virus 40 immunohistochemistry and 60 clinically matched control recipients. The BKVN patients were divided into stable (n = 12) and progressive (n = 18) groups according to allograft kidney function 1 year after diagnosis. Histological rejection scores according to the pathological classification of rejection in renal allografts (Banff classification), histological BKVN stages, and histological polyomavirus load levels (pvl) proposed by the Banff working group were evaluated. The IRR was quantified by histological reflux scores defined with retention and reflux of immunostained Tamm-Horsfall protein in renal tubules and glomeruli. Results Higher reflux scores were observed in the BKVN group compared with that in the control group. No differences in clinical parameters were observed between the BKVN and control groups. Reflux scores and pvl were significantly higher in the progressive group than in the stable BKVN group with no significant difference in BK stage observed between groups. Reflux scores were found to be significantly correlated with pvl. Conclusions Our preliminary study suggested that IRR might be a predisposing and prognostic factor in BKVN. PMID:27500256

  19. BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo

    PubMed Central

    Lai, Michael H.; Wu, Yuejin; Gao, Zhan; Anderson, Mark E.; Dalziel, Julie E.

    2014-01-01

    Large-conductance Ca2+- and voltage-activated K+ (BK) channels play prominent roles in shaping muscle and neuronal excitability. In the cardiovascular system, BK channels promote vascular relaxation and protect against ischemic injury. Recently, inhibition of BK channels has been shown to lower heart rate in intact rodents and isolated hearts, suggesting a novel role in heart function. However, the underlying mechanism is unclear. In the present study, we recorded ECGs from mice injected with paxilline (PAX), a membrane-permeable BK channel antagonist, and examined changes in cardiac conduction. ECGs revealed a 19 ± 4% PAX-induced reduction in heart rate in wild-type but not BK channel knockout (Kcnma1−/−) mice. The heart rate decrease was associated with slowed cardiac pacing due to elongation of the sinus interval. Action potential firing recorded from isolated sinoatrial node cells (SANCs) was reduced by 55 ± 15% and 28 ± 9% by application of PAX (3 μM) and iberiotoxin (230 nM), respectively. Furthermore, baseline firing rates from Kcnma1−/− SANCs were 33% lower than wild-type SANCs. The slowed firing upon BK current inhibition or genetic deletion was due to lengthening of the diastolic depolarization phase of the SANC action potential. Finally, BK channel immunoreactivity and PAX-sensitive currents were identified in SANCs with HCN4 expression and pacemaker current, respectively, and BK channels cloned from SANCs recapitulated similar activation as the PAX-sensitive current. Together, these data localize BK channels to SANCs and demonstrate that loss of BK current decreases SANC automaticity, consistent with slowed sinus pacing after PAX injection in vivo. Furthermore, these findings suggest BK channels are potential therapeutic targets for disorders of heart rate. PMID:25172903

  20. Polyomavirus and Naturally Occuring Neuroglial Tumors in Raccoons (Procyon Lotor).

    PubMed

    Pesavento, Patricia A; Brostoff, Terza; Church, Molly E; Dela Cruz, Florante N; Woolard, Kevin D

    2016-01-01

    Polyomavirus (PyV) infections are widespread in human populations and, although generally associated with silent persistence, rarely cause severe disease. Among diseases convincingly associated with natural PyV infections of humans, there are remarkably different tissue tropisms and outcomes, including progressive multifocal leukoencephalopathy, transient or progressive nephropathy, and cancer. The variable character and unpredictable outcomes of infection attest to large gaps in our basic understanding of PyV biology. In particular, the rich history of research demonstrating the oncogenic potential of PyVs in laboratory animals begs the question of why cancer is not more often associated with infection. Raccoon polyomavirus (RacPyV), discovered in 2010, is consistently identified in neuroglial tumors in free-ranging raccoons in the western United States. Exposure to RacPyV is widespread, and RacPyV is detected in tissues of raccoons without tumors. Studying the relationship of RacPyV with its natural host is a unique opportunity to uncover cogent cellular targets and protein interactions between the virus and its host. Our hypothesis is that RacPyV, as an intact episome, alters cellular pathways within neural progenitor cells and drives oncogenesis.

  1. Molecular Determinants of BK Channel Functional Diversity and Functioning.

    PubMed

    Latorre, Ramon; Castillo, Karen; Carrasquel-Ursulaez, Willy; Sepulveda, Romina V; Gonzalez-Nilo, Fernando; Gonzalez, Carlos; Alvarez, Osvaldo

    2017-01-01

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels play many physiological roles ranging from the maintenance of smooth muscle tone to hearing and neurosecretion. BK channels are tetramers in which the pore-forming α subunit is coded by a single gene (Slowpoke, KCNMA1). In this review, we first highlight the physiological importance of this ubiquitous channel, emphasizing the role that BK channels play in different channelopathies. We next discuss the modular nature of BK channel-forming protein, in which the different modules (the voltage sensor and the Ca(2+) binding sites) communicate with the pore gates allosterically. In this regard, we review in detail the allosteric models proposed to explain channel activation and how the models are related to channel structure. Considering their extremely large conductance and unique selectivity to K(+), we also offer an account of how these two apparently paradoxical characteristics can be understood consistently in unison, and what we have learned about the conduction system and the activation gates using ions, blockers, and toxins. Attention is paid here to the molecular nature of the voltage sensor and the Ca(2+) binding sites that are located in a gating ring of known crystal structure and constituted by four COOH termini. Despite the fact that BK channels are coded by a single gene, diversity is obtained by means of alternative splicing and modulatory β and γ subunits. We finish this review by describing how the association of the α subunit with β or with γ subunits can change the BK channel phenotype and pharmacology.

  2. BK Channels Are Linked to Inositol 1,4,5-Triphosphate Receptors via Lipid Rafts

    PubMed Central

    Weaver, Amy K.; Olsen, Michelle L.; McFerrin, Michael B.; Sontheimer, Harald

    2007-01-01

    Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K+ conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca2+ increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca2+-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP3R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-β-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K+ conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca2+ in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP3Rs negated the effect of methyl-β-cyclodextrin, providing further support that IP3Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca2+-sensitive channels to their second messenger source. PMID:17711864

  3. Assessing Host-Virus Codivergence for Close Relatives of Merkel Cell Polyomavirus Infecting African Great Apes.

    PubMed

    Madinda, Nadège F; Ehlers, Bernhard; Wertheim, Joel O; Akoua-Koffi, Chantal; Bergl, Richard A; Boesch, Christophe; Akonkwa, Dieudonné Boji Mungu; Eckardt, Winnie; Fruth, Barbara; Gillespie, Thomas R; Gray, Maryke; Hohmann, Gottfried; Karhemere, Stomy; Kujirakwinja, Deo; Langergraber, Kevin; Muyembe, Jean-Jacques; Nishuli, Radar; Pauly, Maude; Petrzelkova, Klara J; Robbins, Martha M; Todd, Angelique; Schubert, Grit; Stoinski, Tara S; Wittig, Roman M; Zuberbühler, Klaus; Peeters, Martine; Leendertz, Fabian H; Calvignac-Spencer, Sébastien

    2016-10-01

    It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) codiverged with their animal hosts. In contrast, recent analyses suggested that codivergence may only marginally influence the evolution of PyV. We reassess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing the genetic diversity of these viruses in seven African great ape taxa, we show that they exhibit very strong host specificity. Reconciliation analyses identify more codivergence than noncodivergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support codivergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, our results add to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts. The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double-stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here, we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host specificity and that their evolution largely mirrors that of their hosts, suggesting that codivergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Assessing Host-Virus Codivergence for Close Relatives of Merkel Cell Polyomavirus Infecting African Great Apes

    PubMed Central

    Madinda, Nadège F.; Ehlers, Bernhard; Wertheim, Joel O.; Akoua-Koffi, Chantal; Bergl, Richard A.; Boesch, Christophe; Akonkwa, Dieudonné Boji Mungu; Eckardt, Winnie; Fruth, Barbara; Gillespie, Thomas R.; Gray, Maryke; Hohmann, Gottfried; Karhemere, Stomy; Kujirakwinja, Deo; Langergraber, Kevin; Muyembe, Jean-Jacques; Nishuli, Radar; Pauly, Maude; Petrzelkova, Klara J.; Robbins, Martha M.; Todd, Angelique; Schubert, Grit; Stoinski, Tara S.; Wittig, Roman M.; Zuberbühler, Klaus; Peeters, Martine; Leendertz, Fabian H.

    2016-01-01

    ABSTRACT It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) codiverged with their animal hosts. In contrast, recent analyses suggested that codivergence may only marginally influence the evolution of PyV. We reassess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing the genetic diversity of these viruses in seven African great ape taxa, we show that they exhibit very strong host specificity. Reconciliation analyses identify more codivergence than noncodivergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support codivergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, our results add to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts. IMPORTANCE The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double-stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here, we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host specificity and that their evolution largely mirrors that of their hosts, suggesting that codivergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years. PMID:27440885

  5. Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies.

    PubMed

    Bhattarai, Yogesh; Fernandes, Roxanne; Kadrofske, Mark M; Lockwood, Lizbeth R; Galligan, James J; Xu, Hui

    2014-10-01

    Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  6. Innate Immunity and BK Virus: Prospective Strategies.

    PubMed

    Kariminik, Ashraf; Yaghobi, Ramin; Dabiri, Shahriar

    2016-03-01

    Recent information demonstrated that BK virus reactivation is a dominant complication after kidney transplantation, which occurs because of immunosuppression. BK virus reactivation is the main reason of transplanted kidney losing. Immune response against BK virus is the major inhibitor of the virus reactivation. Therefore, improving our knowledge regarding the main parameters that fight against BK viruses can shed light on to direct new treatment strategies to suppress BK infection. Innate immunity consists of numerous cell systems and also soluble molecules, which not only suppress virus replication, but also activate adaptive immunity to eradicate the infection. Additionally, it appears that immune responses against reactivated BK virus are the main reasons for induction of BK virus-associated nephropathy (BKAN). Thus, improving our knowledge regarding the parameters and detailed mechanisms of innate immunity and also the status of innate immunity of the patients with BK virus reactivation and its complications can introduce new prospective strategies to either prevent or as therapy of the complication. Therefore, this review was aimed to collate the most recent data regarding the roles played by innate immunity against BK virus and also the status of innate immunity in the patients with reactivation BK virus and BKAN.

  7. MW polyomavirus and STL polyomavirus present in tonsillar tissues from children with chronic tonsillar disease.

    PubMed

    Peng, J; Li, K; Zhang, C; Jin, Q

    2016-01-01

    We aimed to explore the frequency of all 13 human polyomaviruses (HPyVs), especially MW polyomavirus (MWPyV) and STL polyomavirus (STLPyV), in tonsillar tissues from Chinese children with chronic tonsillar disease. We examined 99 swabs from mucosal surfaces of palatine tonsils, in which six HPyVs were detected. MWPyV and STLPyV were each detected in two samples. This provides new evidence for the hypothesis that the lymphoid system may play a role in HPyV infection and persistence. We need to define their role in tonsillar disease in the future. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  8. Knockout of the BK β4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability

    PubMed Central

    Wang, Bin; Bugay, Vladislav; Ling, Ling; Chuang, Hui-Hsui; Jaffe, David B.

    2016-01-01

    BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK β4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of β4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the β4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons. PMID:27146987

  9. The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain.

    PubMed

    Douglas, R M; Lai, J C K; Bian, S; Cummins, L; Moczydlowski, E; Haddad, G G

    2006-01-01

    Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent

  10. The Merkel Cell Polyomavirus Minor Capsid Protein

    PubMed Central

    Schowalter, Rachel M.; Buck, Christopher B.

    2013-01-01

    The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types. PMID:23990782

  11. Polymerase chain reaction assay for avian polyomavirus.

    PubMed Central

    Phalen, D N; Wilson, V G; Graham, D L

    1991-01-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV. Images PMID:1647403

  12. Polymerase chain reaction assay for avian polyomavirus.

    PubMed

    Phalen, D N; Wilson, V G; Graham, D L

    1991-05-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  13. Downregulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

    PubMed Central

    Pacheco Otalora, Luis F.; Hernandez, Eder F.; Arshadmansab, Massoud F.; rancisco, Sebastian F; Willis, Michael; Ermolinsky, Boris; Zarei, Masoud; Knaus, Hans-Guenther; Garrido-Sanabria, Emilio R.

    2008-01-01

    In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures. PMID:18295190

  14. Down-regulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy.

    PubMed

    Pacheco Otalora, Luis F; Hernandez, Eder F; Arshadmansab, Massoud F; Francisco, Sebastian; Willis, Michael; Ermolinsky, Boris; Zarei, Masoud; Knaus, Hans-Guenther; Garrido-Sanabria, Emilio R

    2008-03-20

    In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), Western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore-forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel immunofluorescent signals in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures.

  15. Genetic activation of BK currents in vivo generates bidirectional effects on neuronal excitability

    PubMed Central

    Montgomery, Jenna R.; Meredith, Andrea L.

    2012-01-01

    Large-conductance calcium-activated potassium channels (BK) are potent negative regulators of excitability in neurons and muscle, and increasing BK current is a novel therapeutic strategy for neuro- and cardioprotection, disorders of smooth muscle hyperactivity, and several psychiatric diseases. However, in some neurons, enhanced BK current is linked with seizures and paradoxical increases in excitability, potentially complicating the clinical use of agonists. The mechanisms that switch BK influence from inhibitory to excitatory are not well defined. Here we investigate this dichotomy using a gain-of-function subunit (BKR207Q) to enhance BK currents. Heterologous expression of BKR207Q generated currents that activated at physiologically relevant voltages in lower intracellular Ca2+, activated faster, and deactivated slower than wild-type currents. We then used BKR207Q expression to broadly augment endogenous BK currents in vivo, generating a transgenic mouse from a circadian clock-controlled Period1 gene fragment (Tg-BKR207Q). The specific impact on excitability was assessed in neurons of the suprachiasmatic nucleus (SCN) in the hypothalamus, a cell type where BK currents regulate spontaneous firing under distinct day and night conditions that are defined by different complements of ionic currents. In the SCN, Tg-BKR207Q expression converted the endogenous BK current to fast-activating, while maintaining similar current-voltage properties between day and night. Alteration of BK currents in Tg-BKR207Q SCN neurons increased firing at night but decreased firing during the day, demonstrating that BK currents generate bidirectional effects on neuronal firing under distinct conditions. PMID:23112153

  16. Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat.

    PubMed

    Kuntamallappanavar, Guruprasad; Bisen, Shivantika; Bukiya, Anna N; Dopico, Alex M

    2017-02-01

    Large conductance, Ca(2+)i- and voltage-gated K(+) (BK) channels regulate myogenic tone and, thus, arterial diameter. In smooth muscle (SM), BK channels include channel-forming α and auxiliary β1 subunits. BK β1 increases the channel's Ca(2+) sensitivity, allowing BK channels to negatively feedback on depolarization-induced Ca(2+) entry, oppose SM contraction and favor vasodilation. Thus, endothelial-independent vasodilation can be evoked though targeting of SM BK β1 by endogenous ligands, including lithocholate (LCA). Here, we investigated the expression of BK β1 across arteries of the cerebral and peripheral circulations, and the contribution of such expression to channel function and BK β1-mediated vasodilation. Data demonstrate that endothelium-independent, BK β1-mediated vasodilation by LCA is larger in coronary (CA) and basilar (BA) arteries than in anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), and mesenteric (MA) arteries, all arterial segments having a similar diameter. Thus, differential dilation occurs in extracranial arteries which are subjected to similar vascular pressure (CA vs. MA) and in arteries that irrigate different brain regions (BA vs. ACA, MCA, and PCA). SM BK channels from BA and CA displayed increased basal activity and LCA responses, indicating increased BK β1 functional presence. Indeed, in the absence of detectable changes in BK α, BA and CA myocytes showed an increased location of BK β1 in the plasmalemma/subplasmalemma. Moreover, these myocytes distinctly showed increased BK β1 messenger RNA (mRNA) levels. Supporting a major role of enhanced BK β1 transcripts in artery dilation, LCA-induced dilation of MCA transfected with BK β1 complementary DNA (cDNA) was as high as LCA-induced dilation of untransfected BA or CA.

  17. Large $N$ approach to kaon decays and mixing 28 years later: $\\Delta I = 1/2$ rule, $\\hat B_K$ and $\\Delta M_K$

    SciTech Connect

    Buras, Andrzej J.; Gérard, Jean -Marc; Bardeen, William A.

    2014-05-20

    We review and update our results for K → π π decays and K⁰- K¯⁰ mixing obtained by us in the 1980s within an approach based on the dual representation of QCD as a theory of weakly interacting mesons for large N colours. In our analytic approach the dynamics behind the enhancement of ReA0 and suppression of ReA2, the so-called ΔI = 1/2 rule for K → π π decays, has a simple structure: the usual octet enhancement through quark-gluon renormalization group evolution down to the scales O(1 GeV) is continued as a meson evolution down to zero momentum scales at which the factorization of hadronic matrix elements is at work. The inclusion of lowest-lying vector meson contributions in addition to the pseudoscalar ones and of Wilson coefficients in a momentum scheme improves significantly the matching between quark-gluon and meson evolutions. In particular, the anomalous dimension matrix governing the meson evolution exhibits the structure of the known anomalous dimension matrix in the quark-gluon evolution. The recent results on ReA2 and ReA0 from the RBC-UKQC collaboration give support for our approach. In particular, the signs of the two main contractions found numerically by these authors follow uniquely from our analytic approach. At NLO in 1/N we obtain R = ReA0/ReA2= 16.0±1.5 which amounts to an order of magnitude enhancement over the strict large N limit value √2. QCD penguins contribute at 15% level to this result. We also find B^K = 0.73± 0.02, with the smallness of 1/N corrections to the large N value B^K = 3/4 resulting within our approach from an approximate cancellation between pseudoscalar and vector meson one-loop contributions. We summarize the status of ΔMK in this approach.

  18. Characterization of T Antigens, Including Middle T and Alternative T, Expressed by the Human Polyomavirus Associated with Trichodysplasia Spinulosa

    PubMed Central

    van der Meijden, Els; Kazem, Siamaque; Dargel, Christina A.; van Vuren, Nick; Hensbergen, Paul J.

    2015-01-01

    ABSTRACT The polyomavirus tumor (T) antigens play crucial roles in viral replication, transcription, and cellular transformation. They are encoded by partially overlapping open reading frames (ORFs) located in the early region through alternative mRNA splicing. The T expression pattern of the trichodysplasia spinulosa-associated polyomavirus (TSPyV) has not been established yet, hampering further study of its pathogenic mechanisms and taxonomic relationship. Here, we characterized TSPyV T antigen expression in human cell lines transfected with the TSPyV early region. Sequencing of T antigen-encoded reverse transcription-PCR (RT-PCR) products revealed three splice donor and acceptor sites creating six mRNA splice products that potentially encode the antigens small T (ST), middle T (MT), large T (LT), tiny T, 21kT, and alternative T (ALTO). Except for 21kT, these splice products were also detected in skin of TSPyV-infected patients. At least three splice products were confirmed by Northern blotting, likely encoding LT, MT, ST, 21kT, and ALTO. Protein expression was demonstrated for LT, ALTO, and possibly MT, with LT detected in the nucleus and ALTO in the cytoplasm of transfected cells. Splice site and start codon mutations indicated that ALTO is encoded by the same splice product that encodes LT and uses internal start codons for initiation. The genuineness of ALTO was indicated by the identification of acetylated N-terminal ALTO peptides by mass spectrometry. Summarizing, TSPyV exhibits an expression pattern characterized by both MT and ALTO expression, combining features of rodent and human polyomaviruses. This unique expression pattern provides important leads for further study of polyomavirus-related disease and for an understanding of polyomavirus evolution. IMPORTANCE The human trichodysplasia spinulosa-associated polyomavirus (TSPyV) is distinguished among polyomaviruses for combining productive infection with cell-transforming properties. In the research

  19. AP1 enhances polyomavirus DNA replication by promoting T-antigen-mediated unwinding of DNA.

    PubMed Central

    Guo, W; Tang, W J; Bu, X; Bermudez, V; Martin, M; Folk, W R

    1996-01-01

    An early step in the initiation of polyomavirus DNA replication is viral large-T-antigen-mediated unwinding of the origin. We report that components of the AP1 transcription factor, Fos and Jun, interact with T antigen in vitro to enhance unwinding of the viral origin. This provides a biochemical basis for the capacity of AP1 to activate viral DNA replication in vivo. PMID:8763994

  20. Current understanding of iberiotoxin-resistant BK channels in the nervous system

    PubMed Central

    Wang, Bin; Jaffe, David B.; Brenner, Robert

    2014-01-01

    While most large-conductance, calcium-, and voltage-activated potassium channels (BK or Maxi-K type) are blocked by the scorpion venom iberiotoxin, the so-called “type II” subtype has the property of toxin resistance. This property is uniquely mediated by channel assembly with one member of the BK accessory β subunit family, the neuron-enriched β4 subunit. This review will focus on current understanding of iberiotoxin-resistant, β4-containing BK channel properties and their function in the CNS. Studies have shown that β4 dramatically promotes BK channel opening by shifting voltage sensor activation to more negative voltage ranges, but also slows activation to timescales that theoretically preclude BK ability to shape action potentials (APs). In addition, β4 membrane trafficking is regulated through an endoplasmic retention signal and palmitoylation. More recently, the challenge has been to understand the functional role of the iberiotoxin-resistant BK subtype utilizing computational modeling of neurons and neurophysiological approaches. Utilizing iberiotoxin-resistance as a footprint for these channels, they have been identified in dentate gyrus granule neurons and in purkinje neurons of the cerebellum. In these neurons, the role of these channels is largely consistent with slow-gated channels that reduce excitability either through an interspike conductance, such as in purkinje neurons, or by replacing fast-gating BK channels that otherwise facilitate high frequency AP firing, such as in dentate gyrus neurons. They are also observed in presynaptic mossy fiber terminals of the dentate gyrus and posterior pituitary terminals. More recent studies suggest that β4 subunits may also be expressed in some neurons lacking iberiotoxin-resistant BK channels, such as in CA3 hippocampus neurons. Ongoing research using novel, specific blockers and agonists of BK/β4, and β4 knockout mice, will continue to move the field forward in understanding the function of these

  1. Current understanding of iberiotoxin-resistant BK channels in the nervous system.

    PubMed

    Wang, Bin; Jaffe, David B; Brenner, Robert

    2014-01-01

    While most large-conductance, calcium-, and voltage-activated potassium channels (BK or Maxi-K type) are blocked by the scorpion venom iberiotoxin, the so-called "type II" subtype has the property of toxin resistance. This property is uniquely mediated by channel assembly with one member of the BK accessory β subunit family, the neuron-enriched β4 subunit. This review will focus on current understanding of iberiotoxin-resistant, β4-containing BK channel properties and their function in the CNS. Studies have shown that β4 dramatically promotes BK channel opening by shifting voltage sensor activation to more negative voltage ranges, but also slows activation to timescales that theoretically preclude BK ability to shape action potentials (APs). In addition, β4 membrane trafficking is regulated through an endoplasmic retention signal and palmitoylation. More recently, the challenge has been to understand the functional role of the iberiotoxin-resistant BK subtype utilizing computational modeling of neurons and neurophysiological approaches. Utilizing iberiotoxin-resistance as a footprint for these channels, they have been identified in dentate gyrus granule neurons and in purkinje neurons of the cerebellum. In these neurons, the role of these channels is largely consistent with slow-gated channels that reduce excitability either through an interspike conductance, such as in purkinje neurons, or by replacing fast-gating BK channels that otherwise facilitate high frequency AP firing, such as in dentate gyrus neurons. They are also observed in presynaptic mossy fiber terminals of the dentate gyrus and posterior pituitary terminals. More recent studies suggest that β4 subunits may also be expressed in some neurons lacking iberiotoxin-resistant BK channels, such as in CA3 hippocampus neurons. Ongoing research using novel, specific blockers and agonists of BK/β4, and β4 knockout mice, will continue to move the field forward in understanding the function of these

  2. Evaluating sewage-associated JCV and BKV polyomaviruses for sourcing human fecal pollution in a coastal river in Southeast Queensland, Australia.

    PubMed

    Ahmed, W; Wan, C; Goonetilleke, A; Gardner, T

    2010-01-01

    In this study, the host-sensitivity and host-specificity of JC virus (JCV) and BK virus (BKV) polyomaviruses were evaluated by testing wastewater and fecal samples from nine host groups in Southeast Queensland, Australia. The JCV and BKV polyomaviruses were detected in 63 human wastewater samples collected from primary and secondary effluent, suggesting high sensitivity of these viruses in human wastewater. In the 81 animal wastewater and fecal samples tested, 80 were polymerase chain reaction (PCR) negative for the JCV and BKV markers. Only one sample (out of 81 animal wastewater and fecal samples) from pig wastewater was positive. Nonetheless, the overall host-specificity of these viruses to differentiate between human and animal wastewater and fecal samples was 0.99. To our knowledge, this is the first study in Australia that reports on the high specificity of JCV and BKV polyomaviruses. To evaluate the field application of these viral markers for detecting human fecal pollution, 20 environmental samples were collected from a coastal river. In the 20 samples tested, 15% (3/20) and 70% (14/20) samples exceeded the regulatory guidelines for Escherichia coli and enterococci levels for marine waters. In all, five (25%) samples were PCR positive for JCV and BKV, indicating the presence of human fecal pollution in the coastal river investigated. The results suggest that JCV and BKV detection using PCR could be a useful tool for identifying human-sourced fecal pollution in coastal waters.

  3. The Impact of BK Channels on Cellular Excitability Depends on their Subcellular Location

    PubMed Central

    Bock, Tobias; Stuart, Greg J.

    2016-01-01

    Large conductance calcium-activated potassium channels (or BK channels) fulfil a multitude of roles in the central nervous system. At the soma of many neuronal cell types they control the speed of action potential (AP) repolarization and therefore they can have an impact on neuronal excitability. Due to their presence in nerve terminals they also regulate transmitter release. BK channels have also been shown to be present in the dendrites of some neurons where they can regulate the magnitude and duration of dendritic spikes. Here, we investigate the impact of modulating the activation of BK channels at different locations on the cellular excitability of cortical layer 5 pyramidal neurons. We find that while somatic BK channels help to repolarize APs at the soma and mediate the fast after-hyperpolarization, dendritic BK channels are responsible for repolarization of dendritic calcium spikes and thereby regulate somatic AP burst firing. We found no evidence for a role of dendritic BK channels in the regulation of backpropagating AP amplitude or duration. These experiments highlight the diverse roles of BK channels in regulating neuronal excitability and indicate that their functional impact depends on their subcellular location. PMID:27630543

  4. Merkel Cell Polyomavirus: A Newly Discovered Human Virus with Oncogenic Potential

    PubMed Central

    Spurgeon, Megan E.; Lambert, Paul F.

    2012-01-01

    A marked escalation in the rate of discovery of new types of human polyomavirus has occurred over the last five years largely owing to recent technological advances in their detection. Among the newly discovered viruses, Merkel Cell Polyomavirus (MCPyV or MCV) has gained the most attention due to its link with a rare human cancer. Infection with MCPyV is common in the human population, and the virus is detected in several anatomical locations, but most frequently in skin. Study of MCPyV molecular virology has been complicated by the lack of straightforward cell culture models, but recent in vitro studies are making strides towards understanding the virus life cycle, its cellular tropism, and mode of transmission. While MCPyV shares several traditional traits with other human polyomaviruses, the burst of research since its discovery reveals insight into a virus with many unique genetic and mechanistic features. The evidence for a causal link between MCPyV and the rare neuroendocrine cancer, Merkel Cell Carcinoma (MCC), is compelling. A majority of MCCs contain clonally integrated viral DNA, express viral T antigen transcripts and protein, and exhibit an addiction to the viral large T and small t antigen oncoproteins. The MCPyV large T antigen contains MCC tumor-specific mutations that ablate its replication capacity but preserve its oncogenic functions, and the small t antigen promotes an environment favorable for cap-dependent translation. The mechanisms of MCPyV-induced transformation have not been fully elucidated, but the likely etiological role of this new polyomavirus in human cancer provides a strong opportunity to expand knowledge of virus-host interactions and viral oncology. PMID:23217622

  5. Novel polyomavirus associated with brain tumors in free-ranging raccoons, western United States.

    PubMed

    Dela Cruz, Florante N; Giannitti, Federico; Li, Linlin; Woods, Leslie W; Del Valle, Luis; Delwart, Eric; Pesavento, Patricia A

    2013-01-01

    Tumors of any type are exceedingly rare in raccoons. High-grade brain tumors, consistently located in the frontal lobes and olfactory tracts, were detected in 10 raccoons during March 2010-May 2012 in California and Oregon, suggesting an emerging, infectious origin. We have identified a candidate etiologic agent, dubbed raccoon polyomavirus, that was present in the tumor tissue of all affected animals but not in tissues from 20 unaffected animals. Southern blot hybridization and rolling circle amplification showed the episomal viral genome in the tumors. The multifunctional nuclear protein large T-antigen was detectable by immunohistochemical analyses in a subset of neoplastic cells. Raccoon polyomavirus may contribute to the development of malignant brain tumors of raccoons.

  6. Novel Polyomavirus associated with Brain Tumors in Free-Ranging Raccoons, Western United States

    PubMed Central

    Dela Cruz, Florante N.; Giannitti, Federico; Li, Linlin; Woods, Leslie W.; Del Valle, Luis; Delwart, Eric

    2013-01-01

    Tumors of any type are exceedingly rare in raccoons. High-grade brain tumors, consistently located in the frontal lobes and olfactory tracts, were detected in 10 raccoons during March 2010–May 2012 in California and Oregon, suggesting an emerging, infectious origin. We have identified a candidate etiologic agent, dubbed raccoon polyomavirus, that was present in the tumor tissue of all affected animals but not in tissues from 20 unaffected animals. Southern blot hybridization and rolling circle amplification showed the episomal viral genome in the tumors. The multifunctional nuclear protein large T-antigen was detectable by immunohistochemical analyses in a subset of neoplastic cells. Raccoon polyomavirus may contribute to the development of malignant brain tumors of raccoons. PMID:23260029

  7. Mechanism of β4 Subunit Modulation of BK Channels

    PubMed Central

    Wang, Bin; Rothberg, Brad S.; Brenner, Robert

    2006-01-01

    Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C

  8. Early identification of renal transplant recipients with high risk of polyomavirus-associated nephropathy.

    PubMed

    Teutsch, K; Schweitzer, F; Knops, E; Kaiser, R; Pfister, H; Verheyen, J; Göbel, H; Cingöz, T; Di Cristanziano, V

    2015-12-01

    Polyomavirus BK (BKPyV) is ubiquitous among humans. Following primary infection, the virus remains latent predominantly in the hosts' uroepithelial cells. Up to 10 % of renal transplant recipients show a viral reactivation that can lead to polyomavirus-associated nephropathy (PyVAN). In the absence of early treatments, the risk of graft loss is up to 80 %. Monitoring viral load in urine and plasma by real-time PCR after transplantation is the most common diagnostic tool to detect viral reactivation. In the present retrospective study, BKPyV-DNA loads in urine and plasma by quantitative real-time PCR were associated with clinical data, including HLA haplotype, blood parameters and viral genotype, of 40 renal transplant recipients at the University Clinics of Cologne. Seventeen out of 329 patients screened for BKPyV from January 2009 to October 2013 were detected BKPyV positive in urine only, whereas in 23 patients the virus became additionally detectable in plasma. Among these, ten patients progressed to PyVAN. Overall, the present study showed that the detection from the third month onwards after transplantation of a first viruric episode with a median viral load of 1 × 10(8) copies/mL, followed after few days by a first viremic episode with a median viral load of >1 × 10(4) copies/mL, was strongly associated with the development of PyVAN. In conclusion, the viral load and the temporal profile of the first viruric and viremic episode post-transplantation, in combination with specific features of the host immune response, should be considered as relevant clinical determinants of the risk of renal transplant recipients to progress to PyVAN.

  9. Screening for polyomavirus associated nephropathy in renal transplantation with blood viral load measurement.

    PubMed

    Boudreault, Alexandre A; Courtemanche, Chantal; Latulippe, Eva; Côté, Isabelle; Houde, Isabelle; Deschênes, Louise

    2009-08-01

    Polyomavirus associated nephropathy (PVAN) is an important cause of graft failure in the renal transplant population. It has been shown that viremia precedes PVAN, suggesting that measurement of blood viral load could be used for PVAN screening. To verify the utility of BK virus (BKV) blood viral load measurement for PVAN screening in the renal transplant population, establish a threshold value, and determine the sensitivity and specificity of the test. We developed a real-time PCR assay for BKV blood viral load measurement and included this assay in the PVAN screening protocol of the renal transplant recipients of our institution. We report results for 60 patients who had a blood viral load measurement concomitantly with an allograft biopsy with immunohistochemistry for polyomavirus. 14 patients were found to have a PVAN on allograft biopsy together with a viral load above 3.0x10(3)copies/ml. None of the patients with a viral load under 3.0x10(3)copies/ml had a PVAN on allograft biopsy. The area under the receiver operating characteristic (ROC) curve was 0.95 (95% CI: 0.91-1.00) and using a threshold value of 3.0x10(3)copies/ml yielded a sensitivity of 100% (95% CI: 76.8-100%) and a specificity of 89.6% (95% CI: 77.3-96.5%) for PVAN screening. BKV blood viral load measurement is sensitive and specific for PVAN screening when a threshold value is precisely determined.

  10. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

  11. Modulation of BK channel voltage gating by different auxiliary β subunits

    PubMed Central

    Contreras, Gustavo F.; Neely, Alan; Alvarez, Osvaldo; Gonzalez, Carlos; Latorre, Ramon

    2012-01-01

    Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms converge to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming α subunit of BK is associated with one of four alternative β subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, β1 stabilizes the active configuration of the BK voltage sensor having a large effect on BK Ca2+ sensitivity. To determine the extent to which β subunits regulate the BK voltage sensor, we measured gating currents induced by the pore-forming BK α subunit alone and with the different β subunits expressed in Xenopus oocytes (β1, β2IR, β3b, and β4). We found that β1, β2, and β4 stabilize the BK voltage sensor in the active conformation. β3 has no effect on voltage sensor equilibrium. In addition, β4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in α β4 channels explains most of their biophysical properties. For channels composed of the α subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of this charge develops with a time course comparable to that of K+ current activation. In the presence of β1, β2, and β4 this slow component develops in advance of and much more rapidly than ion current activation, suggesting that BK channel opening proceeds in two steps. PMID:23112204

  12. Replication of Oral BK Virus in Human Salivary Gland Cells

    PubMed Central

    Burger-Calderon, Raquel; Madden, Victoria; Hallett, Ryan A.; Gingerich, Aaron D.; Nickeleit, Volker

    2014-01-01

    BK polyomavirus (BKPyV) is the most common viral pathogen among allograft patients. Increasing evidence links BKPyV to the human oral compartment and to HIV-associated salivary gland disease (HIVSGD). To date, few studies have analyzed orally derived BKPyV. This study aimed to characterize BKPyV isolated from throat wash (TW) samples from HIVSGD patients. The replication potential of HIVSGD-derived clinical isolates HIVSGD-1 and HIVSGD-2, both containing the noncoding control region (NCCR) architecture OPQPQQS, were assessed and compared to urine-derived virus. The BKPyV isolates displayed significant variation in replication potential. Whole-genome alignment of the two isolates revealed three nucleotide differences that were analyzed for a potential effect on the viral life cycle. Analysis revealed a negligible difference in NCCR promoter activity despite sequence variation and emphasized the importance of functional T antigen (Tag) for efficient replication. HIVSGD-1 encoded full-length Tag, underwent productive infection in both human salivary gland cells and kidney cells, and expressed viral DNA and Tag protein. Additionally, HIVSGD-1 generated DNase-resistant particles and by far surpassed the replication potential of the kidney-derived isolate in HSG cells. HIVSGD-2 encoded a truncated form of Tag and replicated much less efficiently. Quantitation of infectious virus, via the fluorescent forming unit assay, suggested that HIVSGD BKPyV had preferential tropism for salivary gland cells over kidney cells. Similarly, the results suggested that kidney-derived virus had preferential tropism for kidney cells over salivary gland cells. Evidence of HIVSGD-derived BKPyV oral tropism and adept viral replication in human salivary gland cells corroborated the potential link between HIVSGD pathogenesis and BKPyV. PMID:24173219

  13. Small and middle T antigens contribute to lytic and abortive polyomavirus infection

    SciTech Connect

    Tuerler, H.; Salomon, C.

    1985-02-01

    Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, the authors studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, they measured the proportion of T-antigen-positive cells, the incorporation of (/sup 3/H)thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, they also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, the onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From the experiments, the authors concluded that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells center S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was <10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.

  14. Presynaptic BK Channels Modulate Ethanol-Induced Enhancement of GABAergic Transmission in the Rat Central Amygdala Nucleus

    PubMed Central

    Li, Qiang; Madison, Roger

    2014-01-01

    Large-conductance calcium-activated potassium BK channels are widely expressed in the brain and are involved in the regulation of neuronal functions such as neurotransmitter release. However, their possible role in mediating ethanol-induced GABA release is still unknown. We assessed the role of BK channels in modulating the action of ethanol on inhibitory synaptic transmission mediated via GABAA receptors in the rat central nucleus of the amygdala (CeA). Evoked IPSCs (eIPSCs) mediated by GABAA receptors were isolated from CeA neurons under whole-cell voltage clamp, and their response to selective BK channel antagonists, channel activators, or ethanol was analyzed. Blocking BK channels with the specific BK channel antagonist paxilline significantly increased the mean amplitude of eIPSCs, whereas the activation of BK channels with the channel opener NS1619 reversibly attenuated the mean amplitude of eIPSCs. Ethanol (50 mm) alone enhanced the amplitude of eIPSCs but failed to further enhance eIPSCs in the slices pretreated with paxilline. Bath application of either BK channel blockers significantly increased the frequency of miniature IPSCs (mIPSCs). Similarly, 50 mm ethanol alone also enhanced mIPSC frequency. Increases in mIPSC frequency by either selective BK channel antagonists or ethanol were not accompanied with changes in the amplitude of mIPSCs. Furthermore, following bath application of BK channel blockers for 10 min, ethanol failed to further increase mIPSC frequency. Together, these results suggest that blocking BK channels mimics the effects of ethanol on GABA release and that presynaptic BK channels could serve as a target for ethanol effects in CeA. PMID:25297098

  15. Regulation of viral and cellular promoter activity by polyomavirus early proteins.

    PubMed Central

    Pannuti, A; La Mantia, G; Lania, L

    1987-01-01

    The chloramphenicol-acetyl-transferase (CAT) expression system has been utilized to study the ability of the polyomavirus (Py) early proteins, the 100K large T, the 55K middle T and 22K small T-antigens, to activate a variety of eukaryotic promoters (the SV40 early, the alpha 2(1) collagen, the rabbit beta-globin, the polyomavirus early and the H-2 class I) in both transient and stable expression assays. We have found that either the complete polyomavirus early region or a plasmid expressing only the 55K middle T-antigen are capable of stimulating the expression of all the promoter-CAT plasmids in transient co-transfection experiments in both NIH-3T3 and Rat-2 cells. Conversely, the Py early proteins do not stimulate the transcription of most of the promoter-CAT genes stably introduced in the cell chromosomes, with the exception of H-2 class I promoter, when stimulation of transcription has been observed upon infection with recombinant retrovirus encoding the Py middle T-antigen. Images PMID:3029721

  16. The human polyomaviruses KI and WU: virological background and clinical implications.

    PubMed

    Babakir-Mina, Muhammed; Ciccozzi, Massimo; Perno, Carlo Federico; Ciotti, Marco

    2013-08-01

    In 2007, two novel polyomaviruses KI and WU were uncovered in the respiratory secretions of children with acute respiratory symptoms. Seroepidemiological studies showed that infection by these viruses is widespread in the human population. Following these findings, different biological specimens and body compartments have been screened by real-time PCR in the attempt to establish a pathogenetic role for KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) in human diseases. Although both viruses have been found mainly in respiratory tract samples of immunocompromised patients, a clear causative link with the respiratory disease has not been established. Indeed, the lack of specific clinical or radiological findings, the frequent co-detection with other respiratory pathogens, the detection in subjects without signs or symptoms of respiratory disease, and the variability of the viral loads measured did not allow drawing a definitive conclusion. Prospective studies carried out on a large sample size including both immunocompromised and immunocompetent patients with and without respiratory symptoms are needed. Standardized quantitative real-time PCR methods, definition of a clear clinical cutoff value, timing in the collection of respiratory samples, are also crucial to understand the pathogenic role, if any, of KIPyV and WUPyV in human pathology.

  17. Identification of an avian polyomavirus associated with Adélie penguins (Pygoscelis adeliae).

    PubMed

    Varsani, Arvind; Porzig, Elizabeth L; Jennings, Scott; Kraberger, Simona; Farkas, Kata; Julian, Laurel; Massaro, Melanie; Ballard, Grant; Ainley, David G

    2015-04-01

    Little is known about viruses associated with Antarctic animals, although they are probably widespread. We recovered a novel polyomavirus from Adélie penguin (Pygoscelis adeliae) faecal matter sampled in a subcolony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adélie penguin polyomavirus (AdPyV) has a typical polyomavirus genome organization with three ORFs that encoded capsid proteins on the one strand and two non-structural protein-coding ORFs on the complementary strand. The genome of AdPyV shared ~60 % pairwise identity with all avipolyomaviruses. Maximum-likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino acid sequences showed that the T-Ag of AdPyV clustered with those of avipolyomaviruses, sharing between 48 and 52 % identities. Only three viruses associated with Adélie penguins have been identified at a genomic level, avian influenza virus subtype H11N2 from the Antarctic Peninsula and, respectively, Pygoscelis adeliae papillomavirus and AdPyV from capes Crozier and Royds on Ross Island.

  18. Identification of a Novel Polyomavirus from Patients with Acute Respiratory Tract Infections

    PubMed Central

    Gaynor, Anne M; Nissen, Michael D; Whiley, David M; Mackay, Ian M; Lambert, Stephen B; Wu, Guang; Brennan, Daniel C; Storch, Gregory A; Sloots, Theo P; Wang, David

    2007-01-01

    We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus–specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen. PMID:17480120

  19. Conserved Features in Papillomavirus and Polyomavirus Capsids

    PubMed Central

    Belnap, David M.; Olson, Norman H.; Cladel, Nancy M.; Newcomb, William W.; Brown, Jay C.; Kreider, John W.; Christensen, Neil D.; Baker, Timothy S.

    2014-01-01

    Capsids of papilloma and polyoma viruses (papovavirus family) are composed of 72 pentameric capsomeres arranged on a skewed icosahedral lattice (triangulation number of seven, T = 7). Cottontail rabbit papillo mavirus (CRPV) was reported previously to be a T = 7laevo (left-handed) structure, whereas human wart virus, simian virus 40, and murine polyomavirus were shown to be T = 7dextro (right-handed). The CRPV structure determined by cryoelectron microscopy and image reconstruction was similar to previously determined structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1). CRPV capsids were observed in closed (compact) and open (swollen) forms. Both forms have star-shaped capsomeres, as do BPV-1 and HPV-1, but the open CRPV capsids are ~2 nm larger in radius. The lattice hands of all papillomaviruses examined in this study were found to be T = 7dextro. In the region of maximum contact, papillomavirus capsomeres interact in a manner similar to that found in polyomaviruses. Although papilloma and polyoma viruses have differences in capsid size (~60 versus ~50 nm), capsomere morphology (11 to 12 nm star-shaped versus 8 nm barrel-shaped), and intercapsomere interactions (slightly different contacts between capsomeres), papovavirus capsids have a conserved, 72-pentamer, T = 7dextro structure. These features are conserved despite significant differences in amino acid sequences of the major capsid proteins. The conserved features may be a consequence of stable contacts that occur within capsomeres and flexible links that form among capsomeres. PMID:8656427

  20. Mouse Polyomavirus: Propagation, Purification, Quantification, and Storage.

    PubMed

    Horníková, Lenka; Žíla, Vojtěch; Španielová, Hana; Forstová, Jitka

    2015-08-03

    Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non-enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5-kbp-long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented.

  1. Modulation of BK channels by ethanol

    PubMed Central

    Dopico, Alex M.; Bukiya, Anna N.; Kuntamallappanavar, Guruprasad; Liu, Jianxi

    2017-01-01

    In alcohol-naïve systems, ethanol (<100 mM) exposure of calcium-gated BK channels perturbs physiology and behavior. Brief (several minutes) ethanol exposure usually leads to increased BK current, which results from ethanol interaction with a pocket mapped to the BK channel-forming slo1 protein cytosolic tail domain. The importance of this region in alcohol-induced intoxication has been addressed in Caenorhabditis elegans slo1 mutants. However, ethanol-induced BK activation is not universal as refractoriness and inhibition have been reported. The final effect depends on many factors, including intracellular calcium levels, slo1 isoform, BK beta subunit composition, post-translational modification of BK proteins, channel lipid microenvironment and type of ethanol administration. Studies in Drosophila melanogaster, Caenorhabditis elegans and rodents show that protracted/repeated ethanol administration leads to tolerance to alcohol-induced modification of BK-driven physiology and behavior. Unveiling the mechanisms underlying tolerance is of major importance, as tolerance to alcohol has been proposed as predictor of risk for alcoholism. PMID:27238266

  2. Essential role for smooth muscle BK channels in alcohol-induced cerebrovascular constriction

    NASA Astrophysics Data System (ADS)

    Liu, Pengchong; Xi, Qi; Ahmed, Abu; Jaggar, Jonathan H.; Dopico, Alejandro M.

    2004-12-01

    Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca2+ concentration and caused a reversible constriction of cerebral arteries (EC50 = 27 mM; Emax = 100 mM) that depended on voltage-gated Ca2+ entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca2+ currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (-53%) and amplitude (-32%) of transient Ca2+-activated K+ (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca2+ spark frequency (-49%), a subsarcolemmal Ca2+ signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (-44%). In contrast, ethanol failed to modify Ca2+ waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca2+ spark/BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.

  3. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    NASA Technical Reports Server (NTRS)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  4. A Novel Polyomavirus (Goose Hemorrhagic Polyomavirus) Is the Agent of Hemorrhagic Nephritis Enteritis of Geese

    PubMed Central

    Guerin, Jean-Luc; Gelfi, Jacqueline; Dubois, Luc; Vuillaume, Aimé; Boucraut-Baralon, Corine; Pingret, Jean-Luc

    2000-01-01

    We have identified the etiological agent of hemorrhagic nephritis enteritis of geese (HNEG), a fatal disease of European geese. HNEG has been recognized in almost all goose breeding areas, with an epizootic pattern, and up to now, the infectious agent has remained unknown. In order to identify the causative agent, infected tissues from HNEG-affected geese were inoculated to 1-day-old goslings, which then developed clinical signs typical of HNEG. Tissue homogenates from these birds were subjected to Freon extraction followed by sucrose density gradient ultracentrifugation. The resulting main band was examined by electron microscopy and consisted of spherical, naked, papovavirus-like particles approximately 45 nm in diameter. The virus was isolated and propagated in goose kidney cell primary culture. Tissue- or culture-purified virus allowed the experimental reproduction of the disease in goslings. Random PCR amplification of viral nucleic acid produced a 1,175-bp fragment which was shown to be associated with field samples collected from geese affected by HNEG on commercial farms in France. Sequence analysis of the PCR product revealed a unique open reading frame, showing 63 to 72% amino acid similarity with the major capsid protein (VP1) of several polyomaviruses. Finally, based on phylogenetic analysis, we conclude that the causative agent of HNEG is closely related to but clearly distinct from other polyomaviruses; we thus have named this newly identified virus Goose hemorrhagic polyomavirus. PMID:10775588

  5. Seroprevalence and cross-reactivity of human polyomavirus 9.

    PubMed

    Nicol, Jérôme T J; Touzé, Antoine; Robinot, Rémy; Arnold, Francoise; Mazzoni, Elisa; Tognon, Mauro; Coursaget, Pierre

    2012-08-01

    Many humans have antibodies against simian lymphotropic polyomavirus (LPyV), but its DNA has not been found in humans. Identification of human polyomavirus 9 (HPyV9) led us to compare the seroprevalence and cross-reactivity of LPyV and HpyV9. Results could indicate that humans who have antibodies against LPyV are infected by HPyV9.

  6. Fast activating voltage- and calcium-dependent potassium (BK) conductance promotes bursting in pituitary cells: a dynamic clamp study

    PubMed Central

    Tabak, Joël; Tomaiuolo, Maurizio; Gonzalez-Iglesias, Arturo E.; Milescu, Lorin S.; Bertram, Richard

    2011-01-01

    The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. We demonstrate here that BK channels indeed promote bursting activity in pituitary cells. Blocking BK channels in bursting lacto-somatotroph GH4C1 cells changes their firing activity to spiking, while further adding an artificial BK conductance via dynamic clamp restores bursting. Importantly, this burst-promoting effect requires a relatively fast BK activation/deactivation, as predicted by computational models. We also show that adding a fast activating BK conductance to spiking gonadotrophs converts the activity of these cells to bursting. Together, our results suggest that differences in BK channel expression may underlie the differences in electrical activity and basal hormone secretion levels among pituitary cell types and that the rapid rate of BK channel activation is key to its role in burst promotion. PMID:22090511

  7. BK Knockout by TALEN-Mediated Gene Targeting in Osteoblasts: KCNMA1 Determines the Proliferation and Differentiation of Osteoblasts

    PubMed Central

    Hei, Hongya; Gao, Jianjun; Dong, Jibin; Tao, Jie; Tian, Lulu; Pan, Wanma; Wang, Hongyu; Zhang, Xuemei

    2016-01-01

    Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BK-knockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases. PMID:27329042

  8. Heme Regulates Allosteric Activation of the Slo1 BK Channel

    PubMed Central

    Horrigan, Frank T.; Heinemann, Stefan H.; Hoshi, Toshinori

    2005-01-01

    Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state. PMID:15955873

  9. Heme regulates allosteric activation of the Slo1 BK channel.

    PubMed

    Horrigan, Frank T; Heinemann, Stefan H; Hoshi, Toshinori

    2005-07-01

    Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531-535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G-V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G-V simulated by weakening the coupling of both Ca(2+) binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca(2+)- and voltage-dependent gating as well as intrinsic stability of the open state.

  10. Low Na, High K Diet and the Role of Aldosterone in BK-Mediated K Excretion

    PubMed Central

    Cornelius, Ryan J.; Wen, Donghai; Li, Huaqing; Yuan, Yang; Wang-France, Jun; Warner, Paige C.; Sansom, Steven C.

    2015-01-01

    A low Na, high K diet (LNaHK) is associated with a low rate of cardiovascular (CV) disease in many societies. Part of the benefit of LNaHK relies on its diuretic effects; however, the role of aldosterone (aldo) in the diuresis is not understood. LNaHK mice exhibit an increase in renal K secretion that is dependent on the large, Ca-activated K channel, (BK-α with accessory BK-β4; BK-α/β4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/β4-mediated K secretion in LNaHK mice. We found that the plasma aldo concentration (P[aldo]) was elevated by 10-fold in LNaHK mice compared with control diet (Con) mice. We subjected LNaHK mice to either sham surgery (sham), adrenalectomy (ADX) with low aldo replacement (ADX-LA), or ADX with high aldo replacement (ADX-HA). Compared to sham, the urinary flow, K excretion rate, transtubular K gradient (TTKG), and BK-α and BK-β4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-β4 knockout (β4KO) and WT mice exhibited similar K clearance and TTKG in the ADX-LA groups; however, in sham and ADX-HA, the K clearance and TTKG of β4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in β4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/β4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease. PMID:25607984

  11. Closed state-coupled C-type inactivation in BK channels.

    PubMed

    Yan, Jiusheng; Li, Qin; Aldrich, Richard W

    2016-06-21

    Ion channels regulate ion flow by opening and closing their pore gates. K(+) channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel's activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K(+) together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K(+) channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca(2+)]i) and recovered with depolarized membrane potentials or elevated [Ca(2+)]i Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel's normal closing may represent an early conformational stage of C-type inactivation.

  12. Time-dependent effects of ethanol on BK channel expression and trafficking in hippocampal neurons

    PubMed Central

    Palacio, Stephanie; Velázquez-Marrero, Cristina; Marrero, Héctor G.; Seale, Garrett E.; Yudowski, Guillermo A.; Treistman, Steven N.

    2016-01-01

    Background The large conductance Ca+2 – and voltage-activated K+ channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to EtOH. Methods Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence (TIRF) microscopy, electrophysiology and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time. Results Exposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10-min of EtOH exposure that remained evident after 3-hrs, though not correlated to increases in functional channel expression. In contrast, after 6-hrs of EtOH exposure we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24-hrs EtOH exposure, perimembrane levels of BK had returned to baseline. Conclusion We report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6-hrs of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. Since molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel

  13. Long-term hypoxia increases calcium affinity of BK channels in ovine fetal and adult cerebral artery smooth muscle.

    PubMed

    Tao, Xiaoxiao; Lin, Mike T; Thorington, Glyne U; Wilson, Sean M; Longo, Lawrence D; Hessinger, David A

    2015-04-01

    Acclimatization to high-altitude, long-term hypoxia (LTH) reportedly alters cerebral artery contraction-relaxation responses associated with changes in K(+) channel activity. We hypothesized that to maintain oxygenation during LTH, basilar arteries (BA) in the ovine adult and near-term fetus would show increased large-conductance Ca(2+) activated potassium (BK) channel activity. We measured BK channel activity, expression, and cell surface distribution by use of patch-clamp electrophysiology, flow cytometry, and confocal microscopy, respectively, in myocytes from normoxic control and LTH adult and near-term fetus BA. Electrophysiological data showed that BK channels in LTH myocytes exhibited 1) lowered Ca(2+) set points, 2) left-shifted activation voltages, and 3) longer dwell times. BK channels in LTH myocytes also appeared to be more dephosphorylated. These differences collectively make LTH BK channels more sensitive to activation. Studies using flow cytometry showed that the LTH fetus exhibited increased BK β1 subunit surface expression. In addition, in both fetal groups confocal microscopy revealed increased BK channel clustering and colocalization to myocyte lipid rafts. We conclude that increased BK channel activity in LTH BA occurred in association with increased channel affinity for Ca(2+) and left-shifted voltage activation. Increased cerebrovascular BK channel activity may be a mechanism by which LTH adult and near-term fetal sheep can acclimatize to long-term high altitude hypoxia. Our findings suggest that increasing BK channel activity in cerebral myocytes may be a therapeutic target to ameliorate the adverse effects of high altitude in adults or of intrauterine hypoxia in the fetus.

  14. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    PubMed Central

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  15. Stimulation of BK virus DNA replication by NFI family transcription factors.

    PubMed

    Liang, Bo; Tikhanovich, Irina; Nasheuer, Heinz Peter; Folk, William R

    2012-03-01

    BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.

  16. Two classes of regulatory subunits coassemble in the same BK channel and independently regulate gating

    NASA Astrophysics Data System (ADS)

    Gonzalez-Perez, Vivian; Xia, Xiao-Ming; Lingle, Christopher J.

    2015-09-01

    High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes. However, whether different partners have additive or cooperative functional effects, or whether some combinations of proteins may preclude assembly of others are largely unexplored topics. The large conductance Ca2+-and-voltage activated potassium channel (BK) is well-suited to discern nuanced differences in regulation arising from combinations of subunits. Here we examine whether assembly of two different classes of regulatory proteins, β and γ, in BK channels is exclusive or independent. Our results show that both γ1 and up to four β2-subunits can coexist in the same functional BK complex, with the gating shift caused by β2-subunits largely additive with that produced by the γ1-subunit(s). The multiplicity of β:γ combinations that can participate in a BK complex therefore allow a range of BK channels with distinct functional properties tuned by the specific stoichiometry of the contributing subunits.

  17. [BK virus infections in kidney transplantation].

    PubMed

    Lanot, Antoine; Bouvier, Nicolas; Chatelet, Valérie; Dina, Julia; Béchade, Clémence; Ficheux, Maxence; Henri, Patrick; Lobbedez, Thierry; Hurault de Ligny, Bruno

    2016-04-01

    BK virus is near ubiquitous, with a seroprevalence of around 80% in the general population. Subsequent to an asymptomatic primary infection, BK virus then remains dormant in healthy subjects. Reactivation occurs in immunocompromised people. BKv is pathogenic mainly among patients who have received a kidney transplant, in whom the virus can cause specific tubulo-interstitial nephritis and even result in graft failure among approximately 20 to 30% of nephritic cases. Since the mid 90 s, incidence has increased with the use of new powerful immunosuppressor treatments. The cornerstone of BK virus infection or BK virus-associated nephropathy treatment is a decrease of the immunosuppressive regimen, which must then be offset with the risk of rejection. The use of several adjuvant therapies has been submitted (fluoroquinolones, leflunomide, intravenous immunoglobulins, cidofovir), with no sufficient proof enabling the recommendation of first-line prescription. The high frequency of this infection and its potential harmfulness argue for the use of prevention strategies, at least among patients presenting risk factors. Retransplantation is safe after a first kidney allograft loss caused by BK-virus nephropathy, on condition that a screening for viremia is frequently conducted. Copyright © 2015 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

  18. Reactive oxygen species signaling facilitates FOXO-3a/FBXO-dependent vascular BK channel β1 subunit degradation in diabetic mice.

    PubMed

    Lu, Tong; Chai, Qiang; Yu, Ling; d'Uscio, Livius V; Katusic, Zvonimir S; He, Tongrong; Lee, Hon-Chi

    2012-07-01

    Activity of the vascular large conductance Ca(2+)-activated K(+) (BK) channel is tightly regulated by its accessory β(1) subunit (BK-β(1)). Downregulation of BK-β(1) expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)-dependent F-box-only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β(1) degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β(1) expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator-activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β(1) degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β(1) degradation and protected coronary function in diabetes.

  19. RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.

    PubMed Central

    Brabant, F; Acheson, N H

    1995-01-01

    RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion. PMID:7769704

  20. TRPV1 channels are functionally coupled with BK(mSlo1) channels in rat dorsal root ganglion (DRG) neurons.

    PubMed

    Wu, Ying; Liu, Yongfeng; Hou, Panpan; Yan, Zonghe; Kong, Wenjuan; Liu, Beiying; Li, Xia; Yao, Jing; Zhang, Yuexuan; Qin, Feng; Ding, Jiuping

    2013-01-01

    The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca(2+)). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca(2+) influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the "pain" signal transduction pathway in the peripheral nervous system.

  1. TRPV1 Channels Are Functionally Coupled with BK(mSlo1) Channels in Rat Dorsal Root Ganglion (DRG) Neurons

    PubMed Central

    Yan, Zonghe; Kong, Wenjuan; Liu, Beiying; Li, Xia; Yao, Jing; Zhang, Yuexuan; Qin, Feng; Ding, Jiuping

    2013-01-01

    The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca2+). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca2+ influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the “pain” signal transduction pathway in the peripheral nervous system. PMID:24147119

  2. BK nephropathy in the native kidneys of patients with organ transplants: Clinical spectrum of BK infection.

    PubMed

    Vigil, Darlene; Konstantinov, Nikifor K; Barry, Marc; Harford, Antonia M; Servilla, Karen S; Kim, Young Ho; Sun, Yijuan; Ganta, Kavitha; Tzamaloukas, Antonios H

    2016-09-24

    Nephropathy secondary to BK virus, a member of the Papoviridae family of viruses, has been recognized for some time as an important cause of allograft dysfunction in renal transplant recipients. In recent times, BK nephropathy (BKN) of the native kidneys has being increasingly recognized as a cause of chronic kidney disease in patients with solid organ transplants, bone marrow transplants and in patients with other clinical entities associated with immunosuppression. In such patients renal dysfunction is often attributed to other factors including nephrotoxicity of medications used to prevent rejection of the transplanted organs. Renal biopsy is required for the diagnosis of BKN. Quantitation of the BK viral load in blood and urine are surrogate diagnostic methods. The treatment of BKN is based on reduction of the immunosuppressive medications. Several compounds have shown antiviral activity, but have not consistently shown to have beneficial effects in BKN. In addition to BKN, BK viral infection can cause severe urinary bladder cystitis, ureteritis and urinary tract obstruction as well as manifestations in other organ systems including the central nervous system, the respiratory system, the gastrointestinal system and the hematopoietic system. BK viral infection has also been implicated in tumorigenesis. The spectrum of clinical manifestations from BK infection and infection from other members of the Papoviridae family is widening. Prevention and treatment of BK infection and infections from other Papovaviruses are subjects of intense research.

  3. BK nephropathy in the native kidneys of patients with organ transplants: Clinical spectrum of BK infection

    PubMed Central

    Vigil, Darlene; Konstantinov, Nikifor K; Barry, Marc; Harford, Antonia M; Servilla, Karen S; Kim, Young Ho; Sun, Yijuan; Ganta, Kavitha; Tzamaloukas, Antonios H

    2016-01-01

    Nephropathy secondary to BK virus, a member of the Papoviridae family of viruses, has been recognized for some time as an important cause of allograft dysfunction in renal transplant recipients. In recent times, BK nephropathy (BKN) of the native kidneys has being increasingly recognized as a cause of chronic kidney disease in patients with solid organ transplants, bone marrow transplants and in patients with other clinical entities associated with immunosuppression. In such patients renal dysfunction is often attributed to other factors including nephrotoxicity of medications used to prevent rejection of the transplanted organs. Renal biopsy is required for the diagnosis of BKN. Quantitation of the BK viral load in blood and urine are surrogate diagnostic methods. The treatment of BKN is based on reduction of the immunosuppressive medications. Several compounds have shown antiviral activity, but have not consistently shown to have beneficial effects in BKN. In addition to BKN, BK viral infection can cause severe urinary bladder cystitis, ureteritis and urinary tract obstruction as well as manifestations in other organ systems including the central nervous system, the respiratory system, the gastrointestinal system and the hematopoietic system. BK viral infection has also been implicated in tumorigenesis. The spectrum of clinical manifestations from BK infection and infection from other members of the Papoviridae family is widening. Prevention and treatment of BK infection and infections from other Papovaviruses are subjects of intense research. PMID:27683628

  4. Nitroblue tetrazolium blocks BK channels in cerebrovascular smooth muscle cell membranes

    PubMed Central

    Ye, D; Pospisilik, J A; Mathers, D A

    2000-01-01

    The effects of p-nitroblue tetrazolium (NBT) on large conductance, calcium-activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined. Patch clamp methods were employed to record single BK channel currents from inside-out patches of CVMC membrane maintained at 21–23°C. When applied to the cytoplasmic face of inside-out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean open time of BK channels, without changing channel conductance. NBT altered the frequency distribution of BK channel open times from a two exponential to a single exponential form. In the absence of NBT, mean channel open time increased on membrane depolarization. In the presence of internally applied NBT, mean channel open became essentially independent of membrane potential. Internally applied NBT also reduced the mean closed time of BK channels when measured at membrane potentials in the range −80 mV to +20 mV. The combined effects of internal NBT on mean open and closed times resulted in the suppression of BK channel open probability when measured at positive membrane potentials. When applied to the external membrane face, micromolar concentrations of NBT reduced mean channel open time progressively as the membrane was hyperpolarized, and also reduced open probability at negative membrane potentials. A model is proposed in which NBT alters channel gating by binding to a site at or near to the cytoplasmic membrane face. Externally applied NBT suppressed BK channel open probability at concentrations which also inhibit nitric oxide synthase (NOS). Therefore, the potential role of potassium channel block in NBT actions previously attributed to NOS inhibition is discussed. PMID:10696106

  5. Central role of the BK channel in urinary bladder smooth muscle physiology and pathophysiology.

    PubMed

    Petkov, Georgi V

    2014-09-15

    The physiological functions of the urinary bladder are to store and periodically expel urine. These tasks are facilitated by the contraction and relaxation of the urinary bladder smooth muscle (UBSM), also known as detrusor smooth muscle, which comprises the bladder wall. The large-conductance voltage- and Ca(2+)-activated K(+) (BK, BKCa, MaxiK, Slo1, or KCa1.1) channel is highly expressed in UBSM and is arguably the most important physiologically relevant K(+) channel that regulates UBSM function. Its significance arises from the fact that the BK channel is the only K(+) channel that is activated by increases in both voltage and intracellular Ca(2+). The BK channels control UBSM excitability and contractility by maintaining the resting membrane potential and shaping the repolarization phase of the spontaneous action potentials that determine UBSM spontaneous rhythmic contractility. In UBSM, these channels have complex regulatory mechanisms involving integrated intracellular Ca(2+) signals, protein kinases, phosphodiesterases, and close functional interactions with muscarinic and β-adrenergic receptors. BK channel dysfunction is implicated in some forms of bladder pathologies, such as detrusor overactivity, and related overactive bladder. This review article summarizes the current state of knowledge of the functional role of UBSM BK channels under normal and pathophysiological conditions and provides new insight toward the BK channels as targets for pharmacological or genetic control of UBSM function. Modulation of UBSM BK channels can occur by directly or indirectly targeting their regulatory mechanisms, which has the potential to provide novel therapeutic approaches for bladder dysfunction, such as overactive bladder and detrusor underactivity.

  6. BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus.

    PubMed

    Raffaelli, Giacomo; Saviane, Chiara; Mohajerani, Majid H; Pedarzani, Paola; Cherubini, Enrico

    2004-05-15

    Large conductance calcium- and voltage-activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an 'emergency brake' that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 microM) and iberiotoxin (100 nM) increase the frequency, but not the amplitude, of spontaneously occurring action potential-dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch-clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired-pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired-pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones.

  7. Mortality due to polyomavirus infection in two nightjars (Caprimulgus europaeus).

    PubMed

    Arroube, Ana Sofia; Halami, Mohammad Yahya; Johne, Reimar; Dorrestein, Gerry M

    2009-06-01

    Two nightjars (Caprimulgus europaeus) from a bird park in the Netherlands died suddenly, with no clinical signs, within 1 month of each other. The main pathologic findings at necropsy were splenomegaly and hepatic necrosis. On histologic examination, intranuclear viral inclusion bodies consistent with avian polyomavirus were observed in the liver, spleen, and kidneys. Polymerase chain reaction testing of samples from the liver, spleen, and kidneys detected avian polyomaviral DNA, and sequence analysis showed that the virus had a sequence homology of 99% to psittacine avian polyomavirus strains. To our knowledge, this is the first report of avian polyomavirus infection in the order Caprimulgiformes. Lovebirds (Agapornis species), which were housed near the nightjars, were considered as the possible source of infection.

  8. Blocking the BK Channel Impedes Acquisition of Trace Eyeblink Conditioning

    ERIC Educational Resources Information Center

    Matthews, Elizabeth A.; Disterhoft, John F.

    2009-01-01

    Big-K[superscript +] conductance (BK)-channel mediated fast afterhyperpolarizations (AHPs) following action potentials are reduced after eyeblink conditioning. Blocking BK channels with paxilline increases evoked firing frequency in vitro and spontaneous pyramidal activity in vivo. To examine how increased excitability after BK-channel blockade…

  9. Blocking the BK Channel Impedes Acquisition of Trace Eyeblink Conditioning

    ERIC Educational Resources Information Center

    Matthews, Elizabeth A.; Disterhoft, John F.

    2009-01-01

    Big-K[superscript +] conductance (BK)-channel mediated fast afterhyperpolarizations (AHPs) following action potentials are reduced after eyeblink conditioning. Blocking BK channels with paxilline increases evoked firing frequency in vitro and spontaneous pyramidal activity in vivo. To examine how increased excitability after BK-channel blockade…

  10. Multiorgan WU Polyomavirus Infection in Bone Marrow Transplant Recipient.

    PubMed

    Siebrasse, Erica A; Nguyen, Nang L; Willby, Melisa J; Erdman, Dean D; Menegus, Marilyn A; Wang, David

    2016-01-01

    WU polyomavirus (WUPyV) was detected in a bone marrow transplant recipient with severe acute respiratory distress syndrome who died in 2001. Crystalline lattices of polyomavirus-like particles were observed in the patient's lung by electron microscopy. WUPyV was detected in the lung and other tissues by real-time quantitative PCR and identified in the lung and trachea by immunohistochemistry. A subset of WUPyV-positive cells in the lung had morphologic features of macrophages. Although the role of WUPyV as a human pathogen remains unclear, these results clearly demonstrate evidence for infection of respiratory tract tissues in this patient.

  11. Multiorgan WU Polyomavirus Infection in Bone Marrow Transplant Recipient

    PubMed Central

    Siebrasse, Erica A.; Nguyen, Nang L.; Willby, Melisa J.; Erdman, Dean D.; Menegus, Marilyn A.

    2016-01-01

    WU polyomavirus (WUPyV) was detected in a bone marrow transplant recipient with severe acute respiratory distress syndrome who died in 2001. Crystalline lattices of polyomavirus-like particles were observed in the patient’s lung by electron microscopy. WUPyV was detected in the lung and other tissues by real-time quantitative PCR and identified in the lung and trachea by immunohistochemistry. A subset of WUPyV-positive cells in the lung had morphologic features of macrophages. Although the role of WUPyV as a human pathogen remains unclear, these results clearly demonstrate evidence for infection of respiratory tract tissues in this patient. PMID:26691850

  12. Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    PubMed Central

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J.; Ströh, Luisa; Nitsch, Roger M.; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2016-01-01

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show “recognition holes”; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  13. JC Polyomavirus Infection of Primary Human Renal Epithelial Cells Is Controlled by a Type I IFN-Induced Response

    PubMed Central

    Assetta, Benedetta; De Cecco, Marco; O’Hara, Bethany

    2016-01-01

    ABSTRACT The JC and BK human polyomaviruses (JCPyV and BKPyV, respectively) establish lifelong persistent infections in the kidney. In immunosuppressed individuals, JCPyV causes progressive multifocal leukoencephalopathy (PML), a fatal neurodegenerative disease, and BKPyV causes polyomavirus-associated nephropathy (PVN). In this study, we compared JCPyV and BKPyV infections in primary human renal proximal tubule epithelial (HRPTE) cells. JCPyV established a persistent infection, but BKPyV killed the cells in 15 days. To identify the cellular factors responsible for controlling JCPyV infection and promoting viral persistence, we profiled the transcriptomes of JCPyV- and BKPyV-infected cells at several time points postinfection. We found that infection with both viruses induced interferon production but that interferon-stimulated genes (ISGs) were only activated in the JCPyV-infected cells. Phosphorylated STAT1 and IRF9, which are responsible for inducing ISGs, translocated to the nucleus of JCPyV-infected cells but did not in BKPyV-infected cells. In BKPyV-infected cells, two critical suppressors of cytokine signaling, SOCS3 and SOCS1, were induced. Infection with BKPyV but not JCPyV caused reorganization of PML bodies that are associated with inactivating antiviral responses. Blockade of the interferon receptor and neutralization of soluble interferon alpha (IFN-α) and IFN-β partially alleviated the block to JCPyV infection, leading to enhanced infectivity. Our results show that a type I IFN response contributes to the establishment of persistent infection by JCPyV in HRPTE cells. PMID:27381292

  14. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    SciTech Connect

    Noda, T.; Satake, M.; Robins, T.; Ito, Y.

    1986-10-01

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10/sup 6/ cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.

  15. The regulation of BK channel activity by pre- and post-translational modifications.

    PubMed

    Kyle, Barry D; Braun, Andrew P

    2014-01-01

    Large conductance, Ca(2+)-activated K(+) (BK) channels represent an important pathway for the outward flux of K(+) ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca(2+)]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner.

  16. Extrapolating microdomain Ca(2+) dynamics using BK channels as a Ca(2+) sensor.

    PubMed

    Hou, Panpan; Xiao, Feng; Liu, Haowen; Yuchi, Ming; Zhang, Guohui; Wu, Ying; Wang, Wei; Zeng, Wenping; Ding, Mingyue; Cui, Jianming; Wu, Zhengxing; Wang, Lu-Yang; Ding, Jiuping

    2016-01-18

    Ca(2+) ions play crucial roles in mediating physiological and pathophysiological processes, yet Ca(2+) dynamics local to the Ca(2+) source, either from influx via calcium permeable ion channels on plasmic membrane or release from internal Ca(2+) stores, is difficult to delineate. Large-conductance calcium-activated K(+) (BK-type) channels, abundantly distribute in excitable cells and often localize to the proximity of voltage-gated Ca(2+) channels (VGCCs), spatially enabling the coupling of the intracellular Ca(2+) signal to the channel gating to regulate membrane excitability and spike firing patterns. Here we utilized the sensitivity and dynamic range of BK to explore non-uniform Ca(2+) local transients in the microdomain of VGCCs. Accordingly, we applied flash photolysis of caged Ca(2+) to activate BK channels and determine their intrinsic sensitivity to Ca(2+). We found that uncaging Ca(2+) activated biphasic BK currents with fast and slow components (time constants being τf ≈ 0.2 ms and τs ≈ 10 ms), which can be accounted for by biphasic Ca(2+) transients following light photolysis. We estimated the Ca(2+)-binding rate constant kb (≈1.8 × 10(8)  M(-1) s(-1)) for mSlo1 and further developed a model in which BK channels act as a calcium sensor capable of quantitatively predicting local microdomain Ca(2+) transients in the vicinity of VGCCs during action potentials.

  17. Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

    PubMed Central

    Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

    2014-01-01

    Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

  18. Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice

    PubMed Central

    Sachse, Gregor; Faulhaber, Jörg; Seniuk, Anika; Ehmke, Heimo; Pongs, Olaf

    2014-01-01

    The large conductance voltage- and Ca2+-activated K+ (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1−/−) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1−/− mice independent of genetic background, BKβ1−/− strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1+/+ controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1−/− strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1+/+ strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKβ1 expression increased blood pressure in BKβ1−/− strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways. PMID:24687584

  19. Manipulation of BK channel expression is sufficient to alter auditory hair cell thresholds in larval zebrafish

    PubMed Central

    Rohmann, Kevin N.; Tripp, Joel A.; Genova, Rachel M.; Bass, Andrew H.

    2014-01-01

    Non-mammalian vertebrates rely on electrical resonance for frequency tuning in auditory hair cells. A key component of the resonance exhibited by these cells is an outward calcium-activated potassium current that flows through large-conductance calcium-activated potassium (BK) channels. Previous work in midshipman fish (Porichthys notatus) has shown that BK expression correlates with seasonal changes in hearing sensitivity and that pharmacologically blocking these channels replicates the natural decreases in sensitivity during the winter non-reproductive season. To test the hypothesis that reducing BK channel function is sufficient to change auditory thresholds in fish, morpholino oligonucleotides (MOs) were used in larval zebrafish (Danio rerio) to alter expression of slo1a and slo1b, duplicate genes coding for the pore-forming α-subunits of BK channels. Following MO injection, microphonic potentials were recorded from the inner ear of larvae. Quantitative real-time PCR was then used to determine the MO effect on slo1a and slo1b expression in these same fish. Knockdown of either slo1a or slo1b resulted in disrupted gene expression and increased auditory thresholds across the same range of frequencies of natural auditory plasticity observed in midshipman. We conclude that interference with the normal expression of individual slo1 genes is sufficient to increase auditory thresholds in zebrafish larvae and that changes in BK channel expression are a direct mechanism for regulation of peripheral hearing sensitivity among fishes. PMID:24803460

  20. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    PubMed

    Shruti, Sonal; Urban-Ciecko, Joanna; Fitzpatrick, James A; Brenner, Robert; Bruchez, Marcel P; Barth, Alison L

    2012-01-01

    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  1. The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression

    PubMed Central

    Shruti, Sonal; Urban-Ciecko, Joanna; Fitzpatrick, James A.; Brenner, Robert; Bruchez, Marcel P.; Barth, Alison L.

    2012-01-01

    The large-conductance K+ channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking. PMID:22438928

  2. The regulation of BK channel activity by pre- and post-translational modifications

    PubMed Central

    Kyle, Barry D.; Braun, Andrew P.

    2014-01-01

    Large conductance, Ca2+-activated K+ (BK) channels represent an important pathway for the outward flux of K+ ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca2+]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner. PMID:25202279

  3. A polyomavirus enhancer-binding protein, PEBP5, responsive to 12-O-tetradecanoylphorbol-13-acetate but distinct from AP-1.

    PubMed Central

    Asano, M; Murakami, Y; Furukawa, K; Yamaguchi-Iwai, Y; Satake, M; Ito, Y

    1990-01-01

    Element I, homologous to the adenovirus type 5 E1A enhancer core, is a 10-bp sequence in the A core of the polyomavirus enhancer and was shown previously to be responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that element I by itself was capable of activating polyomavirus DNA replication in COP-5 cells which express the polyomavirus large T antigen. A nuclear factor, polyomavirus enhancer-binding protein 5 (PEBP5), which bound to the entire sequence of element I and was responsive to TPA was identified by an in vitro binding assay. Although the binding site of PEBP5 partly overlaps with that of PEBP1 (PEA1), a member of the AP-1 family, PEBP5 appears to be a distinct factor. Since we previously showed that element I alone was able to activate transcription, our present results suggest that PEBP5 is involved in the regulation of both transcription and replication of DNA. The amount of PEBP5 increased after F9 cells were induced to differentiate by retinoic acid. A relatively large amount of PEBP5 was detected in lymphoid and trophoblast cells. Images PMID:2173774

  4. Evaluating the BK 21 Program. Research Brief

    ERIC Educational Resources Information Center

    Seong, Somi; Popper, Steven W.; Goldman, Charles A.; Evans, David K.; Grammich, Clifford A.

    2008-01-01

    The Brain Korea 21 program (BK21), an effort to improve Korean universities and research, has attracted a great deal of attention in Korea, producing the need to understand how well the program is meeting its goals. RAND developed a logic model for identifying program goals and dynamics, suggested quantitative and qualitative evaluation methods,…

  5. Evaluating the BK 21 Program. Research Brief

    ERIC Educational Resources Information Center

    Seong, Somi; Popper, Steven W.; Goldman, Charles A.; Evans, David K.; Grammich, Clifford A.

    2008-01-01

    The Brain Korea 21 program (BK21), an effort to improve Korean universities and research, has attracted a great deal of attention in Korea, producing the need to understand how well the program is meeting its goals. RAND developed a logic model for identifying program goals and dynamics, suggested quantitative and qualitative evaluation methods,…

  6. Rad51 activates polyomavirus JC early transcription.

    PubMed

    White, Martyn K; Kaminski, Rafal; Khalili, Kamel; Wollebo, Hassen S

    2014-01-01

    The human neurotropic polyomavirus JC (JCV) causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR). NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation--all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

  7. Polyomavirus middle T-antigen NPTY mutants.

    PubMed Central

    Druker, B J; Sibert, L; Roberts, T M

    1992-01-01

    A polyomavirus middle T-antigen (MTAg) mutant containing a substitution of Leu for Pro at amino acid 248 has previously been described as completely transformation defective (B. J. Druker, L. Ling, B. Cohen, T. M. Roberts, and B. S. Schaffhausen, J. Virol. 64:4454-4461, 1990). This mutant had no alterations in associated proteins or associated kinase activities compared with wild-type MTAg. Pro-248 lies in a tetrameric sequence, NPTY, which is reminiscent of the so-called NPXY sequence in the low-density-lipoprotein receptor. In the low-density-lipoprotein receptor, mutations in the NPXY motif but not in the surrounding amino acids abolish receptor function, apparently by decreasing receptor internalization (W. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265:3116-3123, 1990). To determine whether this sequence represents a functional motif in MTAg as well, a series of single amino acid substitutions was constructed in this region of MTAg. All of the mutations of N, P, T, or Y, including the relatively conservative substitution of Ser for Thr at amino acid 249, resulted in a transformation-defective MTAg, whereas mutations outside of this sequence allowed mutants to retain near-wild-type transformation capabilities. Transformation-defective mutants with mutations in the NPTY region behaved similarly to the mutant with the original Pro-248-to-Leu-248 mutation when assayed for associated proteins and activities in vitro; that is, they retained a full complement of wild-type activities and associated proteins. Further, insertion of the tetrameric sequence NPTY downstream of the mutated motif restored transforming abilities to these mutants. Thus, the tetrameric sequence NPTY in MTAg appears to represent a well-defined functional motif of MTAg. Images PMID:1326642

  8. Bromodomain Protein Brd4 Plays a Key Role in Merkel Cell Polyomavirus DNA Replication

    PubMed Central

    Wang, Xin; Li, Jing; Schowalter, Rachel M.; Jiao, Jing; Buck, Christopher B.; You, Jianxin

    2012-01-01

    Merkel cell polyomavirus (MCV or MCPyV) is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4) interacts with MCV large T antigen (LT) and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+) leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus. PMID:23144621

  9. Assembly and Purification of Polyomavirus-Like Particles from Plants.

    PubMed

    Catrice, Emeline V B; Sainsbury, Frank

    2015-10-01

    Polyomaviruses are small DNA viruses that have a history of use in biotechnology. The capsids of a number of species have been developed into experimental prophylactic and therapeutic virus-like particle (VLP) vaccines. In order to explore plants as a host for the expression and purification of polyomavirus-like particles, we have transiently expressed the major capsid protein, VP1, in Nicotiana benthamiana leaves. Deletion of a polybasic motif from the N-terminal region of VP1 resulted in increased expression as well as reduced necrosis of leaf tissue, which was associated with differences in subcellular localisation and reduced DNA binding by the deletion variant (ΔVP1). Self-assembled VLPs were recovered from tissue expressing both wild-type VP1 and ΔVP1 by density gradient ultracentrifugation. VLPs composed of ΔVP1 were more homogenous than wtVPLs and, unlike the latter, did not encapsidate nucleic acid. Such homogenous, empty VLPs are of great interest in biotechnology and nanotechnology. In addition, we show that both MPyV VLP variants assembled in plants can be produced with encapsidated foreign protein. Thus, this study demonstrates the utility of plant-based expression of polyomavirus-like particles and the suitability of this host for further developments in polyomavirus-based technologies.

  10. Relationship between auxiliary gamma subunits and mallotoxin on BK channel modulation

    PubMed Central

    Guan, Xin; Li, Qin; Yan, Jiusheng

    2017-01-01

    The large-conductance, calcium- and voltage-activated K+(BK) channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK channel by shifting its voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα channels by −72 mV. The channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/− mallotoxin) values of −9, −28, or −15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin’s channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin’s binding and action on BKα. PMID:28165042

  11. Collinearly-improved BK evolution meets the HERA data

    DOE PAGES

    Iancu, E.; Madrigal, J. D.; Mueller, A. H.; ...

    2015-10-03

    In a previous publication, we have established a collinearly-improved version of the Balitsky–Kovchegov (BK) equation, which resums to all orders the radiative corrections enhanced by large double transverse logarithms. Here, we study the relevance of this equation as a tool for phenomenology, by confronting it to the HERA data. To that aim, we first improve the perturbative accuracy of our resummation, by including two classes of single-logarithmic corrections: those generated by the first non-singular terms in the DGLAP splitting functions and those expressing the one-loop running of the QCD coupling. The equation thus obtained includes all the next-to-leading order correctionsmore » to the BK equation which are enhanced by (single or double) collinear logarithms. Furthermore, we then use numerical solutions to this equation to fit the HERA data for the electron–proton reduced cross-section at small Bjorken x. We obtain good quality fits for physically acceptable initial conditions. Our best fit, which shows a good stability up to virtualities as large as Q2 = 400 GeV2 for the exchanged photon, uses as an initial condition the running-coupling version of the McLerran–Venugopalan model, with the QCD coupling running according to the smallest dipole prescription.« less

  12. Identification of a novel polyomavirus from vervet monkeys in Zambia.

    PubMed

    Yamaguchi, Hiroki; Kobayashi, Shintaro; Ishii, Akihiro; Ogawa, Hirohito; Nakamura, Ichiro; Moonga, Ladslav; Hang'ombe, Bernard M; Mweene, Aaron S; Thomas, Yuka; Kimura, Takashi; Sawa, Hirofumi; Orba, Yasuko

    2013-06-01

    To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n = 100 each) of yellow baboons and vervet monkeys (VMs) (n = 50 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broad-spectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48 % nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen.

  13. Development of a DNA vaccine targeting Merkel cell polyomavirus.

    PubMed

    Zeng, Qi; Gomez, Bianca P; Viscidi, Raphael P; Peng, Shiwen; He, Liangmei; Ma, Barbara; Wu, T-C; Hung, Chien-Fu

    2012-02-08

    Merkel cell carcinoma (MCC) is a rare but devastating skin disease that is increasing in incidence within the United States. The poor prognosis of MCC patients and limited understanding of MCC pathogenesis warrants innovative treatments to control MCC. Several lines of evidence have pointed to Merkel cell polyomavirus (MCPyV) as the etiological agent of MCC. In particular, the amino terminus of MCPyV large T antigen (LT) (aa1-258) is expressed in all MCPyV-positive tumors and plays an important role in MCC oncogenesis, rendering it an ideal therapeutic target for vaccination. In the current study, we developed a DNA vaccine encoding MCPyV LT aa1-258 (pcDNA3-LT). Within our pcDNA3-LT DNA vaccine, we identified that MCPyV LT aa136-160 likely contains an LT-specific CD4+ T helper epitope. We have also created an LT-expressing B16/LT tumor model using B16, a murine melanoma cell line, to characterize the potency of our DNA vaccine. Using this tumorigenic B16/LT tumor model, we found that pcDNA3-LT DNA vaccine generates antitumor effects mainly mediated by CD4+ T cells against B16/LT tumors in vaccinated C57BL/6 mice. Thus, immunotherapy using pcDNA3-LT DNA vaccine may represent a promising approach for the control of MCPyV-associated lesions. The B16/LT tumor model further serves as a useful model for testing various vaccine strategies against MCC.

  14. Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): Reproducibility of Polyomavirus Immunohistochemistry in Kidney Allografts

    PubMed Central

    Adam, Benjamin; Randhawa, Parmjeet; Chan, Samantha; Zeng, Gang; Regele, Heinz; Kushner, Yael B.; Colvin, Robert B.; Reeve, Jeff; Mengel, Michael

    2014-01-01

    Immunohistochemistry is the gold standard for diagnosing (positive versus negative) polyomavirus BK (BKV) nephropathy and has the potential for disease staging based on staining intensity and quantification of infected cells. This multicenter trial evaluated the reproducibility of BKV immunohistochemistry among 81 pathologists at 60 institutions. Participants stained tissue microarray slides and scored them for staining intensity and percentage of positive nuclei. Staining protocol details and evaluation scores were collected online. Slides were returned for centralized panel re-evaluation and kappa statistics were calculated. Individual assessment of staining intensity and percentage was more reproducible than combined scoring. Inter-institutional reproducibility was moderate for staining intensity (κ=0.49) and percentage (κ=0.42), fair for combined (κ=0.25), and best for simple positive/negative scoring (κ=0.63). Inter-observer reproducibility was substantial for intensity (κ=0.74), percentage (κ=0.66), and positive/negative (κ=0.67), and moderate for combined scoring (κ=0.43). Inter-laboratory reproducibility was fair for intensity (κ=0.37), percentage (κ=0.40), and combined (κ=0.24), but substantial for positive/negative scoring (κ=0.78). BKV RNA copies/cell correlated with staining intensity (r=0.56) and percentage (r=0.62). These results indicate that BKV immunohistochemistry is reproducible between observers but scoring should be simplified to a single-feature schema. Standardization of tissue processing and staining protocols would further improve inter-laboratory reproducibility. PMID:25091177

  15. [Sequencing and analysis of the complete genome sequence of WU polyomavirus in Fuzhou, China].

    PubMed

    Xiu, Wen-qiong; Shen, Xiao-na; Liu, Guang-hua; Xie, Jian-feng; Kang, Yu-lan; Wang, Mei-ai; Zhang, Wen-qing; Weng, Qi-zhu; Yan, Yan-sheng

    2011-03-01

    WU polyomavirus (WUPyV), a new member of the genus Polyomavirus in the family Polyomaviridae, is recently found in patients with respiratory tract infections. In our study, the complete genome of the two WUPyV isolates (FZ18, FZTF) were sequenced and deposited in GenBank (accession nos. FJ890981, FJ890982). The two sequences of the WUPyV isolates in this study varied little from each other. Compared with other complete genome sequences of WUPyV in GenBank (strain B0, S1-S4, CLFF, accession nos. EF444549, EF444550, EF444551, EF444552, EF444553, EU296475 respectively), the sequence length in nucleotides is 5228bp, 1bp shorter than the known sequences. The deleted base pair was at nucleotide position 4536 in the non-coding region of large T antigen (LTAg). The genome of the WUPyV encoded for five proteins. They were three capsid proteins: VP2, VP1, VP3 and LTAg, small T antigen (STAg), respectively. To investigate whether these nucleotide sequences had any unique features, we compared the genome sequence of the 2 WUPyV isolates in Fuzhou, China to those documented in the GenBank database by using PHYLIP software version 3.65 and the neighbor-joining method. The 2 WUPyV strains in our study were clustered together. Strain FZTF was more closed to the reference strain B0 of Australian than strain FZ18.

  16. Quantitative considerations to gather maximum information from viral growth efficiency studies: the example of polyomavirus type BK (BKV).

    PubMed

    Funk, Georg A

    2010-08-01

    This short communication shows how the application of simple mathematical formulae allows researchers to extract maximum information from viral growth efficiency studies at virtually no additional costs (in terms of time or money), thus improving the comparability of results (growth rates, replicative capacities, efficacies of antivirals) between in vitro and in vivo growth efficiency studies. This could help in elucidating kinetic links between the molecular basis of virus function and clinical findings.

  17. Angiotensin II stimulates internalization and degradation of arterial myocyte plasma membrane BK channels to induce vasoconstriction.

    PubMed

    Leo, M Dennis; Bulley, Simon; Bannister, John P; Kuruvilla, Korah P; Narayanan, Damodaran; Jaggar, Jonathan H

    2015-09-15

    Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated K(+) (BK) channel α and auxiliary β1 subunits that modulate arterial contractility. In arterial myocytes, β1 subunits are stored within highly mobile rab11A-positive recycling endosomes. In contrast, BKα subunits are primarily plasma membrane-localized. Trafficking pathways for BKα and whether physiological stimuli that regulate arterial contractility alter BKα localization in arterial myocytes are unclear. Here, using biotinylation, immunofluorescence resonance energy transfer (immunoFRET) microscopy, and RNAi-mediated knockdown, we demonstrate that rab4A-positive early endosomes traffic BKα to the plasma membrane in myocytes of resistance-size cerebral arteries. Angiotensin II (ANG II), a vasoconstrictor, reduced both surface and total BKα, an effect blocked by bisindolylmaleimide-II, concanavalin A, and dynasore, protein kinase C (PKC), internalization, and endocytosis inhibitors, respectively. In contrast, ANG II did not reduce BKα mRNA, and sodium nitroprusside, a nitric oxide donor, did not alter surface BKα protein over the same time course. MG132 and bafilomycin A, proteasomal and lysosomal inhibitors, respectively, also inhibited the ANG II-induced reduction in surface and total BKα, resulting in intracellular BKα accumulation. ANG II-mediated BK channel degradation reduced BK currents in isolated myocytes and functional responses to iberiotoxin, a BK channel blocker, and NS1619, a BK activator, in pressurized (60 mmHg) cerebral arteries. These data indicate that rab4A-positive early endosomes traffic BKα to the plasma membrane in arterial myocytes. We also show that ANG II stimulates PKC-dependent BKα internalization and degradation. These data describe a unique mechanism by which ANG II inhibits arterial myocyte BK currents, by reducing surface channel number, to induce vasoconstriction. Copyright © 2015 the American Physiological Society.

  18. Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

    PubMed Central

    Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

    2013-01-01

    Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

  19. Reduced vascular smooth muscle BK channel current underlies heart failure-induced vasoconstriction in mice

    PubMed Central

    Wan, Elaine; Kushner, Jared S.; Zakharov, Sergey; Nui, Xiao-wei; Chudasama, Neelesh; Kelly, Christopher; Waase, Marc; Doshi, Darshan; Liu, Guoxia; Iwata, Shinichi; Shiomi, Takayuki; Katchman, Alexander; D'Armiento, Jeanine; Homma, Shunichi; Marx, Steven O.

    2013-01-01

    Excessively increased peripheral vasoconstriction is a hallmark of heart failure (HF). Here, we show that in mice with systolic HF post–myocardial infarction, the myogenic tone of third-order mesenteric resistance vessels is increased, the vascular smooth muscle (VSM) membrane potential is depolarized by ∼20 mV, and vessel wall intracellular [Ca2+] is elevated relative to that in sham-operated control mice. Despite the increased [Ca2+], the frequency and amplitude of spontaneous transient outward currents (STOCs), mediated by large conductance, Ca2+-activated BK channels, were reduced by nearly 80% (P<0.01) and 25% (P<0.05), respectively, in HF. The expression of the BK α and β1 subunits was reduced in HF mice compared to controls (65 and 82% lower, respectively, P<0.01). Consistent with the importance of a reduction in BK channel expression and function in mediating the HF-induced increase in myogenic tone are two further findings: a blunting of paxilline-induced increase in myogenic tone in HF mice compared to controls (0.9 vs. 10.9%, respectively), and that HF does not alter the increased myogenic tone of BK β1-null mice. These findings identify electrical dysregulation within VSM, specifically the reduction of BK currents, as a key molecular mechanism sensitizing resistance vessels to pressure-induced vasoconstriction in systolic HF.—Wan, E., Kushner, J. S., Zakharov, S., Nui, X-W., Chudasama, N., Kelly, C., Waase, M., Doshi, D., Liu, G., Iwata, S., Shiomi, T., Katchman, A., D'Armiento, J., Homma, S., Marx, S. O. Reduced vascular smooth muscle BK channel current underlies heart failure-induced vasoconstriction in mice. PMID:23325318

  20. Effect of aldosterone on BK channel expression in mammalian cortical collecting duct.

    PubMed

    Estilo, Genevieve; Liu, Wen; Pastor-Soler, Nuria; Mitchell, Phillip; Carattino, Marcelo D; Kleyman, Thomas R; Satlin, Lisa M

    2008-09-01

    Apical large-conductance Ca(2+)-activated K(+) (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K(+) secretion. Dietary K(+) loading for 10-14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K(+) secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922-F932, 2005). To test whether this adaptation was mediated by a K(+)-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na(+) (LS) or high-Na(+) (HS) diet for 7-10 days to alter circulating levels of aldosterone but not serum K(+) concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, alpha-splice variants, beta-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na(+) (J(Na)) and K(+) (J(K)) transport by microperfusion; kidneys were processed for immunolocalization of BK alpha-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na(+) and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK alpha-, beta(2)-, and beta(4)-subunit mRNA and localization of immunodetectable alpha-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from approximately 1 to 5 nl.min(-1).mm(-1), the increase in J(Na) was greater in LS vs. HS rabbits, yet the flow-stimulated increase in J(K) was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K(+) loading.

  1. NS19504: A Novel BK Channel Activator with Relaxing Effect on Bladder Smooth Muscle Spontaneous Phasic Contractions

    PubMed Central

    Nausch, Bernhard; Rode, Frederik; Jørgensen, Susanne; Nardi, Antonio; Korsgaard, Mads P. G.; Hougaard, Charlotte; Bonev, Adrian D.; Brown, William D.; Dyhring, Tino; Strøbæk, Dorte; Olesen, Søren-Peter; Christophersen, Palle; Grunnet, Morten; Nelson, Mark T.

    2014-01-01

    Large-conductance Ca2+-activated K+ channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader–based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K+ concentration consistent with a K+ channel–mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding. PMID:24951278

  2. Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures.

    PubMed

    Upadhyay, Mohita; Vivekanandan, Perumal

    2015-01-01

    Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses. We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses. All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses. The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the evolutionary lineage of the infected host. Our

  3. Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures

    PubMed Central

    Upadhyay, Mohita; Vivekanandan, Perumal

    2015-01-01

    Background Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses. Methods We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses. Results All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses. Conclusions The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the

  4. Graphs for Isotopes of 97-Bk (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides a graphic representation of nucleon separation energies and residual interaction parameters for isotopes of the chemical element 97-Bk (Berkelium, atomic number Z = 97).

  5. Human endothelial cells allow passage of an archetypal BK virus (BKV) strain--a tool for cultivation and functional studies of natural BKV strains.

    PubMed

    Hanssen Rinaldo, C; Hansen, H; Traavik, T

    2005-07-01

    The ubiquitous human polyomavirus BK (BKV) causes the serious condition BKV-nephropathy in an increasing number of renal-transplant patients. The lack of authentic cell cultures for multiplication of naturally occurring strains has hampered cultivation and functional studies of BKV. Here we demonstrate that the most common urine shed BKV strain, the archetype, multiplies in the human endothelial cell line HUV-EC-C. Additional variants with deletions in the non-coding control region (NCCR) appear upon prolonged propagation. Although the titer produced was low, at the present HUV-EC-C is the only cell line shown to allow propagation of archetypal BKV. HUV-EC-C may therefore be a useful tool for BKV cultivation as well as functional studies.

  6. Progression from Sustained BK Viruria to Sustained BK Viremia with Immunosuppression Reduction Is Not Associated with Changes in the Noncoding Control Region of the BK Virus Genome

    PubMed Central

    Memon, Imran A.; Parikh, Bijal A.; Gaudreault-Keener, Monique; Skelton, Rebecca; Storch, Gregory A.; Brennan, Daniel C.

    2012-01-01

    Changes in the BK virus archetypal noncoding control region (NCCR) have been associated with BK-virus-associated nephropathy (BKVAN). Whether sustained viremia, a surrogate for BKVAN, is associated with significant changes in the BK-NCCR is unknown. We performed PCR amplification and sequencing of (1) stored urine and (2) plasma samples from the time of peak viremia from 11 patients with sustained viremia who participated in a 200-patient clinical trial. The antimetabolite was withdrawn for BK viremia and reduction of the calcineurin inhibitor for sustained BK viremia. DNA sequencing from the 11 patients with sustained viremia revealed 8 insertions, 16 transversions, 3 deletions, and 17 transitions. None were deemed significant. No patient developed clinically evident BKVAN. Our data support, at a genomic level, the effectiveness of reduction of immunosuppression for prevention of progression from viremia to BKVAN. PMID:22701777

  7. The dynamics of herpesvirus and polyomavirus reactivation and shedding in healthy adults: a 14-month longitudinal study

    NASA Technical Reports Server (NTRS)

    Ling, Paul D.; Lednicky, John A.; Keitel, Wendy A.; Poston, David G.; White, Zoe S.; Peng, RongSheng; Liu, Zhensheng; Mehta, Satish K.; Pierson, Duane L.; Rooney, Cliona M.; Vilchez, Regis A.; Smith, E. O'Brian; Butel, Janet S.

    2003-01-01

    Humans are infected with viruses that establish long-term persistent infections. To address whether immunocompetent individuals control virus reactivation globally or independently and to identify patterns of sporadic reactivation, we monitored herpesviruses and polyomaviruses in 30 adults, over 14 months. Epstein-Barr virus (EBV) DNA was quantitated in saliva and peripheral blood mononuclear cells (PBMCs), cytomegalovirus (CMV) was assayed in urine, and JC virus (JCV) and BK virus (BKV) DNAs were assayed in urine and PBMCs. All individuals shed EBV in saliva, whereas 67% had >or=1 blood sample positive for EBV. Levels of EBV varied widely. CMV shedding occurred infrequently but occurred more commonly in younger individuals (P<.03). JCV and BKV virurias were 46.7% and 0%, respectively. JCV shedding was age dependent and occurred commonly in individuals >or=40 years old (P<.03). Seasonal variation was observed in shedding of EBV and JCV, but there was no correlation among shedding of EBV, CMV, and JCV (P>.50). Thus, adults independently control persistent viruses, which display discordant, sporadic reactivations.

  8. [Absorption of 249Bk from the gastrointestinal tract of rats].

    PubMed

    Zalikin, G A; Nisimov, P G

    1988-01-01

    In experiments with albino mongrel female rats a study was made of the absorption of 249Bk from the gastrointestinal tract after a single per os administration. The bulk of 249Bk (96 per cent) administered either intravenously or per os was mainly deposited in the skeleton and liver. The value of 249Bk absorption from the gastrointestinal tract by days 4 and 8 following administration was 0.05 per cent.

  9. Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus Extracts Effectively Inhibit BK Virus and JC Virus Infection of Host Cells.

    PubMed

    Chen, San-Yuan; Teng, Ru-Hsiou; Wang, Meilin; Chen, Pei-Lain; Lin, Mien-Chun; Shen, Cheng-Huang; Chao, Chun-Nun; Chiang, Ming-Ko; Fang, Chiung-Yao; Chang, Deching

    2017-01-01

    The human polyomaviruses BK (BKPyV) and JC (JCPyV) are ubiquitous pathogens long associated with severe disease in immunocompromised individuals. BKPyV causes polyomavirus-associated nephropathy and hemorrhagic cystitis, whereas JCPyV is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy. No effective therapies targeting these viruses are currently available. The goal of this study was to identify Chinese medicinal herbs with antiviral activity against BKPyV and JCPyV. We screened extracts of Chinese medicinal herbs for the ability to inhibit hemagglutination by BKPyV and JCPyV virus-like particles (VLPs) and the ability to inhibit BKPyV and JCPyV binding and infection of host cells. Two of the 40 herbal extracts screened, Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus, had hemagglutination inhibition activity on BKPyV and JCPyV VLPs and further inhibited infection of the cells by BKPyV and JCPyV, as evidenced by reduced expression of viral proteins in BKPyV-infected and JCPyV-infected cells after treatment with Rhodiolae Kirliowii Radix et Rhizoma or Crataegus pinnatifida Fructus extract. The results in this work show that both Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus may be sources of potential antiviral compounds for treating BKPyV and JCPyV infections.

  10. Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus Extracts Effectively Inhibit BK Virus and JC Virus Infection of Host Cells

    PubMed Central

    Chen, San-Yuan; Teng, Ru-Hsiou; Wang, Meilin; Chen, Pei-Lain; Lin, Mien-Chun; Chao, Chun-Nun; Chiang, Ming-Ko

    2017-01-01

    The human polyomaviruses BK (BKPyV) and JC (JCPyV) are ubiquitous pathogens long associated with severe disease in immunocompromised individuals. BKPyV causes polyomavirus-associated nephropathy and hemorrhagic cystitis, whereas JCPyV is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy. No effective therapies targeting these viruses are currently available. The goal of this study was to identify Chinese medicinal herbs with antiviral activity against BKPyV and JCPyV. We screened extracts of Chinese medicinal herbs for the ability to inhibit hemagglutination by BKPyV and JCPyV virus-like particles (VLPs) and the ability to inhibit BKPyV and JCPyV binding and infection of host cells. Two of the 40 herbal extracts screened, Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus, had hemagglutination inhibition activity on BKPyV and JCPyV VLPs and further inhibited infection of the cells by BKPyV and JCPyV, as evidenced by reduced expression of viral proteins in BKPyV-infected and JCPyV-infected cells after treatment with Rhodiolae Kirliowii Radix et Rhizoma or Crataegus pinnatifida Fructus extract. The results in this work show that both Rhodiolae Kirliowii Radix et Rhizoma and Crataegus pinnatifida Fructus may be sources of potential antiviral compounds for treating BKPyV and JCPyV infections. PMID:28757888

  11. Merkel cell polyomavirus is present in common warts and carcinoma in situ of the skin.

    PubMed

    Mertz, Kirsten D; Pfaltz, Madeleine; Junt, Tobias; Schmid, Mirka; Fernandez Figueras, Maria Teresa; Pfaltz, Katrin; Barghorn, André; Kempf, Werner

    2010-10-01

    Polyomaviruses have been linked to diseases of immunosuppressed patients. We sought to determine the prevalence of Merkel cell polyomavirus in benign epithelial skin neoplasms and nonmelanoma skin cancer of immunosuppressed renal transplant recipients and long-term dialysis patients. Merkel cell polyomavirus DNA was detected by polymerase chain reaction (PCR) in 2 (10%) of 20 patients, in carcinomas in situ (Bowen's disease). In one of our patients with Merkel cell polyomavirus-positive carcinoma in situ, 9 (39.1%) of 23 skin lesions at various anatomical locations tested positive for Merkel cell polyomavirus sequences by PCR, including all of his common warts (4/4), half of his carcinoma in situ lesions (3/6), and 2 of his 3 seborrheic keratoses. In a second cohort of immunosuppressed renal transplant recipients, Merkel cell polyomavirus DNA was found in 1 (6.3%) of 16 common warts and in 2 (9.5%) of 21 carcinomas in situ. In immunocompetent individuals, Merkel cell polyomavirus DNA was found in 2 (6.7%) of 30 common warts and in 2 (8.3%) of 24 carcinomas in situ. DNA of other human polyomaviruses was not detected in any of the investigated skin neoplasms. We conclude that common warts and carcinomas in situ can be positive for Merkel cell polyomavirus in immunosuppressed as well as immunocompetent individuals. Remarkably, some of the Merkel cell polyomavirus-positive common warts did not contain human papillomavirus. Furthermore, Merkel cell polyomavirus can be found in various skin neoplasms of the same individual.

  12. Complete Genome Sequence of a Polyomavirus Recovered from a Pomona Leaf-Nosed Bat (Hipposideros pomona) Metagenome Data Set

    PubMed Central

    Cantalupo, Paul G.; Buck, Christopher B.

    2017-01-01

    ABSTRACT We report here the complete genome sequence of a polyomavirus found in a nasal/rectal metagenome of Hipposideros pomona (Pomona leaf-nosed bat). Interestingly, the genetic organization and phylogenetic relationships of the new virus suggest greater similarity to recently discovered fish-associated polyomaviruses rather than to polyomavirus species previously observed in bats. PMID:28104645

  13. Polyomaviruses KI and WU in immunocompromised patients with respiratory disease.

    PubMed

    Mourez, Thomas; Bergeron, Anne; Ribaud, Patricia; Scieux, Catherine; de Latour, Régis Peffault; Tazi, Abdellatif; Socié, Gérard; Simon, François; LeGoff, Jérôme

    2009-01-01

    Polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified, mainly in respiratory specimens from children. Among 200 patients with respiratory disorders admitted to Saint Louis Hospital, Paris, France, KIPyV was detected in 8% and WUPyV in 1%. KIPyV was significantly more frequent among human stem cell transplant patients (17.8% vs. 5.1%; p = 0.01).

  14. Polyomaviruses KI and WU in Immunocompromised Patients with Respiratory Disease

    PubMed Central

    Mourez, Thomas; Bergeron, Anne; Ribaud, Patricia; Scieux, Catherine; Peffault de Latour, Régis; Tazi, Abdellatif; Socié, Gérard; Simon, François

    2009-01-01

    Polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified, mainly in respiratory specimens from children. Among 200 patients with respiratory disorders admitted to Saint Louis Hospital, Paris, France, KIPyV was detected in 8% and WUPyV in 1%. KIPyV was significantly more frequent among human stem cell transplant patients (17.8% vs. 5.1%; p = 0.01). PMID:19116066

  15. Role of the Human Polyomavirus, BKV, in Prostate Cancer

    DTIC Science & Technology

    2004-08-01

    polyomavirus and late regions, and encodes five major proteins nephropathy (Arthur et al., 1986; Hiraoka et al., 1991; (Figure 1; Seif et al., 1979...specimens was first ascertained by PCR (45 cycles) with the f-globin primers. stained with hematoxylin and cosin to confirm diagnosis . The The... pregnancy , or HIV infection, high throughout the rest of the time course. levels of BKV replication are observed in the kidneys and The next step in the BKV

  16. Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of BK channels.

    PubMed

    Zuidema, Mozow Y; Yang, Yan; Wang, Meifang; Kalogeris, Theodore; Liu, Yajun; Meininger, Cynthia J; Hill, Michael A; Davis, Michael J; Korthuis, Ronald J

    2010-11-01

    The objectives of this study were to determine the role of calcium-activated, small (SK), intermediate (IK), and large (BK) conductance potassium channels in initiating the development of an anti-inflammatory phenotype elicited by preconditioning with an exogenous hydrogen sulfide (H(2)S) donor, sodium hydrosulfide (NaHS). Intravital microscopy was used to visualize rolling and firmly adherent leukocytes in vessels of the small intestine of mice preconditioned with NaHS (in the absence and presence of SK, IK, and BK channel inhibitors, apamin, TRAM-34, and paxilline, respectively) or SK/IK (NS-309) or BK channel activators (NS-1619) 24 h before ischemia-reperfusion (I/R). I/R induced marked increases in leukocyte rolling and adhesion, effects that were largely abolished by preconditioning with NaHS, NS-309, or NS-1619. The postischemic anti-inflammatory effects of NaHS-induced preconditioning were mitigated by BKB channel inhibitor treatment coincident with NaHS, but not by apamin or TRAM-34, 24 h before I/R. Confocal imaging and immunohistochemistry were used to demonstrate the presence of BKα subunit staining in both endothelial and vascular smooth muscle cells of isolated, pressurized mesenteric venules. Using patch-clamp techniques, we found that BK channels in cultured endothelial cells were activated after exposure to NaHS. Bath application of the same concentration of NaHS used in preconditioning protocols led to a rapid increase in a whole cell K(+) current; specifically, the component of K(+) current blocked by the selective BK channel antagonist iberiotoxin. The activation of BK current by NaHS could also be demonstrated in single channel recording mode where it was independent of a change in intracellular Ca(+) concentration. Our data are consistent with the concept that H(2)S induces the development of an anti-adhesive state in I/R in part mediated by a BK channel-dependent mechanism.

  17. The glycosylation of the extracellular loop of β2 subunits diversifies functional phenotypes of BK Channels.

    PubMed

    Huang, Zhi-Gang; Liu, Hao-Wen; Yan, Zhen-Zhen; Wang, Sheng; Wang, Lu-Yang; Ding, Jiu-Ping

    2017-03-04

    Large-conductance Ca(2+)- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming α subunit (Slo) and 4 types of auxiliary β subunits or just a pore-forming α subunit. Although multiple N-linked glycosylation sites in the extracellular loop of β subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp recordings, we demonstrated that 3 sites in the extracellular loop of β2 subunit are N-glycosylated (N-X-T/S at N88, N96 and N119). Glycosylation of these sites strongly and differentially regulate gating kinetics, outward rectification, toxin sensitivity and physical association between the α and β2 subunits. We constructed a model and used molecular dynamics (MD) to simulate how the glycosylation facilitates the association of α/β2 subunits and modulates the dimension of the extracellular cavum above the pore of the channel, ultimately to modify biophysical and pharmacological properties of BK channels. Our results suggest that N-glycosylation of β2 subunits plays crucial roles in imparting functional heterogeneity of BK channels, and is potentially involved in the pathological phenotypes of carbohydrate metabolic diseases.

  18. Functional regulation of BK potassium channels by γ1 auxiliary subunits.

    PubMed

    Gonzalez-Perez, Vivian; Xia, Xiao-Ming; Lingle, Christopher J

    2014-04-01

    Many K(+) channels are oligomeric complexes with intrinsic structural symmetry arising from the homo-tetrameric core of their pore-forming subunits. Allosteric regulation of tetramerically symmetric proteins, whether by intrinsic sensing domains or associated auxiliary subunits, often mirrors the fourfold structural symmetry. Here, through patch-clamp recordings of channel population ensembles and also single channels, we examine regulation of the Ca(2+)- and voltage-activated large conductance Ca(2+)-activated K(+) (BK) channel by associated γ1-subunits. Through expression of differing ratios of γ1:α-subunits, the results reveal an all-or-none functional regulation of BK channels by γ-subunits: channels either exhibit a full gating shift or no shift at all. Furthermore, the γ1-induced shift exhibits a state-dependent labile behavior that recapitulates the fully shifted or unshifted behavior. The γ1-induced shift contrasts markedly to the incremental shifts in BK gating produced by 1-4 β-subunits and adds a new layer of complexity to the mechanisms by which BK channel functional diversity is generated.

  19. Functional regulation of BK potassium channels by γ1 auxiliary subunits

    PubMed Central

    Gonzalez-Perez, Vivian; Xia, Xiao-Ming; Lingle, Christopher J.

    2014-01-01

    Many K+ channels are oligomeric complexes with intrinsic structural symmetry arising from the homo-tetrameric core of their pore-forming subunits. Allosteric regulation of tetramerically symmetric proteins, whether by intrinsic sensing domains or associated auxiliary subunits, often mirrors the fourfold structural symmetry. Here, through patch-clamp recordings of channel population ensembles and also single channels, we examine regulation of the Ca2+- and voltage-activated large conductance Ca2+-activated K+ (BK) channel by associated γ1-subunits. Through expression of differing ratios of γ1:α-subunits, the results reveal an all-or-none functional regulation of BK channels by γ-subunits: channels either exhibit a full gating shift or no shift at all. Furthermore, the γ1-induced shift exhibits a state-dependent labile behavior that recapitulates the fully shifted or unshifted behavior. The γ1-induced shift contrasts markedly to the incremental shifts in BK gating produced by 1–4 β-subunits and adds a new layer of complexity to the mechanisms by which BK channel functional diversity is generated. PMID:24639523

  20. Habituation of reflexive and motivated behavior in mice with deficient BK channel function

    PubMed Central

    Typlt, Marei; Mirkowski, Magdalena; Azzopardi, Erin; Ruth, Peter; Pilz, Peter K. D.; Schmid, Susanne

    2013-01-01

    Habituation is considered the most basic form of learning. It describes the decrease of a behavioral response to a repeated non-threatening sensory stimulus and therefore provides an important sensory filtering mechanism. While some neuronal pathways mediating habituation are well described, underlying cellular/molecular mechanisms are not yet fully understood. In general, there is an agreement that short-term and long-term habituation are based on different mechanisms. Historically, a distinction has also been made between habituation of motivated versus reflexive behavior. In recent studies in invertebrates the large conductance voltage- and calcium-activated potassium (BK) channel has been implicated to be a key player in habituation by regulating synaptic transmission. Here, we tested mice deficient for the pore forming α-subunit of the BK channel for short-term and long-term habituation of the acoustic startle reflex (reflexive behavior) and of the exploratory locomotor behavior in the open field box (motivated behavior). Short-term habituation of startle was completely abolished in the BK knock-out mice, whereas neither long-term habituation of startle nor habituation of motivated behavior was affected by the BK deficiency. Our results support a highly preserved mechanism for short-term habituation of startle across species that is distinct from long-term habituation mechanisms. It also supports the notion that there are different mechanisms underlying habituation of motivated behavior versus reflexive behavior. PMID:24312024

  1. CRL4A(CRBN) E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis.

    PubMed

    Liu, Jiye; Ye, Jia; Zou, Xiaolong; Xu, Zhenghao; Feng, Yan; Zou, Xianxian; Chen, Zhong; Li, Yuezhou; Cang, Yong

    2014-05-21

    Ion channels regulate membrane excitation, and mutations of ion channels often cause serious neurological disorders including epilepsy. Compared with extensive analyses of channel protein structure and function, much less is known about the fine tuning of channel activity by post-translational modification. Here we report that the large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are targeted by the E3 ubiquitin ligase CRL4A(CRBN) for polyubiquitination and retained in the endoplasmic reticulum (ER). Inactivation of CRL4A(CRBN) releases deubiquitinated BK channels from the ER to the plasma membrane, leading to markedly enhanced channel activity. Mice with CRL4A(CRBN) mutation in the brain or treated with a CRL4A(CRBN) inhibitor are very sensitive to seizure induction, which can be attenuated by blocking BK channels. Finally, the mutant mice develop spontaneous epilepsy when aged. Therefore, ubiquitination of BK channels before their cell surface expression is an important step to prevent systemic neuronal excitability and epileptogenesis.

  2. BK channel opening involves side-chain reorientation of multiple deep-pore residues

    PubMed Central

    Chen, Xixi; Yan, Jiusheng; Aldrich, Richard W.

    2014-01-01

    Three deep-pore locations, L312, A313, and A316, were identified in a scanning mutagenesis study of the BK (Ca2+-activated, large-conductance K+) channel S6 pore, where single aspartate substitutions led to constitutively open mutant channels (L312D, A313D, and A316D). To understand the mechanisms of the constitutive openness of these mutant channels, we individually mutated these three sites into the other 18 amino acids. We found that charged or polar side-chain substitutions at each of the sites resulted in constitutively open mutant BK channels, with high open probability at negative voltages, as well as a loss of voltage and Ca2+ dependence. Given the fact that multiple pore residues in BK displayed side-chain hydrophilicity-dependent constitutive openness, we propose that BK channel opening involves structural rearrangement of the deep-pore region, where multiple residues undergo conformational changes that may increase the exposure of their side chains to the polar environment of the pore. PMID:24367115

  3. Trichodysplasia spinulosa: a polyomavirus infection specifically targeting follicular keratinocytes in immunocompromised patients.

    PubMed

    Rouanet, J; Aubin, F; Gaboriaud, P; Berthon, P; Feltkamp, M C; Bessenay, L; Touzé, A; Nicol, J T J; Franck, F; D'Incan, M

    2016-03-01

    Trichodysplasia spinulosa (TS) is a rare skin disease, caused by a specific polyomavirus, occurring in immunocompromised patients. The pathophysiological mechanisms of TS are not yet fully understood. By using polymerase chain reaction and skin biopsy immunostaining we report evidence, in a paediatric case, of follicular keratinocytes being the primary target of trichodysplasia spinulosa-associated polyomavirus.

  4. Diagnostic methods for and clinical pictures of polyomavirus primary infections in children, Finland.

    PubMed

    Chen, Tingting; Tanner, Laura; Simell, Ville; Hedman, Lea; Mäkinen, Marjaana; Sadeghi, Mohammadreza; Veijola, Riitta; Hyöty, Heikki; Ilonen, Jorma; Knip, Mikael; Toppari, Jorma; Simell, Olli; Söderlund-Venermo, Maria; Hedman, Klaus

    2014-04-01

    We used comprehensive serodiagnostic methods (IgM, IgG, and IgG avidity) and PCR to study Merkel cell polyomavirus and trichodysplasia spinulosa-associated polyomavirus infections in children observed from infancy to adolescence. Comparing seroconversion intervals with previous and subsequent intervals, we found that primary infections with these 2 viruses were asymptomatic in childhood.

  5. β1-subunit–induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel

    PubMed Central

    Castillo, Juan P.; Sánchez-Rodríguez, Jorge E.; Hyde, H. Clark; Zaelzer, Cristian A.; Aguayo, Daniel; Sepúlveda, Romina V.; Luk, Louis Y. P.; Kent, Stephen B. H.; Gonzalez-Nilo, Fernando D.; Bezanilla, Francisco; Latorre, Ramón

    2016-01-01

    Large-conductance Ca2+- and voltage-activated K+ (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit–induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex. PMID:27217576

  6. β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel.

    PubMed

    Castillo, Juan P; Sánchez-Rodríguez, Jorge E; Hyde, H Clark; Zaelzer, Cristian A; Aguayo, Daniel; Sepúlveda, Romina V; Luk, Louis Y P; Kent, Stephen B H; Gonzalez-Nilo, Fernando D; Bezanilla, Francisco; Latorre, Ramón

    2016-06-07

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

  7. BK potassium channels control transmitter release at CA3−CA3 synapses in the rat hippocampus

    PubMed Central

    Raffaelli, Giacomo; Saviane, Chiara; Mohajerani, Majid H; Pedarzani, Paola; Cherubini, Enrico

    2004-01-01

    Large conductance calcium- and voltage-activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an ‘emergency brake’ that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 μm) and iberiotoxin (100 nm) increase the frequency, but not the amplitude, of spontaneously occurring action potential-dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch-clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired-pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired-pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones. PMID:15034127

  8. Examining Merkel Cell Polyomavirus Minor Capsid Proteins | Center for Cancer Research

    Cancer.gov

    Merkel cell polyomavirus (MCV or MCPyV) is a recently discovered member of the viral family Polyomaviridae. It is a skin-dwelling polyomavirus species that appears to cause a rare but highly lethal form of skin cancer called Merkel cell carcinoma (MCC). Despite MCC being uncommon, chronic MCV infection of human skin is widespread, and most infected people have no known symptoms. The surface of polyomavirus virions is made up of pentameric knobs of the major capsid protein VP1. VP1 enables attachment of the virus to the cell surface, permitting infectious entry and delivery of the viral genome to host cells. The VP1 protein of previously studied polyomaviruses, such as simian virus 40 and murine polyomavirus, associates with two minor capsid proteins, VP2 and VP3, which are considered to play important roles during the infectious entry process.

  9. Goose Hemorrhagic polyomavirus detection in geese using real-time PCR assay.

    PubMed

    Leon, Olivier; Corrand, Léni; Bich, Tran Ngoc; Le Minor, Odile; Lemaire, Mylène; Guérin, Jean-Luc

    2013-12-01

    Goose hemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis enteritis of geese (HNEG), a lethal disease of goslings. Although death is the most common outcome, geese that recover from HNEG are persistently infected. Here, we present the development of real-time SYBR Green real-time PCR targeted to GHPV and its use to assess the prevalence of GHPV infection in French geese flocks. When compared with classical end-point PCR, real-time PCR revealed a much better sensitivity and equivalent specificity. Real-time PCR could, therefore, be considered a gold standard for the detection of GHPV. Results of field investigations evidenced a very high prevalence of GHPV infections in French geese, largely associated with healthy carriage.

  10. Characterization of novel polyomaviruses from Bornean and Sumatran orang-utans.

    PubMed

    Groenewoud, Marlous J; Fagrouch, Zahra; van Gessel, Sabine; Niphuis, Henk; Bulavaite, Aiste; Warren, Kristin S; Heeney, Jonathan L; Verschoor, Ernst J

    2010-03-01

    Serological screening of sera from orang-utans demonstrated a high percentage of sera that cross-reacted with antigens of the polyomavirus (PyV) simian virus 40. Analysis of archival DNA samples from 71 Bornean and eight Sumatran orang-utans with a broad-spectrum PCR assay resulted in the detection of PyV infections in 11 animals from both species. Sequence analysis of the amplicons revealed considerable differences between the PyVs from Bornean and Sumatran orang-utans. The genome from two PyVs, one from each species, was therefore amplified and sequenced. Both viral genomes revealed a characteristic PyV architecture, but lacked an obvious agnogene. Neighbour-joining analysis positioned the viruses in a large cluster together with viruses from bats, bovines, rodents and several primate PyVs from chimpanzees, African green monkeys, squirrel monkeys and the human Merkel cell PyV.

  11. The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

    PubMed Central

    Femling, Jon K.; Cherny, Vladimir V.; Morgan, Deri; Rada, Balázs; Davis, A. Paige; Czirják, Gabor; Enyedi, Peter; England, Sarah K.; Moreland, Jessica G.; Ligeti, Erzsébet; Nauseef, William M.; DeCoursey, Thomas E.

    2006-01-01

    Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and

  12. [The blastomogenic effect of 249Bk in rats].

    PubMed

    Moskalev, Iu I; Zalikin, G A; Zhorova, E S; Nisimov, P G

    1984-01-01

    It is shown that 249Bk nitrate injected intraperitoneally in a wide range of doses to albino mongrel female rats is preferentially accumulated in the bony tissue (39.8%) and liver (18.4%). Incorporation of 249Bk to rats results in development of osteosarcomas, neoplasms of hemopoietic and lymphoid tissues, mammary tumours, thyroid and pituitary glands.

  13. SUPERNOVA 2008bk AND ITS RED SUPERGIANT PROGENITOR

    SciTech Connect

    Van Dyk, Schuyler D.; Elias-Rosa, Nancy; and others

    2012-01-15

    We have obtained limited photometric and spectroscopic data for supernova (SN) 2008bk in NGC 7793, primarily at {approx}> 150 days after explosion. We find that it is a Type II-Plateau (II-P) SN that most closely resembles the low-luminosity SN 1999br in NGC 4900. Given the overall similarity between the observed light curves and colors of SNe 2008bk and 1999br, we infer that the total visual extinction to SN 2008bk (A{sub V} = 0.065 mag) must be almost entirely due to the Galactic foreground, similar to what has been assumed for SN 1999br. We confirm the identification of the putative red supergiant (RSG) progenitor star of the SN in high-quality g'r'i' images we had obtained in 2007 at the Gemini-South 8 m telescope. Little ambiguity exists in this progenitor identification, qualifying it as the best example to date, next to the identification of the star Sk -69 Degree-Sign 202 as the progenitor of SN 1987A. From a combination of photometry of the Gemini images with that of archival, pre-SN, Very Large Telescope JHK{sub s} images, we derive an accurate observed spectral energy distribution (SED) for the progenitor. We find from nebular strong-intensity emission-line indices for several H II regions near the SN that the metallicity in the environment is likely subsolar (Z Almost-Equal-To 0.6 Z{sub Sun }). The observed SED of the star agrees quite well with synthetic SEDs obtained from model RSG atmospheres with effective temperature T{sub eff} = 3600 {+-} 50 K. We find, therefore, that the star had a bolometric luminosity with respect to the Sun of log (L{sub bol}/L{sub Sun} ) = 4.57 {+-} 0.06 and radius R{sub *} = 496 {+-} 34 R{sub Sun} at {approx}6 months prior to explosion. Comparing the progenitor's properties with theoretical massive-star evolutionary models, we conclude that the RSG progenitor had an initial mass in the range of 8-8.5 M{sub Sun }. This mass is consistent with, albeit at the low end of, the inferred range of initial masses for SN II

  14. Osteopenia Due to Enhanced Cathepsin K Release by BK Channel Ablation in Osteoclasts

    PubMed Central

    Missbach-Guentner, Jeannine; Kabagema, Clement; Flockerzie, Katarina; Kuscher, Gerd Marten; Stuehmer, Walter; Neuhuber, Winfried; Ruth, Peter; Alves, Frauke; Sausbier, Matthias

    2011-01-01

    Background The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified. Methodology/Principal Findings We found, that in juvenile bone the large conductance, voltage and Ca2+-activated (BK) K+ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K+ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca2+ and triiodthyronine as well as osteoclastogenesis were not altered in BK−/− females. Conclusion/Significance Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity. PMID:21695131

  15. Calcium-Activated Potassium (BK) Channels Are Encoded by Duplicate slo1 Genes in Teleost Fishes

    PubMed Central

    Deitcher, David L.; Bass, Andrew H.

    2009-01-01

    Calcium-activated, large conductance potassium (BK) channels in tetrapods are encoded by a single slo1 gene, which undergoes extensive alternative splicing. Alternative splicing generates a high level of functional diversity in BK channels that contributes to the wide range of frequencies electrically tuned by the inner ear hair cells of many tetrapods. To date, the role of BK channels in hearing among teleost fishes has not been investigated at the molecular level, although teleosts account for approximately half of all extant vertebrate species. We identified slo1 genes in teleost and nonteleost fishes using polymerase chain reaction and genetic sequence databases. In contrast to tetrapods, all teleosts examined were found to express duplicate slo1 genes in the central nervous system, whereas nonteleosts that diverged prior to the teleost whole-genome duplication event express a single slo1 gene. Phylogenetic analyses further revealed that whereas other slo1 duplicates were the result of a single duplication event, an independent duplication occurred in a basal teleost (Anguilla rostrata) following the slo1 duplication in teleosts. A third, independent slo1 duplication (autotetraploidization) occurred in salmonids. Comparison of teleost slo1 genomic sequences to their tetrapod orthologue revealed a reduced number of alternative splice sites in both slo1 co-orthologues. For the teleost Porichthys notatus, a focal study species that vocalizes with maximal spectral energy in the range electrically tuned by BK channels in the inner ear, peripheral tissues show the expression of either one (e.g., vocal muscle) or both (e.g., inner ear) slo1 paralogues with important implications for both auditory and vocal physiology. Additional loss of expression of one slo1 paralogue in nonneural tissues in P. notatus suggests that slo1 duplicates were retained via subfunctionalization. Together, the results predict that teleost fish achieve a diversity of BK channel subfunction via

  16. BK Virus in Kidney Transplant Recipients: The Influence of Immunosuppression

    PubMed Central

    Barraclough, Katherine A.; Isbel, Nicole M.; Staatz, Christine E.; Johnson, David W.

    2011-01-01

    The incidence of BK virus infection in kidney transplant recipients has increased over recent decades, coincident with the use of more potent immunosuppression. More importantly, posttransplant BK virus replication has emerged as an important cause of graft damage and subsequent graft loss. Immunosuppression has been accepted as a major risk for BK virus replication. However, the specific contribution of individual immunosuppressive medications to this risk has not been well established. The purpose of this paper is to provide an overview of the recent literature on the influence of the various immunosuppressant drugs and drug combinations on posttransplant BK virus replication. Evidence supporting the various immunosuppression reduction strategies utilised in the management of BK virus will also be briefly discussed. PMID:21766009

  17. BK and Kv3.1 potassium channels control different aspects of deep cerebellar nuclear neurons action potentials and spiking activity.

    PubMed

    Pedroarena, Christine M

    2011-12-01

    Deep cerebellar nuclear neurons (DCNs) display characteristic electrical properties, including spontaneous spiking and the ability to discharge narrow spikes at high frequency. These properties are thought to be relevant to processing inhibitory Purkinje cell input and transferring well-timed signals to cerebellar targets. Yet, the underlying ionic mechanisms are not completely understood. BK and Kv3.1 potassium channels subserve similar functions in spike repolarization and fast firing in many neurons and are both highly expressed in DCNs. Here, their role in the abovementioned spiking characteristics was addressed using whole-cell recordings of large and small putative-glutamatergic DCNs. Selective BK channel block depolarized DCNs of both groups and increased spontaneous firing rate but scarcely affected evoked activity. After adjusting the membrane potential to control levels, the spike waveforms under BK channel block were indistinguishable from control ones, indicating no significant BK channel involvement in spike repolarization. The increased firing rate suggests that lack of DCN-BK channels may have contributed to the ataxic phenotype previously found in BK channel-deficient mice. On the other hand, block of Kv3.1 channels with low doses of 4-aminopyridine (20 μM) hindered spike repolarization and severely depressed evoked fast firing. Therefore, I propose that despite similar characteristics of BK and Kv3.1 channels, they play different roles in DCNs: BK channels control almost exclusively spontaneous firing rate, whereas DCN-Kv3.1 channels dominate the spike repolarization and enable fast firing. Interestingly, after Kv3.1 channel block, BK channels gained a role in spike repolarization, demonstrating how the different function of each of the two channels is determined in part by their co-expression and interplay.

  18. Discovery of a polyomavirus in European badgers (Meles meles) and the evolution of host range in the family Polyomaviridae

    PubMed Central

    Hill, Sarah C.; Murphy, Aisling A.; Cotten, Matthew; Palser, Anne L.; Benson, Phillip; Lesellier, Sandrine; Gormley, Eamonn; Richomme, Céline; Grierson, Sylvia; Bhuachalla, Deirdre Ni; Chambers, Mark; Kellam, Paul; Boschiroli, María-Laura

    2015-01-01

    Polyomaviruses infect a diverse range of mammalian and avian hosts, and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here, we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterized in the family Mustelidae, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187 bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance amongst polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups. PMID:25626684

  19. Discovery of a polyomavirus in European badgers (Meles meles) and the evolution of host range in the family Polyomaviridae.

    PubMed

    Hill, Sarah C; Murphy, Aisling A; Cotten, Matthew; Palser, Anne L; Benson, Phillip; Lesellier, Sandrine; Gormley, Eamonn; Richomme, Céline; Grierson, Sylvia; Bhuachalla, Deirdre Ni; Chambers, Mark; Kellam, Paul; Boschiroli, María-Laura; Ehlers, Bernhard; Jarvis, Michael A; Pybus, Oliver G

    2015-06-01

    Polyomaviruses infect a diverse range of mammalian and avian hosts, and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here, we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterized in the family Mustelidae, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187 bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance amongst polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups.

  20. AN ECHO OF SUPERNOVA 2008bk

    SciTech Connect

    Van Dyk, Schuyler D.

    2013-08-01

    I have discovered a prominent light echo around the low-luminosity Type II-plateau supernova (SN) 2008bk in NGC 7793, seen in archival images obtained with the Wide Field Channel of the Advanced Camera for Surveys on board the Hubble Space Telescope (HST). The echo is a partial ring, brighter to the north and east than to the south and west. The analysis of the echo I present suggests that it is due to the SN light pulse scattered by a sheet, or sheets, of dust located Almost-Equal-To 15 pc from the SN. The composition of the dust is assumed to be of standard Galactic diffuse interstellar grains. The visual extinction of the dust responsible for the echo is A{sub V} Almost-Equal-To 0.05 mag in addition to the extinction due to the Galactic foreground toward the host galaxy. That the SN experienced much less overall extinction implies that it is seen through a less dense portion of the interstellar medium in its environment. The late-time HST photometry of SN 2008bk also clearly demonstrates that the progenitor star has vanished.

  1. Developmental expression of BK channels in chick cochlear hair cells

    PubMed Central

    2009-01-01

    Background Cochlear hair cells are high-frequency sensory receptors. At the onset of hearing, hair cells acquire fast, calcium-activated potassium (BK) currents, turning immature spiking cells into functional receptors. In non-mammalian vertebrates, the number and kinetics of BK channels are varied systematically along the frequency-axis of the cochlea giving rise to an intrinsic electrical tuning mechanism. The processes that control the appearance and heterogeneity of hair cell BK currents remain unclear. Results Quantitative PCR results showed a non-monotonic increase in BK α subunit expression throughout embryonic development of the chick auditory organ (i.e. basilar papilla). Expression peaked near embryonic day (E) 19 with six times the transcript level of E11 sensory epithelia. The steady increase in gene expression from E11 to E19 could not explain the sudden acquisition of currents at E18-19, implicating post-transcriptional mechanisms. Protein expression also preceded function but progressed in a sequence from diffuse cytoplasmic staining at early ages to punctate membrane-bound clusters at E18. Electrophysiology data confirmed a continued refinement of BK trafficking from E18 to E20, indicating a translocation of BK clusters from supranuclear to subnuclear domains over this critical developmental age. Conclusions Gene products encoding BK α subunits are detected up to 8 days before the acquisition of anti-BK clusters and functional BK currents. Therefore, post-transcriptional mechanisms seem to play a key role in the delayed emergence of calcium-sensitive currents. We suggest that regulation of translation and trafficking of functional α subunits, near voltage-gated calcium channels, leads to functional BK currents at the onset of hearing. PMID:20003519

  2. Determination of real oxidation potentials of the Bk /SUP IV/ -Bk /SUP III/ pair in phosphoric acid solutions

    SciTech Connect

    Perevalov, S.A.; Kulyako, Y.M.; Lebedev, I.A.; Myasoedov, B.F.

    1986-03-01

    The authors measure the oxidation potential of the Bk(IV)-Bk(III) pair in H3PO4 solutions by a direct spectroelectrchemical method. When the phosphoric acid concentration is increased from 3 to 10 M, its value decreases from 1.123 to 1.065 V (with respect to a normal hydrogen electrode).

  3. The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels

    PubMed Central

    Hou, Shangwei; Xu, Rong; Heinemann, Stefan H.; Hoshi, Toshinori

    2008-01-01

    Carbon monoxide (CO) is a lethal gas, but it is also increasingly recognized as a physiological signaling molecule capable of regulating a variety of proteins. Among them, large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels, important in vasodilation and neuronal firing, have been suggested to be directly stimulated by CO. However, the molecular mechanism of the stimulatory action of CO on the Slo1 BK channel has not been clearly elucidated. We report here that CO reliably and repeatedly activates Slo1 BK channels in excised membrane patches in the absence of Ca2+ in a voltage-sensor-independent manner. The stimulatory action of CO on the Slo1 BK channel requires an aspartic acid and two histidine residues located in the cytoplasmic RCK1 domain, and the effect persists under the conditions known to inhibit the conventional interaction between CO and heme in other proteins. We propose that CO acts as a partial agonist for the high-affinity divalent cation sensor in the RCK1 domain of the Slo1 BK channel. PMID:18316727

  4. The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels.

    PubMed

    Hou, Shangwei; Xu, Rong; Heinemann, Stefan H; Hoshi, Toshinori

    2008-03-11

    Carbon monoxide (CO) is a lethal gas, but it is also increasingly recognized as a physiological signaling molecule capable of regulating a variety of proteins. Among them, large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels, important in vasodilation and neuronal firing, have been suggested to be directly stimulated by CO. However, the molecular mechanism of the stimulatory action of CO on the Slo1 BK channel has not been clearly elucidated. We report here that CO reliably and repeatedly activates Slo1 BK channels in excised membrane patches in the absence of Ca(2+) in a voltage-sensor-independent manner. The stimulatory action of CO on the Slo1 BK channel requires an aspartic acid and two histidine residues located in the cytoplasmic RCK1 domain, and the effect persists under the conditions known to inhibit the conventional interaction between CO and heme in other proteins. We propose that CO acts as a partial agonist for the high-affinity divalent cation sensor in the RCK1 domain of the Slo1 BK channel.

  5. WNK4 inhibits Ca2+-activated big-conductance potassium channels (BK) via mitogen-activated protein kinase-dependent pathway

    PubMed Central

    Yue, Peng; Zhang, Chengbiao; Lin, Dao-Hong; Sun, Peng; Wang, Wen-Hui

    2013-01-01

    We used the perforated whole-cell recording technique to examine the effect of With-No-Lysine Kinase 4 (WNK4) on the Ca2+ activated big-conductance K channels (BK) in HEK293T cells transfected with BK–α subunit (BK-α). Expression of WNK4 inhibited BK channels and decreased the outward K currents. Coexpression of SGK1 abolished the inhibitory effect of WNK4 on BK channels and restored the outward K currents. Expression of WNK4S1169D//1196D, in which both SGK1-phosphorylation sites (serine 1169 and 1196) were mutated to aspartate, had no effect on BK channels. Moreover, coexpression of SGK1 had no additional effect on K currents in the cells transfected with BKα + WNK4 S1169D//1196D, suggesting that SGK1 reversed WNK4-induced inhibition of BK channels by stimulating WNK4 phosphorylation. Expression of WNK4 but not WNK4 S1169D//1196D increased the phosphorylation of ERK and p38 mitogen-activated protein kinase (MAPK); an effect was abolished by coexpression of SGK1. The role of ERK and p38 MAPK in mediating the effect of WNK4 on BK channels was further suggested by the finding that inhibition of ERK and P38 MAPK completely abolished the inhibitory effect of WNK4 on BK channels. In contrast, inhibition of MAPK failed to abolish the inhibitory effect of WNK4 on ROMK channels in both HEK cells and Xenopus oocytes. Expression of dominant negative dynaminK44A (DynK44A) or treatment of the cells with dynasore, a dynamin inhibitor, not only increased K currents but also largely abolished the inhibitory effect of WNK4 on BK channels. However, inhibition of MAPK still increased the outward K currents in the cells transfected with BKα+WNK4 and treated with dynasore. Similar results were obtained in experiments performed in the native tissue in which inhibition of ERK and p38 MAPK increased BK channel activity in the cortical collecting duct (CCD) treated with dynasore. We concluded that WNK4 inhibited BK channels by stimulating ERK and p38 MAPK and that activation of MAPK

  6. Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca2+ sparks in airway myocytes

    PubMed Central

    Lifshitz, Lawrence M.; Carmichael, Jeffrey D.; Lai, F. Anthony; Sorrentino, Vincenzo; Bellvé, Karl; Fogarty, Kevin E.

    2011-01-01

    Short-lived, localized Ca2+ events mediate Ca2+ signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca2+-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca2+ signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca2+ signaling system composed of Ca2+ sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca2+-activated K+ (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca2+ sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca2+ spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca2+] resulting from Ca2+ sparks, and the kinetic model of BK channels, we estimate that an average Ca2+ spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca2+ sparks using explicit knowledge of the spatial relationship between RYRs (the Ca2+ source) and BK channels (the Ca2+ target). PMID:21746845

  7. A single-amino-acid substitution in polyomavirus VP1 correlates with plaque size and hemagglutination behavior.

    PubMed Central

    Freund, R; Garcea, R L; Sahli, R; Benjamin, T L

    1991-01-01

    The plaque size and hemagglutination characteristics of five cloned wild-type strains of polyomavirus were determined. The strains fell into two groups, those with large or small plaques, each with distinctive hemagglutination behavior at different temperatures and pHs. The nucleotide sequence of VP1, the major capsid protein of the virus, was determined for each of the viral strains. The PTA (large-plaque) and RA (small-plaque) strains differed only at residue 92 of VP1, where there is a glutamic acid or glycine, respectively (R. Freund, A. Calderone, C. J. Dawe, and T. L. Benjamin, J. Virol. 65:335-341, 1991). The same amino acid difference in VP1 correlated with plaque size and hemagglutination properties of the other sequenced viruses. Mutagenesis converting amino acid 92 from glutamic acid to glycine converted the plaque size and hemagglutination behavior of the large-plaque PTA strain to that of a small-plaque strain. Furthermore, PTA and RA VP1 proteins produced in Escherichia coli behaved as their parental viruses did in hemagglutination assays. These results demonstrate that amino acid residue 92 of VP1 is involved in determining the plaque size and hemagglutination behavior of polyomavirus and strongly suggest that this region of the VP1 polypeptide interacts directly with cell receptors. Images PMID:1845896

  8. B_K on 2+1 flavor Iwasaki DWF lattices

    NASA Astrophysics Data System (ADS)

    Cohen, Saul

    2006-12-01

    We present current results from an ongoing calculation of BK on 2+1 flavor domain wall fermion lattices with β 2¢ 13 generated with the Iwasaki gauge action (inverse lattice spacing a 1 £ ¤ ¡ 1¢ 6 GeV). Nonperturbative renormalization and chiral fits to the partially quenched 2+1 flavor form are discussed. A new kind of source for large lattice volumes is introduced.

  9. Gene Regulation and Quality Control in Murine Polyomavirus Infection

    PubMed Central

    Carmichael, Gordon G.

    2016-01-01

    Murine polyomavirus (MPyV) infects mouse cells and is highly oncogenic in immunocompromised hosts and in other rodents. Its genome is a small, circular DNA molecule of just over 5000 base pairs and it encodes only seven polypeptides. While seemingly simply organized, this virus has adopted an unusual genome structure and some unusual uses of cellular quality control pathways that, together, allow an amazingly complex and varied pattern of gene regulation. In this review we discuss how MPyV leverages these various pathways to control its life cycle. PMID:27763514

  10. A detached binary system BK Pegasi

    NASA Astrophysics Data System (ADS)

    Demircan, Osman; Kaya, Yalcin; Tufekcioglu, Zeki

    1994-12-01

    The UBV photometry of the detached F-type eclipsing binary BK Pegasi is presented. The light curve solution by a simple spherical model assumption suggests that a slightly hotter, larger and more luminous primary was eclipsed during the primary minimum. Combined with the radial velocity curves in Popper (1983), it was used to determine anew the system's parameters. The age and the metal abundance of the system were estimated as (3.3 +/- 0.2) x 109 yr and z = 0.028 +/- 0.003 from a comparison with the new grids of stellar models and the isochrones by Schaller et al. (1993). The distance of the system was estimated as 290 pc.

  11. Seroprevalence of human polyomavirus 9 and cross-reactivity to African green monkey-derived lymphotropic polyomavirus.

    PubMed

    Trusch, Franziska; Klein, Marcus; Finsterbusch, Tim; Kühn, Joachim; Hofmann, Jörg; Ehlers, Bernhard

    2012-04-01

    Human polyomavirus 9 (HPyV9) was discovered recently in immunocompromised patients and shown to be genetically closely related to B-lymphotropic polyomavirus (LPyV). No serological data are available for HPyV9, but human antibodies against LPyV have been reported previously. To investigate the seroepidemiology of HPyV9 and the sero-cross-reactivity between HPyV9 and LPyV, a capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV9 and LPyV. VP1 of an avian polyomavirus was used as control. For HPyV9, a seroprevalence of 47 % was determined in healthy adults and adolescents (n = 328) and 20 % in a group of children (n =101). In both groups, the seroreactivities for LPyV were less frequent and the ELISA titres of LPyV were lower. Of the HPyV9-reactive sera, 47 % reacted also with LPyV, and the titres for both PyVs correlated. Sera from African green monkeys, the natural hosts of LPyV, reacted also with both HPyV9 and LPyV, but here the HPyV9 titres were lower. This potential sero-cross-reactivity between HPyV9 and LPyV was confirmed by competition assays, and it was hypothesized that the reactivity of human sera against LPyV may generally be due to cross-reactivity between HPyV9 and LPyV. The HPyV9 seroprevalence of liver transplant recipients and patients with neurological dysfunctions did not differ from that of age-matched controls, but a significantly higher seroprevalence was determined in renal and haematopoietic stem-cell transplant recipients, indicating that certain immunocompromised patient groups may be at a higher risk for primary infection with or for reactivation of HPyV9.

  12. p53 gene mutational rate, Gleason score, and BK virus infection in prostate adenocarcinoma: Is there a correlation?

    PubMed

    Russo, Giuseppe; Anzivino, Elena; Fioriti, Daniela; Mischitelli, Monica; Bellizzi, Anna; Giordano, Antonio; Autran-Gomez, Anamaria; Di Monaco, Franco; Di Silverio, Franco; Sale, Patrizio; Di Prospero, Laura; Pietropaolo, Valeria

    2008-12-01

    Prostate cancer represents the second leading cause of cancer deaths in Western countries. Viral infections could play a role in prostate carcinogenesis. Human polyomavirus BK (BKV) is a possible candidate because of its transforming properties. In this study, BKV sequences in urine, blood, fresh, and paraffin-embedded prostate cancer samples from 26 patients were searched using Q-PCR analysis. T antigen (TAg) and p53 localization in neoplastic cells were evaluated by immunohistochemical analysis. Also, the presence of mutations in 5-9 exons of p53 gene was analyzed. Results showed that BKV-DNA was found in urine (54%), plasma (31%), and in fresh prostate cancer specimens (85%). The analysis of p53 gene evidenced several mutations in high Gleason patients, according to tumor advanced stage. Immunohistochemical analysis results evidenced the localization of p53 and TAg into cytoplasm, whereas in TAg-negative tumors, p53 was nuclear. This study suggests that BKV acts as cofactor in the pathogenesis of prostate cancer. These observations emphasize previous studies regarding the cellular pathways that may be deregulated by BKV.

  13. Phylogenetic reconstruction and polymorphism analysis of BK virus VP2 gene isolated from renal transplant recipients in China

    PubMed Central

    WANG, ZHANG-YANG; HONG, WEI-LONG; ZHU, ZHE-HUI; CHEN, YUN-HAO; YE, WEN-LE; CHU, GUANG-YU; LI, JIA-LIN; CHEN, BI-CHENG; XIA, PENG

    2015-01-01

    BK polyomavirus (BKV) is important pathogen for kidney transplant recipients, as it is frequently re-activated, leading to nephropathy. The aim of this study was to investigate the phylogenetic reconstruction and polymorphism of the VP2 gene in BKV isolated from Chinese kidney transplant recipients. Phylogenetic analysis was carried out in the VP2 region from 135 BKV-positive samples and 28 reference strains retrieved from GenBank. The unweighted pair-group method with arithmetic mean (UPGMA) grouped all strains into subtypes, but failed to subdivide strains into subgroups. Among the plasma and urine samples, all plasma (23/23) and 82 urine samples (82/95) were identified to contain subtype I; the other 10 urine samples contained subtype IV. A 86-bp fragment was identified as a highly conserved sequence. Following alignment with 36 published BKV sequences from China, 92 sites of polymorphism were identified, including 11 single nucleotide polymorphisms (SNPs) prevalent in Chinese individuals and 30 SNPs that were specific to the two predominant subtypes I and IV. The limitations of the VP2 gene segment in subgrouping were confirmed by phylogenetic analysis. The conserved sequence and polymorphism identified in this study may be helpful in the detection and genotyping of BKV. PMID:26640547

  14. Increased BK viremia and progression to BK-virus nephropathy following high-dose intravenous immunoglobulin for acute cellular rejection.

    PubMed

    Boonyapredee, Maytee; Knight, Kendral; Little, Dustin

    2014-06-01

    BK virus nephropathy and cellular rejection are common causes of allograft dysfunction in renal transplant recipients. The two can be difficult to distinguish on allograft biopsy and can be present simultaneously. Management of the patient with coexistent BK infection and rejection is complicated by the conflicting ideals of decreasing immunosuppression to treat the former and increasing immunosuppression to treat the latter. The authors present the case of a 57-year-old renal transplant recipient who underwent allograft biopsy 8 weeks post-transplant for evaluation of increased serum creatinine in the setting of BK viremia (BKV). Biopsy revealed Banff classification 1b acute cellular rejection, with insufficient evidence to diagnose BK virus-associated nephropathy. The patient was administered intravenous immune globulin (IVIG), with no other changes in immunosuppressive therapy. Plasma and urine BK increased exponentially following IVIG administration, and allograft function further deteriorated. Repeat biopsy showed overt BK viral nephropathy, and BKV and creatinine decreased only after reduction in immunosuppression and initiation of leflunomide. Although case series have suggested a potential role for IVIG in the setting of BK infection, further study is needed to define the safety and efficacy of this approach. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

  15. Rubidium efflux as a tool for the pharmacological characterisation of compounds with BK channel opening properties.

    PubMed

    McKay, Neil G; Kirby, Robert W; Lawson, Kim

    2008-01-01

    This chapter describes a method of assaying rubidium (Rb(+)) efflux as a measure of potassium channel activity. In this assay, rubidium acts as a tracer for potassium movement across the cell membrane. HEK 293 cells expressing the alpha subunit of the human brain large-conductance, voltage-activated, calcium-sensitive potassium channel (BK channel) are loaded with Rb(+), washed, and then incubated under experimental conditions. The cell supernatant is removed, and the remaining cell monolayer lysed. These two samples contain Rb(+) that has moved out of the cell and Rb(+) that remains in the cell, respectively. Measurement of the Rb(+) content of these samples by flame atomic absorption spectrometry allows calculation of the percentage Rb(+) efflux and, depending on the experimental design, provides pharmacological data about the control and test compounds used. In this chapter, we describe the protocol and steps for optimisation and illustrate this with data obtained using NS1619, a well-characterised BK channel opener.

  16. BK channels modulate pre- and postsynaptic signaling at reciprocal synapses in retina

    PubMed Central

    Grimes, William N.; Li, Wei; Chávez, Andrés E.; Diamond, Jeffrey S.

    2009-01-01

    In the mammalian retina, A17 amacrine cells provide reciprocal inhibitory feedback to rod bipolar cells, thereby shaping the time course of visual signaling in vivo. Previous results indicate that A17 feedback can be triggered by Ca2+ influx through Ca2+ permeable AMPARs and can occur independently of voltage-gated Ca2+ (Cav) channels, whose presence and functional role in A17 dendrites have not been explored. Here, we combine electrophysiology, calcium imaging and immunohistochemistry to show that L-type Cav channels in rat A17 amacrine cells are located at the sites of reciprocal synaptic feedback, but their contribution to GABA release is diminished by large-conductance Ca2+-activated potassium (BK) channels, which suppress postsynaptic depolarization in A17s and limit Cav channel activation. We also show that BK channels, by limiting GABA release from A17s, regulate the flow of excitatory synaptic transmission through the rod pathway. PMID:19363492

  17. Alcohol modulation of BK channel gating depends on β subunit composition.

    PubMed

    Kuntamallappanavar, Guruprasad; Dopico, Alex M

    2016-11-01

    In most mammalian tissues, Ca(2+)i/voltage-gated, large conductance K(+) (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca(2+)i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca(2+)i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca(2+)i while inhibiting current at high Ca(2+)i, the potentiation-inhibition crossover occurring at 20 µM Ca(2+)i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca(2+)i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca(2+) affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca(2+) binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd

  18. Limited variation during circulation of a polyomavirus in the human population involves the COCO-VA toggling site of Middle and Alternative T-antigen(s).

    PubMed

    Kazem, Siamaque; Lauber, Chris; van der Meijden, Els; Kooijman, Sander; Kravchenko, Alexander A; Feltkamp, Mariet C W; Gorbalenya, Alexander E

    2016-01-01

    We have recently shown that the trichodysplasia spinulosa-associated polyomavirus (TSPyV) belongs to a large monophyletic group of mammalian polyomaviruses that experienced accelerated codon-constrained Val-Ala (COCO-VA) toggling at a protein site common to both Middle and Alternative T-antigens (MT/ALTO). Here we analyzed thirteen, mostly newly sequenced TSPyV genomes, representing ~40% of reported TS disease cases world-wide. We found two deletions and 30 variable sites (≤0.6%) that included only four sites with non-synonymous substitutions (NSS). One NSS site was under positive selection in the exon shared by Small and Middle T antigens, while three others were segregated in MT/ALTO. Two MT/ALTO sites covaried with five sites elsewhere in the genome and determined separation of twelve TSPyVs into two most populous phylogenetic lineages. The other, most distant TSPyV was distinguished by NSS at the COCO-VA site, observed for the first time during intra-species evolution. Our findings reveal a connection between micro- and macro-evolution of polyomaviruses.

  19. Enhancer dependence of polyomavirus persistence in mouse kidneys.

    PubMed Central

    Rochford, R; Moreno, J P; Peake, M L; Villarreal, L P

    1992-01-01

    We previously showed that alterations in the enhancer sequence of polyomavirus DNA can alter both the level and the organ specificity of viral DNA replication during the acute phase of infection of newborn mice (R. Rochford, B. A. Campbell, and L. P. Villarreal, J. Virol. 64:476-485, 1990). In this study, we examined whether these enhancer sequence alterations can also affect polyomavirus replication during the persistent phase of infection in vivo. After infection of newborn mice with a mixture of three enhancer variants, the individual organs could select for enhancer-specific viral DNA replication during both the acute and the persistent phases of infection. Contrary to expectations, the ability of some variants to establish a high-level acute infection in some organs (e.g., the pancreas) did not necessarily lead to a persistent infection in those organs. Thus, enhancers can affect acute and persistent infections differently. In addition, some enhancer variants tended to establish a high-level persistent infection in the kidneys immediately following an acute infection; however, in all cases considerable histopathology was associated with these elevated long-term infections, and these mice were always runty. A persistent infection in the kidneys thus appears able to exist in two distinguishable states, a high-level pathological state and a low-level nonpathological state, which can be affected by the viral enhancer sequence. Images PMID:1316448

  20. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  1. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  2. Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels.

    PubMed

    Hristov, Kiril L; Parajuli, Shankar P; Provence, Aaron; Petkov, Georgi V

    2016-12-01

    In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of -20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca(2+) imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca(2+) concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability. Copyright © 2016 the American Physiological Society.

  3. The role of BK-type Ca2+-dependent K+ channels in spike broadening during repetitive firing in rat hippocampal pyramidal cells

    PubMed Central

    Shao, Li-Rong; Halvorsrud, Ragnhild; Borg-Graham, Lyle; Storm, Johan F

    1999-01-01

    The role of large-conductance Ca2+-dependent K+ channels (BK-channels; also known as maxi-K- or slo-channels) in spike broadening during repetitive firing was studied in CA1 pyramidal cells, using sharp electrode intracellular recordings in rat hippocampal slices, and computer modelling. Trains of action potentials elicited by depolarizing current pulses showed a progressive, frequency-dependent spike broadening, reflecting a reduced rate of repolarization. During a 50 ms long 5 spike train, the spike duration increased by 63·6 ± 3·4% from the 1st to the 3rd spike. The amplitude of the fast after-hyperpolarization (fAHP) also rapidly declined during each train. Suppression of BK-channel activity with (a) the selective BK-channel blocker iberiotoxin (IbTX, 60 nM), (b) the non-peptidergic BK-channel blocker paxilline (2–10 μM), or (c) calcium-free medium, broadened the 1st spike to a similar degree (≈60%). BK-channel suppression also caused a similar change in spike waveform as observed during repetitive firing, and eliminated (occluded) most of the spike broadening during repetitive firing. Computer simulations using a reduced compartmental model with transient BK-channel current and 10 other active ionic currents, produced an activity-dependent spike broadening that was strongly reduced when the BK-channel inactivation mechanism was removed. These results, which are supported by recent voltage-clamp data, strongly suggest that in CA1 pyramidal cells, fast inactivation of a transient BK-channel current (ICT), substantially contributes to frequency-dependent spike broadening during repetitive firing. PMID:10562340

  4. Calcium- and voltage-gated potassium (BK) channel activators in the 5β-cholanic acid-3α-ol analogue series with modifications in the lateral chain.

    PubMed

    Bukiya, Anna N; Patil, Shivaputra A; Li, Wei; Miller, Duane D; Dopico, Alex M

    2012-10-01

    Large conductance, calcium- and voltage-gated potassium (BK) channels regulate various physiological processes and represent an attractive target for drug discovery. Numerous BK channel activators are available. However, these agents usually interact with the ubiquitously distributed channel-forming subunit and thus cannot selectively target a particular tissue. We performed a structure-activity relationship study of lithocholic acid (LCA), a cholane that activates BK channels via the accessory BK β1 subunit. The latter protein is highly abundant in smooth muscle but scarce in most other tissues. Modifications to the LCA lateral chain length and functional group yielded two novel smooth muscle BK channel activators in which the substituent at C24 has a small volume and a net negative charge. Our data provide detailed structural information that will be useful to advance a pharmacophore in search of β1 subunit-selective BK channel activators. These compounds are expected to evoke smooth muscle relaxation, which would be beneficial in the pharmacotherapy of prevalent human disorders associated with increased smooth muscle contraction, such as systemic hypertension, cerebral or coronary vasospasm, bronchial asthma, bladder hyperactivity, and erectile dysfunction.

  5. Ureteral stent placement and BK viremia in kidney transplant recipients.

    PubMed

    Kayler, L; Zendejas, I; Schain, D; Magliocca, J

    2013-04-01

    BK virus (BKV) infection is an important cause of kidney transplant dysfunction. A possible association of double-J ureteral stent placement and BK viremia has been suggested in previous studies; however, risk factors for BK are incompletely understood. We aimed to determine if stent placement is an independent risk factor for BK viremia. Data were collected on consecutive kidney-only transplant recipients between December 1, 2006 and June 30, 2010. All patients had at least 12 months of follow-up. Of 600 consecutive kidney transplants, BK viremia within the first post-transplant year was detected in 93 patients (15.5%); in 70 of these cases, the peak BKV polymerase chain reaction was ≥10,000 copies/mL. By multivariate analysis, significant risk factors for BK viremia were recipient age (P = 0.02) and stent placement (P = 0.03). Stents were placed in 49.2% and removed at a median of 46 days (range: 11-284) post transplantation; removals occurred within 0-30, 30-60, 60-90, 90-120, 120-150, and >150 days post transplantation in 18.4%, 67.2%, 10.5%, 2.4%, 1.0%, and 0.3% of cases, respectively. No association was found of BK viremia with stent duration >46 days (P = 0.70) or by the 6-level groupings (P = 0.92). Although we observed a significant association of BK viremia with stent placement, no dose-dependent effect was seen. © 2013 John Wiley & Sons A/S.

  6. Polyomavirus nephropathy of the native kidney in a patient with rheumatoid arthritis and pulmonary fibrosis.

    PubMed

    Krystel-Whittemore, Melissa; McCarthy, Ellen T; Damjanov, Ivan; Fields, Timothy A

    2015-08-28

    Polyomavirus nephropathy is commonly seen in the renal allograft setting but is uncommon in native kidneys. This paper describes polyomavirus nephropathy that developed in the native kidneys of a patient following immunosuppressive therapy for rheumatoid arthritis/Sjögren's syndrome associated lung disease. The patient presented with dyspnoea and a slow steady rise in serum creatinine. Owing to chronic immunosuppression, calcineurin-inhibitor toxicity was suspected. However, renal biopsy revealed polyomavirus nephropathy. The treatment of choice, lowered immunosuppression, was complicated by exacerbation of the patient's lung disease. This case highlights features of polyomavirus nephropathy in the native kidney, as well as the difficulty in its treatment when immunosuppressive treatment is necessary for medical comorbidities.

  7. Rapid detection of trichodysplasia spinulosa-associated polyomavirus in skin biopsy specimen.

    PubMed

    Urbano, Paulo Roberto P; Pannuti, Cláudio Sérgio; Pierrotti, Ligia C; David-Neto, Elias; Romano, Camila Malta

    2014-07-24

    Trichodysplasia spinulosa-associated polyomavirus (TSV) is responsible for a rare skin cancer. Using metagenomic approaches, we determined the complete genome sequence of a TSV first detected in Brazil in spicules of an immunocompromised patient suspected to have trichodysplasia spinulosa.

  8. Human polyomaviruses 6, 7, 9, 10 and Trichodysplasia spinulosa-associated polyomavirus in HIV-infected men.

    PubMed

    Wieland, Ulrike; Silling, Steffi; Hellmich, Martin; Potthoff, Anja; Pfister, Herbert; Kreuter, Alexander

    2014-04-01

    Recently, several novel human polyomaviruses (HPyVs) have been detected. HPyV6, 7, 9 and 10 are not associated with any disease so far. Trichodysplasia spinulosa (TS)-associated polyomavirus (TSPyV) can cause the rare skin disease TS. We have evaluated cutaneous DNA prevalence and viral loads of five HPyVs in HIV-infected men compared to healthy male controls. 449 forehead swabs were analysed by HPyV-specific real-time PCR. HPyV6, HPyV7, TSPyV and HPyV10 were found significantly more frequently on the skin of 210 HIV-infected compared to 239 HIV-negative men (HPyV6, 39.0 vs 27.6 %; HPyV7, 21.0 vs 13.4 %; TSPyV, 3.8 vs 0.8 %; HPyV10, 9.3 vs 3.4 %; P<0.05, respectively). HPyV9 was not detected. Multiple infections were more frequent in HIV-positive men, but HPyV-DNA loads did not differ significantly in both groups. In contrast to HPyV6, 7 and 10, TSPyV and HPyV9 do not seem to be a regular part of the human skin microbiome.

  9. Ultrastructural and molecular confirmation of the trichodysplasia spinulosa-associated polyomavirus in biopsies of patients with trichodysplasia spinulosa.

    PubMed

    Elaba, Zendee; Hughey, Lauren; Isayeva, Tatyana; Weeks, Beth; Solovan, Caius; Solovastru, Laura; Andea, Aleodor

    2012-11-01

    Trichodysplasia spinulosa (TS) is a rare and only recently characterized cutaneous disease occurring in immunocompromised patients. The disease is characterized by spiny follicular papules on clinical examination and by the presence of viral inclusions at ultrastructural examination. In the last year, this virus has been identified as a new member of the polyomavirus family and designated as TS-associated polyomavirus (TSPyV). We report two organ transplant patients with this disease in which we were able to identify the TSPyV at ultrastructural and molecular level from formalin-fixed paraffin-embedded biopsies of lesional skin. Similar to prior described cases, the patients presented with follicular papules which were concentrated on the central face and associated with alopecia. Histopathology of both cases showed dilated follicular infundibula plugged with cornified eosinophilic cells containing large trichohyaline granules. Transmission electron microscopy on paraffin-embedded tissue in case 1 showed 28-nm intracellular viral particles morphologically consistent with polyoma virus. For both cases the presence of TSPyV was confirmed by polymerase chain reaction with virus-specific primers followed by identification by direct sequencing. These two cases show the presence of the newly described TSPyV in TS further establishing its association with this distinctive disease.

  10. Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels.

    PubMed

    Zhang, Guohui; Geng, Yanyan; Jin, Yakang; Shi, Jingyi; McFarland, Kelli; Magleby, Karl L; Salkoff, Lawrence; Cui, Jianmin

    2017-03-06

    Large conductance Ca(2+)-activated K(+) channels (BK channels) gate open in response to both membrane voltage and intracellular Ca(2+) The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca(2+) sensor. How these voltage and Ca(2+) sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca(2+) activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca(2+) sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's β1 and β2 subunits.

  11. BK channels: multiple sensors, one activation gate.

    PubMed

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

    Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca(2+) activated BK channels, a K(+) channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate.

  12. How Polyomaviruses Exploit the ERAD Machinery to Cause Infection

    PubMed Central

    Dupzyk, Allison; Tsai, Billy

    2016-01-01

    To infect cells, polyomavirus (PyV) traffics from the cell surface to the endoplasmic reticulum (ER) where it hijacks elements of the ER-associated degradation (ERAD) machinery to penetrate the ER membrane and reach the cytosol. From the cytosol, the virus transports to the nucleus, enabling transcription and replication of the viral genome that leads to lytic infection or cellular transformation. How PyV exploits the ERAD machinery to cross the ER membrane and access the cytosol, a decisive infection step, remains enigmatic. However, recent studies have slowly unraveled many aspects of this process. These emerging insights should advance our efforts to develop more effective therapies against PyV-induced human diseases. PMID:27589785

  13. JC Polyomavirus (JCV) and Monoclonal Antibodies: Friends or Potential Foes?

    PubMed Central

    Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Clementi, Massimo

    2013-01-01

    Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS), observed in immunodeficient patients and caused by JC virus ((JCV), also called JC polyomavirus (JCPyV)). After the HIV pandemic and the introduction of immunomodulatory therapy, the PML incidence significantly increased. The correlation between the use of natalizumab, a drug used in multiple sclerosis (MS), and the PML development of particular relevance. The high incidence of PML in natalizumab-treated patients has highlighted the importance of two factors: the need of PML risk stratification among natalizumab-treated patients and the need of effective therapeutic options. In this review, we discuss these two needs under the light of the major viral models of PML etiopathogenesis. PMID:23878587

  14. Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels

    PubMed Central

    Hei, Hongya; Li, Fangping; Wang, Yunman; Peng, Wen; Zhang, Xuemei

    2015-01-01

    Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms. PMID:26672753

  15. Interactions among the major and minor coat proteins of polyomavirus.

    PubMed Central

    Barouch, D H; Harrison, S C

    1994-01-01

    Murine polyomavirus contains two related minor coat proteins, VP2 and VP3, in addition to the major coat protein, VP1. The sequence of VP3 is identical to that of the carboxy-terminal two-thirds of VP2. VP2 may serve a role in uncoating of the virus, and both minor coat proteins may be important for viral assembly. In this study, we show that VP3 and a series of deletion mutants of VP3 can be expressed in Escherichia coli as fusion proteins to glutathione S-transferase and partially solubilized with a mild detergent. Using an in vitro binding assay, we demonstrate that a 42-amino-acid fragment near the carboxy terminus of VP3 (residues 140 to 181) is sufficient for binding to purified VP1 pentamers. This binding interaction is rapid, saturable, and specific for the common carboxy terminus of VP2 and VP3. The VP1-VP3 complex can be coimmunoprecipitated with an antibody specific to VP1, and a purified VP3 fragment can selectively extract VP1 from a crude cell lysate. The stoichiometry of the binding reaction suggests that each VP1 pentamer in the virus binds either one VP2 or one VP3, with the VP1-VP2/3 complex stabilized by hydrophobic interactions. These results, taken together with studies from other laboratories on the expression of polyomavirus capsid proteins in mouse and insect cells (S. E. Delos, L. Montross, R. B. Moreland, and R. L. Garcea, Virology, 194:393-398, 1993; J. Forstova, N. Krauzewicz, S. Wallace, A. J. Street, S. M. Dilworth, S. Beard, and B. E. Griffin, J. Virol. 67:1405-1413, 1993), support the idea that a VP1-VP2/3 complex forms in the cytoplasm and, after translocation into the nucleus, acts as the unit for viral assembly. Images PMID:8189532

  16. BK virus infection in human immunodeficiency virus-infected patients.

    PubMed

    Ledesma, J; Muñoz, P; Garcia de Viedma, D; Cabrero, I; Loeches, B; Montilla, P; Gijon, P; Rodriguez-Sanchez, B; Bouza, E

    2012-07-01

    The aim of this study is to evaluate the prevalence of BK virus (BKV) infection in HIV-positive patients receiving highly active antiretroviral therapy (HAART) in our hospital. The presence of BKV was analysed in urine and plasma samples from 78 non-selected HIV-infected patients. Clinical data were recorded using a pre-established protocol. We used a nested PCR to amplify a specific region of the BKV T-large antigen. Positive samples were quantified using real-time PCR. Mean CD4 count in HIV-infected patients was 472 cells/mm3 and median HIV viral load was <50 copies/mL. BKV viraemia was detected in only 1 HIV-positive patient, but 57.7% (45 out of 78) had BKV viruria, which was more common in patients with CD4 counts>500 cells/mm3 (74.3% vs 25.7%; p=0.007). Viruria was present in 21.7% of healthy controls (5 out of 23 samples, p=0.02). All viral loads were low (<100 copies/mL), and we could not find any association between BKV infection and renal or neurological manifestations. We provide an update on the prevalence of BKV in HIV-infected patients treated with HAART. BKV viruria was more common in HIV-infected patients; however, no role for BKV has been demonstrated in this population.

  17. Atomic determinants of BK channel activation by polyunsaturated fatty acids

    PubMed Central

    Tian, Yutao; Aursnes, Marius; Hansen, Trond Vidar; Tungen, Jørn Eivind; Galpin, Jason D.; Leisle, Lilia; Ahern, Christopher A.; Xu, Rong; Heinemann, Stefan H.; Hoshi, Toshinori

    2016-01-01

    Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA–channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion–dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery. PMID:27849612

  18. Post-transcriptional regulation of BK channel splice variant stability by miR-9 underlies neuroadaptation to alcohol

    PubMed Central

    Pietrzykowski, Andrzej Z.; Friesen, Ryan M.; Martin, Gilles E.; Puig, Sylvie I.; Nowak, Cheryl L.; Wynne, Patricia M.; Siegelmann, Hava T.; Treistman, Steven N.

    2008-01-01

    Summary Tolerance represents a critical component of addiction. The large conductance calcium-and voltage-activated potassium channel (BK) is a well-established alcohol target, and an important element in behavioral and molecular alcohol tolerance. We tested whether microRNA, a newly-discovered class of gene expression regulators, plays a role in the development of tolerance. We show that in adult mammalian brain alcohol upregulates microRNA (miR-9) and mediates post-transcriptional reorganization in BK mRNA splice variants by miR-9-dependent destabilization of BK mRNAs containing 3’UTRs with a miR-9 Recognition Element (MRE). Different splice variants encode BK isoforms with different alcohol sensitivities. Computational modeling indicates that this miR-9 dependent mechanism contributes to alcohol tolerance. Moreover, this mechanism can be extended to regulation of additional miR-9 targets relevant to alcohol abuse. Our results describe a novel mechanism of multiplex regulation of stability of alternatively spliced mRNA by miRNA in drug adaptation and neuronal plasticity. PMID:18667155

  19. BK viruria and viremia in children with systemic lupus erythematosus.

    PubMed

    Gupta, Nirupama; Nguyen, Cuong Q; Modica, Renee F; Elder, Melissa E; Garin, Eduardo H

    2017-04-11

    BK virus (BKV) is a ubiquitous polyoma virus that lies dormant in the genitourinary tract once acquired in early childhood. In states of cellular immunodeficiency, the virus can reactivate to cause hemorrhagic cystitis and nephritis. Children with systemic lupus erythematosus (SLE) have an increased risk of developing infectious complications secondary to their immunocompromised state from the administration of several immuno-modulatory drugs. Currently, there are no data regarding the prevalence of BK viruria or viremia in children with SLE. We conducted a prospective cohort study involving children with SLE of 18 years and younger. We obtained urine and blood samples at baseline and every 3 months up to 1 year for BK virus detection by real-time, quantitative polymerase chain reaction analysis. A comprehensive review of demographic information, clinical characteristics and medication history was also obtained. Thirty-two pediatric patients (26 females and 6 males) with SLE were enrolled. Median age at the time of SLE diagnosis and enrollment into study was 13.6 years and 16.0 years old, respectively. The prevalence at enrollment was 3.1% (1/32) for BK viruria and 6.2% (2/32) for BK viremia. During the study period, 3 patients had viruria, 5 had viremia and 4 had both viruria and viremia. Of the 12 patients with BKV reactivation, only one was positive for microscopic hematuria, all others were asymptomatic. A total of nine of 97(9.2%) urine samples and 10 of 96(10.4%) blood samples were positive for BK virus. The most commonly utilized biologics in this cohort group were Rituximab (90.6%), Abatacept (12.5%), and Belimumab (9.3%). The type of medication exposure and clinical characteristics did not statistically differ between the groups that did or did not have BK viruria and/or viremia. Our study suggests that pediatric patients with SLE have BK viremia and/or viruria at a higher rate than the general healthy population, although the significance of the

  20. BK channel inactivation gates daytime excitability in the circadian clock

    PubMed Central

    Whitt, Joshua P.; Montgomery, Jenna R.; Meredith, Andrea L.

    2016-01-01

    Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K+ channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain's intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal ‘ball-and-chain' domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime ‘upstate'. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm. PMID:26940770

  1. A BK (Slo1) channel journey from molecule to physiology

    PubMed Central

    Contreras, Gustavo F; Castillo, Karen; Enrique, Nicolás; Carrasquel-Ursulaez, Willy; Castillo, Juan Pablo; Milesi, Verónica; Neely, Alan; Alvarez, Osvaldo; Ferreira, Gonzalo; González, Carlos; Latorre, Ramón

    2013-01-01

    Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca2+ and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca2+ sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression. PMID:24025517

  2. BK channel inactivation gates daytime excitability in the circadian clock.

    PubMed

    Whitt, Joshua P; Montgomery, Jenna R; Meredith, Andrea L

    2016-03-04

    Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K(+) channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain's intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal 'ball-and-chain' domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime 'upstate'. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm.

  3. Antibodies to merkel cell polyomavirus T antigen oncoproteins reflect tumor burden in merkel cell carcinoma patients.

    PubMed

    Paulson, Kelly G; Carter, Joseph J; Johnson, Lisa G; Cahill, Kevin W; Iyer, Jayasri G; Schrama, David; Becker, Juergen C; Madeleine, Margaret M; Nghiem, Paul; Galloway, Denise A

    2010-11-01

    Merkel cell polyomavirus (MCPyV) is a common infectious agent that is likely involved in the etiology of most Merkel cell carcinomas (MCC). Serum antibodies recognizing the MCPyV capsid protein VP1 are detectable at high titer in nearly all MCC patients and remain stable over time. Although antibodies to the viral capsid indicate prior MCPyV infection, they provide limited clinical insight into MCC because they are also detected in more than half of the general population. We investigated whether antibodies recognizing MCPyV large and small tumor-associated antigens (T-Ag) would be more specifically associated with MCC. Among 530 population control subjects, these antibodies were present in only 0.9% and were of low titer. In contrast, among 205 MCC cases, 40.5% had serum IgG antibodies that recognize a portion of T-Ag shared between small and large T-Ags. Among cases, titers of T-Ag antibodies fell rapidly (∼8-fold per year) in patients whose cancer did not recur, whereas they rose rapidly in those with progressive disease. Importantly, in several patients who developed metastases, the rise in T-Ag titer preceded clinical detection of disease spread. These results suggest that antibodies recognizing T-Ag are relatively specifically associated with MCC, do not effectively protect against disease progression, and may serve as a clinically useful indicator of disease status.

  4. Differential regulation of SK and BK channels by Ca2+ signals from Ca2+ channels and ryanodine receptors in guinea-pig urinary bladder myocytes

    PubMed Central

    Herrera, Gerald M; Nelson, Mark T

    2002-01-01

    Small-conductance (SK) and large-conductance (BK) Ca2+-activated K+ channels are key regulators of excitability in urinary bladder smooth muscle (UBSM) of guinea-pig. The overall goal of this study was to define how SK and BK channels respond to Ca2+ signals from voltage-dependent Ca2+ channels (VDCCs) in the surface membrane and from ryanodine-sensitive Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane. To characterize the role of SK channels in UBSM, the effects of the SK channel blocker apamin on phasic contractions were examined. Apamin caused a dose-dependent increase in the amplitude of phasic contractions over a broad concentration range (10−10 to 10−6m). To determine the effects of Ca2+ signals from VDCCs and RyRs to SK and BK channels, whole cell membrane current was measured in isolated myocytes bathed in physiological solutions. Depolarization (-70 to +10 mV for 100 ms) of isolated myocytes caused an inward Ca2+ current (ICa), followed by an outward current. The outward current was reduced in a dose-dependent manner by apamin (10−10 to 10−6m), and designated ISK. ISK had a mean amplitude of 53.8 ± 6.1 pA or ∼1.4 pA pF−1 at +10 mV. The amplitude of ISK correlated with the peak ICa. Blocking ICa abolished ISK. In contrast, ISK was insensitive to the RyR blocker ryanodine (10 μM). These data indicate that Ca2+ signals from VDCCs, but not from RyRs, activate SK channels. BK channel currents (IBK) were isolated from other currents by using the BK channel blockers tetraethylammonium ions (TEA+; 1 mm) or iberiotoxin (200 nm). Voltage steps evoked transient and steady-state IBK components. Transient BK currents have previously been shown to result from BK channel activation by local Ca2+ release through RyRs (‘Ca2+ sparks’). Transient BK currents were inhibited by ryanodine (10 μM), as expected, and had a mean amplitude of 152.6 pA at +10 mV. The mean number of transient BK currents during a

  5. A molecular case-control study of the Merkel cell polyomavirus in colon cancer.

    PubMed

    Campello, Cesare; Comar, Manola; D'Agaro, Pierlanfranco; Minicozzi, Anna; Rodella, Luca; Poli, Albino

    2011-04-01

    To explore the putative role of the Merkel cell polyomavirus in human colon cancer, a prospective molecular case-control study was undertaken in patients and their relatives enrolled during a screening program. Fresh tissue samples from 64 cases of colon cancer (mean age 69.9 ± 11.0 years; 40 males) and fresh biopsies from 80 relatives (mean age 53.7 ± 8.6 years; 43 male; 55 son/daughter, 23 brother/sister, 2 parents) were analyzed by PCR and sequencing. Pre-cancerous lesions, namely adenomas and polyps, were detected in 15 (18.8%) and 9 (11.2%) of the controls, respectively. In addition, 144 blood samples were examined. Merkel cell polyomavirus DNA was detected in 6.3% of cases and 8.8% of controls. This difference was not statistically significant in the logistic regression analysis, after adjustment for age. Whereas blood samples from both cases and controls tested negative, the DNA Merkel cell polyomavirus was identified in 12.5% of adenoma/polyp tissues. No statistically significant difference was found when prevalence rates of Merkel cell polyomavirus in normal, pre-cancerous and cancer tissues were compared. Sequence analysis of the viral LT3 and VP1 regions showed high homology (>99%) with those of strains circulating worldwide, especially with genotypes detected in France. The findings of this survey are consistent with the hypothesis that the Merkel cell polyomavirus, in addition to other human polyomaviruses, can be recovered frequently from the gastrointestinal tract, because it is transmitted throughout the fecal-oral route. Moreover, the study does not indicate a role for Merkel cell polyomavirus in the genesis of colon cancer.

  6. Intrinsic Electrostatic Potential in the BK Channel Pore: Role in Determining Single Channel Conductance and Block

    PubMed Central

    Carvacho, Ingrid; Gonzalez, Wendy; Torres, Yolima P.; Brauchi, Sebastian; Alvarez, Osvaldo; Gonzalez-Nilo, Fernando D.; Latorre, Ramon

    2008-01-01

    The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017–9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current–voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, Δφ, caused by the charge neutralization using TEA+ block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 ± 2 mV for Δφ at the TEA+ site when turning off the extracellular charges, and 17 ± 2 mV for the Δφ at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Δφ using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329. PMID:18227273

  7. Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration

    PubMed Central

    Knight, Laura M.; Stakaityte, Gabriele; Wood, Jennifer, J.; Abdul-Sada, Hussein; Griffiths, David A.; Howell, Gareth J.; Wheat, Rachel; Blair, G. Eric; Steven, Neil M.; Macdonald, Andrew; Blackbourn, David J.

    2014-01-01

    ABSTRACT Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule

  8. Unique action of sodium tanshinone II-A sulfonate (DS-201) on the Ca(2+) dependent BK(Ca) activation in mouse cerebral arterial smooth muscle cells.

    PubMed

    Tan, Xiaoqiu; Yang, Yan; Cheng, Jun; Li, Pengyun; Inoue, Isao; Zeng, Xiaorong

    2011-04-10

    Sodium tanshinone II-A sulfonate (DS-201) is a water-soluble derivative of tanshinone IIA, a main active constituent of Salvia miltiorrhiza which has been used for treatments of cardio- and cerebro-vascular diseases. DS-201 activates large conductance Ca(2+)-sensitive K(+) channels (BK(Ca)) in arterial smooth muscle cells, and reduces the vascular tone. Here we investigated the effect of DS-201 on the BK(Ca) channel kinetics by analyzing single channel currents. Smooth muscle cells were freshly isolated from mouse cerebral arteries. Single channel currents of BK(Ca) were recorded by patch clamp. DS-201 increased the total open probability (NPo) of BK(Ca) in a concentration-dependent manner. But this action required intracellular Ca(2+), and the effect depended on the Ca(2+) concentration ([Ca(2+)](free)). DS-201 activated BK(Ca) with the half maximal effective concentration (EC(50)) of 111.5μM at 0.01μM [Ca(2+)](free), and 68.5μM at 0.1μM [Ca(2+)](free.) The effect of DS-201 on NPo was particularly strong in the range of [Ca(2+)](free) between 0.1 and 1μM. Analysis of the channel kinetics revealed that DS-201 had only the effect on the channel closing without affecting the channel opening, which was a striking contrast to the effect of [Ca(2+)](free), that is characterized by changing the channel opening without changing the channel closing. DS-201 may be bound to the open state of BK(Ca), and have an inhibitory effect on the transition from the open to closed state. By this way DS-201 may enhance the activity of BK(Ca), and exhibit a strong vasodilating effect against vasoconstriction in the range of [Ca(2+)](free) between 0.1 and 1μM. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. BK potassium channels facilitate high-frequency firing and cause early spike frequency adaptation in rat CA1 hippocampal pyramidal cells

    PubMed Central

    Gu, Ning; Vervaeke, Koen; Storm, Johan F

    2007-01-01

    Neuronal potassium (K+) channels are usually regarded as largely inhibitory, i.e. reducing excitability. Here we show that BK-type calcium-activated K+ channels enhance high-frequency firing and cause early spike frequency adaptation in neurons. By combining slice electrophysiology and computational modelling, we investigated functions of BK channels in regulation of high-frequency firing in rat CA1 pyramidal cells. Blockade of BK channels by iberiotoxin (IbTX) selectively reduced the initial discharge frequency in response to strong depolarizing current injections, thus reducing the early spike frequency adaptation. IbTX also blocked the fast afterhyperpolarization (fAHP), slowed spike rise and decay, and elevated the spike threshold. Simulations with a computational model of a CA1 pyramidal cell confirmed that the BK channel-mediated rapid spike repolarization and fAHP limits activation of slower K+ channels (in particular the delayed rectifier potassium current (IDR)) and Na+ channel inactivation, whereas M-, sAHP- or SK-channels seem not to be important for the early facilitating effect. Since the BK current rapidly inactivates, its facilitating effect diminishes during the initial discharge, thus producing early spike frequency adaptation by an unconventional mechanism. This mechanism is highly frequency dependent. Thus, IbTX had virtually no effect at spike frequencies < 40 Hz. Furthermore, extracellular field recordings demonstrated (and model simulations supported) that BK channels contribute importantly to high-frequency burst firing in response to excitatory synaptic input to distal dendrites. These results strongly support the idea that BK channels play an important role for early high-frequency, rapidly adapting firing in hippocampal pyramidal neurons, thus promoting the type of bursting that is characteristic of these cells in vivo, during behaviour. PMID:17303637

  10. B_K in unquenched QCD using improved staggered fermions

    NASA Astrophysics Data System (ADS)

    Kim, Jongjeong

    2006-12-01

    We present preliminary results for BK calculated using improved staggered fermions with a mixed action (HYP-smeared staggered valence quarks and AsqTad staggered sea quarks). We investigate £¡ ¢ a2¤ effect due to non- the effect of non-degenerate quarks on BK and attempt to estimate the Goldstone pions in loops. We fit the data to continuum partially quenched chiral perturbation theory. We find that the quality of fit for BK improves if we include non-degenerate quark mass combinations. We also observe, however, that the fitting curve deviates from the data points in the light quark mass region. This may indicate the need to include taste-breaking in pion loops.

  11. Genome Sequences of Polyomaviruses from the Wild-Living Red Colobus (Piliocolobus badius) and Western Chimpanzee (Pan troglodytes verus).

    PubMed

    Ben Salem, Nicole; Leendertz, Fabian H; Ehlers, Bernhard

    2016-10-13

    We identified with PCR and sequencing the full genomes of the recently discovered Pan troglodytes verus polyomavirus 8 and Piliocolobus badius polyomavirus 2 in a western chimpanzee and a western red colobus free-ranging in Taï National Park of Côte d'Ivoire. Copyright © 2016 Ben Salem et al.

  12. Relation between BK-α/β4-mediated potassium secretion and ENaC-mediated sodium reabsorption.

    PubMed

    Wen, Donghai; Cornelius, Ryan J; Rivero-Hernandez, Dianelys; Yuan, Yang; Li, Huaqing; Weinstein, Alan M; Sansom, Steven C

    2014-07-01

    The large-conductance, calcium-activated BK-α/β4 potassium channel, localized to the intercalated cells of the distal nephron, mediates potassium secretion during high-potassium, alkaline diets. Here we determine whether BK-α/β4-mediated potassium transport is dependent on epithelial sodium channel (ENaC)-mediated sodium reabsorption. We maximized sodium-potassium exchange in the distal nephron by feeding mice a low-sodium, high-potassium diet. Wild-type and BK-β4 knockout mice were maintained on a low-sodium, high-potassium, alkaline diet or a low-sodium, high-potassium, acidic diet for 7-10 days. Wild-type mice maintained potassium homeostasis on the alkaline, but not acid, diet. BK-β4 knockout mice could not maintain potassium homeostasis on either diet. During the last 12 h of diet, wild-type mice on either a regular, alkaline, or an acid diet, or knockout mice on an alkaline diet, were administered amiloride (an ENaC inhibitor). Amiloride enhanced sodium excretion in all wild-type and knockout groups to similar values; however, amiloride diminished potassium excretion by 59% in wild-type but only by 33% in knockout mice on an alkaline diet. Similarly, amiloride decreased the trans-tubular potassium gradient by 68% in wild-type but only by 42% in knockout mice on an alkaline diet. Amiloride treatment equally enhanced sodium excretion and diminished potassium secretion in knockout mice on an alkaline diet and wild-type mice on an acid diet. Thus, the enhanced effect of amiloride on potassium secretion in wild-type compared to knockout mice on the alkaline diet clarify a BK- α/β4-mediated potassium secretory pathway in intercalated cells driven by ENaC-mediated sodium reabsorption linked to bicarbonate secretion.

  13. n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses.

    PubMed Central

    Shadan, F F; Cowsert, L M; Villarreal, L P

    1994-01-01

    Small DNA viruses are dependent on the interaction of early proteins (such as large T antigen) with host p53 and Rb to bring about the G1-to-S cell cycle transition. The large DNA viruses are less dependent on host regulatory genes since additional early viral proteins (such as viral DNA polymerase, DNA metabolic enzymes, and other replication proteins) are involved in DNA synthesis. A highly conserved domain of large T antigen (similar to the p53-binding region) exclusively identifies papovavirus, parvovirus, and papillomaviruses from all other larger DNA viruses and implies a conserved interaction with host regulatory genes. In this report, we show that 3 to 6 mM butyrate, a general cell cycle blocker implicated in inhibition of the G1-to-S transition, inhibits DNA replication of polyomavirus and human papillomavirus type 11 but not the replication of larger DNA viruses such as adenovirus types 2 and 5, herpes simplex virus type 1, Epstein-Barr virus, and cytomegalovirus, which all bypass the butyrate-mediated cell cycle block. This butyrate effect on polyomavirus replication is not cell type specific, nor does it depend on the p53 or Rb gene, as inhibition was seen in fibroblasts with intact or homozygous deleted p53 or Rb, 3T6 cells, keratinocytes, C2C12 myoblasts, and 3T3-L1 adipocytes. In addition, butyrate did not inhibit expression of polyomavirus T antigen. The antiviral effect of butyrate involves a form of imprinted state, since pretreatment of cells with 3 mM butyrate inhibits human papillomavirus type 11 DNA replication for at least 96 h after its removal. Butyrate, therefore, serves as a molecular tool in dissecting the life cycle of smaller DNA viruses from that of the larger DNA viruses in relation to the cell cycle. Images PMID:8035479

  14. Does polyomavirus infection interfere with bladder cancer fluorescence in situ hybridization?

    PubMed

    Hossain, Deloar; Hull, David; Kalantarpour, Fatemeh; Maitlen, Rebecca; Qian, Junqi; Bostwick, David G

    2014-03-01

    Urine cytology is a proven and widely used screening tool for the detection of urothelial carcinoma. However, morphologic features of polyomavirus infected cells, characterized by nuclear inclusions (decoy cells) are a known source of diagnostic confusion with malignancy. Fluorescence in situ hybridization (FISH) is now routinely used to support the cytological diagnosis of urothelial carcinoma and monitor for recurrence. We sought to determine whether polyomavirus infection could result in positive FISH results (aneuploidy). This study deals with retrospective study of 100 polyomavirus-infected urine samples from patients with no history of urothelial carcinoma or organ transplantation. All cases were stained with Papanicolaou and acid hematoxylin stain. One slide from each sample was de-stained and FISH was performed using chromosome enumeration probes 3, 7, 17, and locus-specific probe 9p21. Adequate cells for FISH analysis (25 cells) were present in 81 cases; 19 cases were insufficient due to loss of cells during de-staining and FISH preparation process. All polyomavirus-infected cells (decoy cells) exhibited a normal chromosome pattern. Four cases were FISH positive, but there were no positive decoy cells. Decoy cells did not exhibit aneuploidy by FISH. The presence of decoy cells does not exclude the possibility of concurrent urothelial carcinoma. Acid hematoxylin stain appeared to supplement the Papanicolou stain in identifying and confirming the presence of polyomavirus infection.

  15. Tumorigenic activity of Merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice

    PubMed Central

    Spurgeon, Megan E.; Cheng, Jingwei; Bronson, Roderick T.; Lambert, Paul F.; DeCaprio, James A.

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contain wild type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiological role in human cancer. PMID:25596282

  16. Emerging From the Unknown: Structural and Functional Features of Agnoprotein of Polyomaviruses

    PubMed Central

    Saribas, A. Sami; Coric, Pascale; Hamazaspyan, Anahit; Davis, William; Axman, Rachel; White, Martyn K.; Abou-Gharbia, Magid; Childers, Wayne; Condra, Jon H.; Bouaziz, Serge; Safak, Mahmut

    2016-01-01

    Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe-rich domain. Structural NMR studies revealed that this domain adopts an alpha-helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB-1, p53, Ku70, FEZ1, HP1α, PP2A, AP-3, PCNA, and α-SNAP; and viral proteins, including small t antigen, large T antigen, HIV-1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein. PMID:26831433

  17. Absence of Merkel cell polyomavirus in primary parotid high-grade neuroendocrine carcinomas regardless of cytokeratin 20 immunophenotype.

    PubMed

    Chernock, Rebecca D; Duncavage, Eric J; Gnepp, Douglas R; El-Mofty, Samir K; Lewis, James S

    2011-12-01

    High-grade neuroendocrine carcinoma of the salivary glands is a rare malignancy that can be difficult to distinguish from metastatic neuroendocrine (Merkel cell) carcinoma of the skin, which often occurs on the head and neck and may metastasize to lymph nodes in or adjacent to salivary glands, particularly the parotid gland. As the 2 tumors have morphologic and immunophenotypic overlap, additional diagnostic tools may be clinically useful. Merkel cell carcinoma is known to harbor Merkel cell polyomavirus in up to 80% of cases. However, the presence or absence of this virus in salivary gland neuroendocrine carcinomas has not been investigated. We evaluated 7 primary salivary gland high-grade neuroendocrine carcinomas (all from the parotid) for the virus by both immunohistochemistry (CM2B4 clone) and real-time polymerase chain reaction directed against the conserved small T antigen. Five of the tumors had small cell morphology, and 2 had large cell morphology. All were either chromogranin and/or synaptophysin positive. Four of the 5 small cell (80%) and 1 of the 2 large cell (50%) carcinomas were cytokeratin 20 positive. All but 1 case had cervical lymph node metastases at presentation. Merkel cell polyomavirus T antigen was not detected in any of the 7 tumors, either by immunohistochemistry or by polymerase chain reaction with adequate controls. These observations suggest that primary parotid high-grade neuroendocrine carcinoma arises from a biological pathway that is different from that of cutaneous Merkel cell carcinomas. Furthermore, viral testing may aid in distinguishing the 2 tumor types, as a positive result would favor a metastasis.

  18. Clinical and pathological features of kidney transplant patients with concurrent polyomavirus nephropathy and rejection-associated endarteritis.

    PubMed

    McGregor, Stephanie M; Chon, W James; Kim, Lisa; Chang, Anthony; Meehan, Shane M

    2015-12-24

    To describe the clinicopathologic features of concurrent polyomavirus nephropathy (PVN) and endarteritis due to rejection in renal allografts. We searched our electronic records database for cases with transplant kidney biopsies demonstrating features of both PVN and acute rejection (AR). PVN was defined by the presence of typical viral cytopathic effect on routine sections and positive polyomavirus SV40 large-T antigen immunohistochemistry. AR was identified by endarteritis (v1 by Banff criteria). All cases were subjected to chart review in order to determine clinical presentation, treatment course and outcomes. Outcomes were recorded with a length of follow-up of at least one year or time to nephrectomy. Of 94 renal allograft recipients who developed PVN over an 11-year period at our institution, we identified 7 (7.4%) with viral cytopathic changes, SV40 large T antigen staining, and endarteritis in the same biopsy specimen, indicative of concurrent PVN and AR. Four arose after reduction of immunosuppression (IS) (for treatment of PVN in 3 and tuberculosis in 1), and 3 patients had no decrease of IS before developing simultaneous concurrent disease. Treatment consisted of reduced oral IS and leflunomide for PVN, and anti-rejection therapy. Three of 4 patients who developed endarteritis in the setting of reduced IS lost their grafts to rejection. All 3 patients with simultaneous PVN and endarteritis cleared viremia and were stable at 1 year of follow up. Patients with endarteritis and PVN arising in a background of reduced IS had more severe rejection and poorer outcome. Concurrent PVN and endarteritis may be more frequent than is currently appreciated and may occur with or without prior reduction of IS.

  19. Clinical and pathological features of kidney transplant patients with concurrent polyomavirus nephropathy and rejection-associated endarteritis

    PubMed Central

    McGregor, Stephanie M; Chon, W James; Kim, Lisa; Chang, Anthony; Meehan, Shane M

    2015-01-01

    AIM: To describe the clinicopathologic features of concurrent polyomavirus nephropathy (PVN) and endarteritis due to rejection in renal allografts. METHODS: We searched our electronic records database for cases with transplant kidney biopsies demonstrating features of both PVN and acute rejection (AR). PVN was defined by the presence of typical viral cytopathic effect on routine sections and positive polyomavirus SV40 large-T antigen immunohistochemistry. AR was identified by endarteritis (v1 by Banff criteria). All cases were subjected to chart review in order to determine clinical presentation, treatment course and outcomes. Outcomes were recorded with a length of follow-up of at least one year or time to nephrectomy. RESULTS: Of 94 renal allograft recipients who developed PVN over an 11-year period at our institution, we identified 7 (7.4%) with viral cytopathic changes, SV40 large T antigen staining, and endarteritis in the same biopsy specimen, indicative of concurrent PVN and AR. Four arose after reduction of immunosuppression (IS) (for treatment of PVN in 3 and tuberculosis in 1), and 3 patients had no decrease of IS before developing simultaneous concurrent disease. Treatment consisted of reduced oral IS and leflunomide for PVN, and anti-rejection therapy. Three of 4 patients who developed endarteritis in the setting of reduced IS lost their grafts to rejection. All 3 patients with simultaneous PVN and endarteritis cleared viremia and were stable at 1 year of follow up. Patients with endarteritis and PVN arising in a background of reduced IS had more severe rejection and poorer outcome. CONCLUSION: Concurrent PVN and endarteritis may be more frequent than is currently appreciated and may occur with or without prior reduction of IS. PMID:26722657

  20. Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism.

    PubMed

    Zhou, Yu; Lingle, Christopher J

    2014-11-01

    Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca(2+)- and voltage-activated K(+) (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed-open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC50 for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10(6) M(-1)s(-1). Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller

  1. Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism

    PubMed Central

    Zhou, Yu

    2014-01-01

    Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca2+- and voltage-activated K+ (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed–open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC50 for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 106 M−1s−1. Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller

  2. Identification of a Novel Cetacean Polyomavirus from a Common Dolphin (Delphinus delphis) with Tracheobronchitis

    PubMed Central

    Anthony, Simon J.; St. Leger, Judy A.; Navarrete-Macias, Isamara; Nilson, Erica; Sanchez-Leon, Maria; Liang, Eliza; Seimon, Tracie; Jain, Komal; Karesh, William; Daszak, Peter; Briese, Thomas; Lipkin, W. Ian

    2013-01-01

    A female short-beaked common dolphin calf was found stranded in San Diego, California in October 2010, presenting with multifocal ulcerative lesions in the trachea and bronchi. Viral particles suggestive of polyomavirus were detected by EM, and subsequently confirmed by PCR and sequencing. Full genome sequencing (Ion Torrent) revealed a circular dsDNA genome of 5,159 bp that was shown to form a distinct lineage within the genus Polyomavirus based on phylogenetic analysis of the early and late transcriptomes. Viral infection and distribution in laryngeal mucosa was characterised using in-situ hybridisation, and apoptosis observed in the virus-infected region. These results demonstrate that polyomaviruses can be associated with respiratory disease in cetaceans, and expand our knowledge of their diversity and clinical significance in marine mammals. PMID:23874559

  3. Identification of a novel cetacean polyomavirus from a common dolphin (Delphinus delphis) with Tracheobronchitis.

    PubMed

    Anthony, Simon J; St Leger, Judy A; Navarrete-Macias, Isamara; Nilson, Erica; Sanchez-Leon, Maria; Liang, Eliza; Seimon, Tracie; Jain, Komal; Karesh, William; Daszak, Peter; Briese, Thomas; Lipkin, W Ian

    2013-01-01

    A female short-beaked common dolphin calf was found stranded in San Diego, California in October 2010, presenting with multifocal ulcerative lesions in the trachea and bronchi. Viral particles suggestive of polyomavirus were detected by EM, and subsequently confirmed by PCR and sequencing. Full genome sequencing (Ion Torrent) revealed a circular dsDNA genome of 5,159 bp that was shown to form a distinct lineage within the genus Polyomavirus based on phylogenetic analysis of the early and late transcriptomes. Viral infection and distribution in laryngeal mucosa was characterised using in-situ hybridisation, and apoptosis observed in the virus-infected region. These results demonstrate that polyomaviruses can be associated with respiratory disease in cetaceans, and expand our knowledge of their diversity and clinical significance in marine mammals.

  4. α decay of 97249Bk and levels in 95245Am

    NASA Astrophysics Data System (ADS)

    Ahmad, I.; Greene, J. P.; Kondev, F. G.; Zhu, S.; Carpenter, M. P.; Janssens, R. V. F.; Boll, R. A.; Ezold, J. G.; Van Cleve, S. M.; Browne, E.

    2013-05-01

    α decay of 249Bk has been investigated by measuring its α and γ-ray spectra, both in singles and in coincidence modes. The α spectrum of a freshly purified 249Bk sample was measured with a high-resolution, double-focusing magnetic spectrometer. γ singles, γ-γ coincidence, and γ-α coincidence spectra were also recorded. The absolute intensity of the 327.45-keV γ ray has been determined to be (1.44±0.08)×10-5% per 249Bk decay. Assignments of previously known single-particle states were confirmed. A new rotational band was identified in the α singles spectrum and Am K x rays have been observed in its decay. This single-particle state, with an energy of 154 keV, has been assigned to the 3/2-[521] Nilsson state. This is the lowest excitation energy for this orbital in any Am nucleus. More precise energies and intensities of the 249Bk α groups and γ-ray transitions are provided.

  5. Hydroxyproline in the major capsid protein VP1 of polyomavirus

    SciTech Connect

    Ludlow, J.W.; Consigli, R.A.

    1989-06-01

    Amino acid analysis of (/sup 3/H)proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of /sup 3/H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from (/sup 35/S)methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.

  6. Diagnostic assays for polyomavirus JC and progressive multifocal leukoencephalopathy.

    PubMed

    White, Martyn K; Sariyer, Ilker K; Gordon, Jennifer; Delbue, Serena; Pietropaolo, Valeria; Berger, Joseph R; Khalili, Kamel

    2016-03-01

    Progressive multifocal leukoencephalopathy (PML) is a devastating and often fatal demyelinating disease of the central nervous system for which effective therapies are lacking. It is caused by the replication of polyomavirus JC (JCV) in the oligodendrocytes and astrocytes leading to their cytolytic death and loss of myelin from the subcortical white matter. While the virus is very common in human populations worldwide, the incidence of the disease is very low and confined almost exclusively to individuals with some form of immunological dysfunction. However, the number of people who constitute the at-risk population is growing larger and includes individuals with HIV-1/AIDS and patients receiving immunomodulatory therapies such as multiple sclerosis patients treated with natalizumab. Further adding to the public health significance of this disease are the difficulties encountered in the diagnosis of PML and the lack of useful biomarkers for PML progression. In this review, we examine the diagnostic assays that are available for different aspects of the JCV life cycle, their usefulness and drawbacks, and the prospects for improvements. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Diagnostic Assays for Polyomavirus JC and Progressive Multifocal Leukoencephalopathy

    PubMed Central

    White, Martyn K.; Sariyer, Ilker K.; Gordon, Jennifer; Delbue, Serena; Pietropaolo, Valeria; Berger, Joseph R.; Khalili, Kamel

    2016-01-01

    SUMMARY Progressive multifocal leukoencephalopathy (PML) is a devastating and often fatal demyelinating disease of the central nervous system (CNS) for which effective therapies are lacking. It is caused by the replication of polyomavirus JC (JCV) in the oligodendrocytes and astrocytes leading to their cytolytic death and loss of myelin from the subcortical white matter. While the virus is very common in human populations worldwide, the incidence of the disease is very low and confined almost exclusively to individuals with some form of immunological dysfunction. However, the number of people who constitute the at-risk population is growing larger and includes individuals with HIV-1/AIDS and patients receiving immunomodulatory therapies such as multiple sclerosis patients treated with natalizumab. Further adding to the public health significance of this disease are the difficulties encountered in the diagnosis of PML and the lack of useful biomarkers for PML progression. In this review, we examine the diagnostic assays that are available for different aspects of the JCV life cycle, their usefulness and drawbacks, and the prospects for improvements. PMID:26663440

  8. Immunohistochemical detection of KI polyomavirus in lung and spleen.

    PubMed

    Siebrasse, Erica A; Nguyen, Nang L; Smith, Colin; Simmonds, Peter; Wang, David

    2014-11-01

    Little is known about the tissue tropism of KI polyomavirus (KIPyV), and there are no studies to date describing any specific cell types it infects. The limited knowledge of KIPyV tropism has hindered study of this virus and understanding of its potential pathogenesis in humans. We describe tissues from two immunocompromised patients that stained positive for KIPyV antigen using a newly developed immunohistochemical assay targeting the KIPyV VP1 (KVP1) capsid protein. In the first patient, a pediatric bone marrow transplant recipient, KVP1 was detected in lung tissue. Double immunohistochemical staining demonstrated that approximately 50% of the KVP1-positive cells were CD68-positive cells of the macrophage/monocyte lineage. In the second case, an HIV-positive patient, KVP1 was detected in spleen and lung tissues. These results provide the first identification of a specific cell type in which KVP1 can be detected and expand our understanding of basic properties and in vivo tropism of KIPyV.

  9. JC polyomavirus mutants escape antibody-mediated neutralization.

    PubMed

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  10. Small-molecule inhibitors of JC polyomavirus infection

    PubMed Central

    Yatawara, Achani; Gaidos, Gabriel; Rupasinghe, Chamila N.; O’Hara, Bethany A.; Pellegrini, Maria; Atwood, Walter J.; Mierke, Dale F.

    2015-01-01

    The JC polyomavirus (JCPyV) infects approximately 50% of the human population. In healthy individuals the infection remains dormant and asymptomatic, but in immuno-suppressed patients it can cause progressive multifocal leukoencephalopathy (PML), a potentially fatal demyelinating disease. Currently, there are no drugs against JCPyV infection, nor for the treatment of PML. Here, we report the development of small molecule inhibitors of JCPyV that target the initial interaction between the virus and host cell and thereby block viral entry. Utilizing a combination of computational and NMR-based screening techniques, we target the LSTc tetrasaccharide binding site within the VP1 pentameric coat protein of JCPyV. Four of the compounds from the screen effectively block viral infection in our in vitro assays using SVG-A cells. For the most potent compound, we used saturation transfer difference NMR to determine the mode of binding to purified pentamers of JCPyV VP1. Collectively these results demonstrate the viability of this class of compounds for eventual development of JCPyV-antiviral therapeutics. PMID:25522925

  11. The Raccoon Polyomavirus Genome and Tumor Antigen Transcription Are Stable and Abundant in Neuroglial Tumors

    PubMed Central

    Brostoff, Terza; Dela Cruz, Florante N.; Church, Molly E.; Woolard, Kevin D.

    2014-01-01

    ABSTRACT Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. IMPORTANCE The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor

  12. The raccoon polyomavirus genome and tumor antigen transcription are stable and abundant in neuroglial tumors.

    PubMed

    Brostoff, Terza; Dela Cruz, Florante N; Church, Molly E; Woolard, Kevin D; Pesavento, Patricia A

    2014-11-01

    Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor tissue or in

  13. Polyomavirus Infection in Gouldian Finches (Erythrura gouldiae) and Other Pet Birds of the Family Estrildidae.

    PubMed

    Circella, E; Caroli, A; Marino, M; Legretto, M; Pugliese, N; Bozzo, G; Cocciolo, G; Dibari, D; Camarda, A

    2017-02-18

    A syndrome characterized by apathy, diarrhoea and high mortality of nestlings was observed in a flock of pet birds of the family Estrildidae. Enlargement of the liver, pulmonary congestion and urate accretions in the kidney were observed. Microscopically, there was glomerular atrophy, oedema and congestion of the lungs and necrosis and fibrosis of the liver. Cowdry type B intranuclear inclusion bodies were detected in the tissues. Polyomavirus was detected by polymerase chain reaction. The entire genome of the virus was amplified and sequenced, revealing 99 % identity to the sequence of finch polyomavirus isolated from the Eurasian bullfinch (family Fringillidae).

  14. Occurrence, genotypic characterization, and patterns of shedding of human polyomavirus JCPyV and BKPyV in urine samples of healthy individuals in São Paulo, Brazil.

    PubMed

    Urbano, Paulo Roberto Palma; Oliveira, Renato Reis; Romano, Camila Malta; Pannuti, Claudio Sergio; Fink, Maria Cristina Domingues da Silva

    2016-01-01

    The objective of this study was to evaluate the prevalence, genotypic characterization, and determination of the patterns of shedding of human polyomavirus JC (JCPyV) and BK (BKPyV) in consecutive urine samples collected from healthy adults. Urine samples collected monthly over a 6 month period were screened by polymerase chain reaction (PCR) with two sets of primers complementary to the VP1 protein region specific for the JCPyV or BKPyV genome. The viral load of JCPyV and BKPyV in positive samples was determined by quantitative real time PCR. Seventy-one healthy individuals (ages between 18 and 65) were included in the study. Polyomavirus DNA urinary shedding was identified in 44 (62%) of the 71 individuals evaluated: BKPyV only in 16 (22.5%); JCPyV only in 19 (26.7%); and both in 9 (12.7%). Among the 28 individuals shedding JCPyV, the shedding was nearly continuous in 13 (46.4%) and sporadic in 15 (53.6%), whereas all BKPyV shedding was sporadic. A total of 45 (19 BKPyV and 26 JCPyV) strains were identified. Of the BKPyV strains, individuals were observed that excreted all genotypes except genotype 3 and the JCPyV strains, excretion of 5 different genotypes. Evaluating the age of individuals who excrete JCPyV and BKPyV, mostly are young adults, with a slight increase with increasing age and observing the viral load can not draw any parallel between the increase or decrease of age or excreted genotype as there was a wide variation both in the excretion of BKPyV and JCPyV. The high occurrence of isolated or simultaneous urinary shedding of JCPyV and BKPyV in healthy individuals merits further study.

  15. A study of the low-luminosity Type II-Plateau supernova 2008bk

    NASA Astrophysics Data System (ADS)

    Lisakov, S. M.; Dessart, Luc; Hillier, D. John; Waldman, Roni; Livne, Eli

    2017-04-01

    Supernova (SN) 2008bk is a well-observed low-luminosity Type II event visually associated with a low-mass red-supergiant progenitor. To model SN 2008bk, we evolve a 12 M⊙ star from the main sequence until core collapse, when it has a total mass of 9.88 M⊙, a He-core mass of 3.22 M⊙ and a radius of 502 R⊙. We then artificially trigger an explosion that produces 8.29 M⊙ of ejecta with a total energy of 2.5 × 1050 erg and ∼0.009 M⊙ of 56Ni. We model the subsequent evolution of the ejecta with non-local thermodynamic equilibrium time-dependent radiative transfer. Although somewhat too luminous and energetic, this model reproduces satisfactorily the multiband light curves and multi-epoch spectra of SN 2008bk, confirming the suitability of a low-mass massive star progenitor. As in other low-luminosity SNe II, the structured Hα profile at the end of the plateau phase is probably caused by Ba II 6496.9 Å rather than asphericity. We discuss the sensitivity of our results to changes in progenitor radius and mass, as well as chemical mixing. A 15 per cent increase in progenitor radius causes a 15 per cent increase in luminosity and a 0.2 mag V-band brightening of the plateau but leaves its length unaffected. An increase in ejecta mass by 10 per cent lengthens the plateau by ∼10 d. Chemical mixing introduces slight changes to the bolometric light curve, limited to the end of the plateau, but has a large impact on colours and spectra at nebular times.

  16. Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring

    PubMed Central

    Miranda, Pablo; Giraldez, Teresa; Holmgren, Miguel

    2016-01-01

    Large-conductance voltage- and calcium-activated K+ (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the “gating ring,” which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. In contrast to Ca2+ that binds to both RCK domains, Mg2+, Cd2+, or Ba2+ interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg2+ and Ba2+ selectively induce structural movement of the RCK2 domain, whereas Cd2+ causes motions of RCK1, in all cases substantially smaller than those elicited by Ca2+. By combining divalent species interacting with unique sites, we demonstrate that RCK1 and RCK2 domains move independently when their specific binding sites are occupied. Moreover, binding of chemically distinct cations to both RCK domains is additive, emulating the effect of fully occupied Ca2+ binding sites. PMID:27872281

  17. Fatal avian polyomavirus infection during quarantine in adult wild-caught red-faced lovebirds (Agapornis pullaria).

    PubMed

    Enders, F; Gravendyck, M; Gerlach, H; Kaleta, E F

    1997-01-01

    An outbreak of avian polyomavirus infection is reported in a group of six wild-caught red-faced lovebirds (Agapornis pullaria), all of which died during quarantine. The birds had not shown any previous symptoms. Histologic examination of the lungs, kidneys, livers, and spleens revealed the presence of basophilic intranuclear inclusions. Avian polyomavirus was isolated from the liver and the spleen. Neutralizing antibodies were detected in the sera from other lovebirds (Agapornis personata, Agapornis taranta) that had contact with the A. pullaria. A serologic comparison showed a close relationship with budgerigar polyomavirus.

  18. Cysteine residue 911 in C-terminal tail of human BK(Ca)α channel subunit is crucial for its activation by carbon monoxide.

    PubMed

    Telezhkin, Vsevolod; Brazier, Stephen P; Mears, Ruth; Müller, Carsten T; Riccardi, Daniela; Kemp, Paul J

    2011-06-01

    The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

  19. Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO*

    PubMed Central

    Yi, Li; Morgan, Jeffrey T.; Ragsdale, Stephen W.

    2010-01-01

    Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca2+ and voltage-activated K+ channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca2+-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (Kd = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (Kd = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions. PMID:20427280

  20. Genetic diversity in twenty variants of the avian polyomavirus.

    PubMed

    Phalen, D N; Wilson, V G; Gaskin, J M; Derr, J N; Graham, D L

    1999-01-01

    To determine if different pathotypes of the avian polyomavirus (APV) exist and to compare the genomes of APVs originating from different geographic areas, dates, and species of birds, the partial sequences of 18 APVs were determined. New viral sequences were compared with three published APV sequences. Two of the new viruses had identical sequences. Forty point mutations were found at 31 loci. A 27-bp deletion was found in the VP2 and VP3 open reading frames of one virus. A duplication of the putative origin of replication and adjacent enhancer region was previously reported in one APV. Smaller duplications involving the origin in one APV and a second enhancer region in another were discovered. All duplications were in tissue culture-adapted viruses, suggesting they occurred during the isolation process. Excluding duplications and the deletion, maximum variation between viruses was small (11 bp). A maximum parsimony tree was constructed that contained three major branches. The three earliest isolates were on separate branches. The European viruses were confined to branch I, but APVs from the United States were on all three branches. Lovebird, budgerigar, and macaw APVs were also on each of the three branches, suggesting that species-specific pathotypes have not developed. Most nonsynonymous mutations occurred in a small portion of the VP2 and VP3 open reading frames, demonstrating a selection for these mutations. That a glycine at VP2 221 will inhibit virus replication in chicken embryo fibroblasts (CEFs) has been previously reported. In contrast, six of seven of the new APVs isolated in CEFs had a glycine at VP2 221.

  1. 5-HT2 receptors facilitate JC polyomavirus entry.

    PubMed

    Assetta, Benedetta; Maginnis, Melissa S; Gracia Ahufinger, Irene; Haley, Sheila A; Gee, Gretchen V; Nelson, Christian D S; O'Hara, Bethany A; Allen Ramdial, Stacy-ann A; Atwood, Walter J

    2013-12-01

    The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT2 receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT2 receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT2 receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.

  2. Interspecific adaptation by binary choice at de novo polyomavirus T antigen site through accelerated codon-constrained Val-Ala toggling within an intrinsically disordered region.

    PubMed

    Lauber, Chris; Kazem, Siamaque; Kravchenko, Alexander A; Feltkamp, Mariet C W; Gorbalenya, Alexander E

    2015-05-26

    It is common knowledge that conserved residues evolve slowly. We challenge generality of this central tenet of molecular biology by describing the fast evolution of a conserved nucleotide position that is located in the overlap of two open reading frames (ORFs) of polyomaviruses. The de novo ORF is expressed through either the ALTO protein or the Middle T antigen (MT/ALTO), while the ancestral ORF encodes the N-terminal domain of helicase-containing Large T (LT) antigen. In the latter domain the conserved Cys codon of the LXCXE pRB-binding motif constrains codon evolution in the overlapping MT/ALTO ORF to a binary choice between Val and Ala codons, termed here as codon-constrained Val-Ala (COCO-VA) toggling. We found the rate of COCO-VA toggling to approach the speciation rate and to be significantly accelerated compared to the baseline rate of chance substitution in a large monophyletic lineage including all viruses encoding MT/ALTO and three others. Importantly, the COCO-VA site is located in a short linear motif (SLiM) of an intrinsically disordered region, a typical characteristic of adaptive responders. These findings provide evidence that the COCO-VA toggling is under positive selection in many polyomaviruses, implying its critical role in interspecific adaptation, which is unprecedented for conserved residues.

  3. WU Polyomavirus in Respiratory Epithelial Cells from Lung Transplant Patient with Job Syndrome

    PubMed Central

    Siebrasse, Erica A.; Pastrana, Diana V.; Nguyen, Nang L.; Wang, Annie; Roth, Mark J.; Holland, Steven M.; Freeman, Alexandra F.; McDyer, John; Buck, Christopher B.

    2015-01-01

    We detected WU polyomavirus (WUPyV) in a bronchoalveolar lavage sample from lungs transplanted into a recipient with Job syndrome by using immunoassays specific for the WUPyV viral protein 1. Co-staining for an epithelial cell marker identified most WUPyV viral protein 1–positive cells as respiratory epithelial cells. PMID:25531075

  4. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication.

    PubMed Central

    Tang, W J; Berger, S L; Triezenberg, S J; Folk, W R

    1987-01-01

    The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 E1A enhancer core AGGAAGTGACT; acquisition of an A----G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with immunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein. Images PMID:3037332

  5. Avian Polyomavirus Genome Sequences Recovered from Parrots in Captive Breeding Facilities in Poland

    PubMed Central

    Dayaram, Anisha; Piasecki, Tomasz; Chrząstek, Klaudia; White, Robyn; Julian, Laurel; van Bysterveldt, Katherine

    2015-01-01

    Eight genomes of avian polyomaviruses (APVs) were recovered and sequenced from deceased Psittacula eupatria, Psittacula krameri, and Melopsittacus undulatus from various breeding facilities in Poland. Of these APV-positive samples, six had previously tested positive for beak and feather disease virus (BFDV) and/or parrot hepatitis B virus (PHBV). PMID:26404592

  6. Trichodysplasia spinulosa: a benign adnexal proliferation with follicular differentiation associated with polyomavirus.

    PubMed

    Lee, Yvonne Y; Tucker, Simon C; Prow, Natalie A; Setoh, Yin Xiang; Banney, Leith A

    2014-05-01

    Trichodysplasia spinulosa is a rare polyomavirus-associated cutaneous eruption occurring in the setting of immunosuppression. Clinically it is characterised by multiple centrofacial folliculocentric papules with spinous protuberances. The histopathology is distinct and treatment with antiviral agents appears to be the most effective.

  7. The Cre/loxP recombination system for production of infectious mouse polyomavirus.

    PubMed

    Hron, Tomáš; Spanielová, Hana; Suchanová, Jiřina; Forstová, Jitka

    2013-09-01

    Murine polyomavirus mutants are frequently produced for experimental as well as therapy purposes. Commonly used methods for preparation of mutant viral genomes from recombinant vectors are laborious and give variable yields and quality. We describe an efficient and reproducible Cre/loxP-mediated recombination system that generates polyomavirus genomes from recombinant plasmid in vivo. We designed and constructed two variants of recombinant vectors containing the wild-type polyomavirus genome flanked by loxP homologous sites. The loxP sites were introduced either into the intronic region of early genes or between the two poly(A) signal sites of convergent transcriptional units. After cotransfection of the recombinant plasmids with the Cre-expressing vector into mouse 3T6 cells, we obtained infectious virus from the genome variant containing loxP site in the intronic region, but we failed to isolate any infectious virus from the viral genome containing loxP site between poly(A) signals. We show that the Cre/loxP-based method of polyomavirus production is simple, expedient, and reproducible and works with satisfactory efficiency.

  8. Rapid Detection of Trichodysplasia Spinulosa-Associated Polyomavirus in Skin Biopsy Specimen

    PubMed Central

    Urbano, Paulo Roberto P.; Pannuti, Cláudio Sérgio; Pierrotti, Ligia C.; David-Neto, Elias

    2014-01-01

    Trichodysplasia spinulosa-associated polyomavirus (TSV) is responsible for a rare skin cancer. Using metagenomic approaches, we determined the complete genome sequence of a TSV first detected in Brazil in spicules of an immunocompromised patient suspected to have trichodysplasia spinulosa. PMID:25059864

  9. Merkel Cell Polyomavirus DNA in Respiratory Specimens from Children and Adults

    PubMed Central

    Lambert, Stephen B.; Whiley, David M.; Nissen, Michael D.; Sloots, Theo P.

    2009-01-01

    Merkel cell polyomavirus (MCPyV) DNA was detected in 7 (1.3%) of 526 respiratory tract samples from patients in Australia with upper or lower respiratory tract symptoms. Partial T antigen and major capsid protein sequences of MCPyV identified in respiratory secretions showed high homology (99%–100%) to those found in Merkel cell carcinoma. PMID:19239774

  10. Human bocavirus and KI/WU polyomaviruses in pediatric intensive care patients.

    PubMed

    van de Pol, Alma C; Wolfs, Tom F W; Jansen, Nicolaas J G; Kimpen, Jan L L; van Loon, Anton M; Rossen, John W A

    2009-03-01

    We evaluated the prevalence of human bocavirus and KI and WU polyomaviruses in pediatric intensive care patients with and without lower respiratory tract infection (LRTI). The prevalence of these viruses was 5.1%, 0%, and 2.6%, respectively, in children with LRTI and 4.8%, 4.8%, and 2.4%, respectively, in those without LRTI.

  11. Prevalence and Pathogenicity of WU and KI Polyomaviruses in Children, the Netherlands

    PubMed Central

    Rossen, John W. A.; van Ewijk, Bart E.; Wilbrink, Berry; van Esch, Petra C.H.M.; Wolfs, Tom F.W.; van der Ent, Cornelis K.

    2008-01-01

    A longitudinal study in 2004 and 2005 detected polyomaviruses WU and KI in 44% and 17% of children with and without respiratory symptoms, respectively, in the Netherlands. In some children both viruses were detected for long periods. In several symptomatic children no other respiratory pathogen was detected. PMID:18976572

  12. Human Bocavirus and KI/WU Polyomaviruses in Pediatric Intensive Care Patients

    PubMed Central

    Wolfs, Tom F.W.; Jansen, Nicolaas J.G.; Kimpen, Jan L.L.; van Loon, Anton M.; Rossen, John W.A.

    2009-01-01

    We evaluated the prevalence of human bocavirus and KI and WU polyomaviruses in pediatric intensive care patients with and without lower respiratory tract infection (LRTI). The prevalence of these viruses was 5.1%, 0%, and 2.6%, respectively, in children with LRTI and 4.8%, 4.8%, and 2.4%, respectively, in those without LRTI. PMID:19239763

  13. Evaluation of Fluoroquinolones for the Prevention of BK Viremia after Renal Transplantation

    PubMed Central

    Waikar, Sushrut S.; Martin, Spencer; Roberts, Keri; Chen, Jie; Borgi, Lea; Sheashaa, Hussein; Dyer, Christine; Malek, Sayeed K.; Tullius, Stefan G.; Vadivel, Nidyanandh; Grafals, Monica; Abdi, Reza; Najafian, Nader; Milford, Edgar; Chandraker, Anil

    2010-01-01

    Background and objectives: Nearly 30% of renal transplant recipients develops BK viremia, a prerequisite for BK nephropathy. Case reports have evaluated treatment options for BK virus, but no controlled studies have assessed prophylactic therapies. Fluoroquinolone antibiotics were studied for prevention of BK viremia after renal transplantation. Design, setting, participants, & measurements: This retrospective analysis evaluated adult renal transplant recipients with at least one BK viral load (blood) between 90 and 400 days after transplantation. Six to 12 months of co-trimoxazole was used for Pneumocystis prophylaxis. In sulfa-allergic/-intolerant patients, 6 to 12 months of atovaquone with 1 month of a fluoroquinolone was used. Fluoroquinolones can inhibit BK DNA topoisomerase. The two groups studied were those that received 30 days of levofloxacin or ciprofloxacin after transplantation and those that did not. The primary endpoint was BK viremia rates at 1 year. Of note, of the 160 patients not receiving fluoroquinolone prophylaxis, 40 received a fluoroquinolone for treatment of a bacterial infection within 3 months after transplantation. Subgroup analysis evaluating these 40 patients against the 120 who had no exposure to fluoroquinolones was completed. Results: A 1-month fluoroquinolone course after transplantation was associated with significantly lower rates of BK viremia at 1 year compared with those with no fluoroquinolone. In the subgroup analysis, exposure to fluoroquinolone for treatment of bacterial infections within 3 months after transplantation was associated with significantly lower 1-year rates of BK viremia. Conclusions: This analysis demonstrates that fluoroquinolones are effective at preventing BK viremia after renal transplantation. PMID:20507960

  14. Biopsy-Proven BK Virus-Associated Nephropathy: Clinico-Pathologic Correlations.

    PubMed

    Jahdali, Sarah; Al Oudah, Noura; Alsaad, Khaled O; Kfoury, Hala; Qurashi, Salem; Al Sayyari, Abdulla

    2017-06-01

    Our objective was to study the clinico-pathologic correlations in BK virus nephropathy. We conducted a retrospective study of all patients with biopsy-proven polyoma (BK) virus infection. We compared their survival and renal outcomes versus BK virus-negative patients with biopsy-proven graft rejection. Histopathologic characterization by a blinded nephropathologist was performed. BK nephropathy was found in 10 patients biopsied for graft dysfunction. All virus-positive patients received antithymocyte globulin induction therapy compared with only 59.3% of the BK-negative group (P = .06). The percentage of patients in the BK-negative group who received acyclovir was significantly higher than that in the BK-positive group (P = .01). After a mean observation period of 6.8 ± 3.2 years, 70% of the BK group had functioning grafts compared with 68% in the BK-negative group (P = .9) with similar 3-year graft survival in the 2 groups (80% and 90%; P = .8). Within the BK group, graft survival was better in the older group (P = .005) and in those with deceased donor kidney grafts (P = .016). Patients in the BK-negative group were heavier (mean weight of 64.3 ± 12.1 vs 46.7 ± 20.6 kg; P = .003). None of the histopathologic features studied had any effect on renal prognosis. The risk factors for developing BK nephropathy were use of antithymocyte globulin, lower weight, and not using acyclovir as early prophylaxis. Within the BK nephropathy group, better graft survival was observed in deceased donor kidney recipients and in older patients. The viral load and polyoma virus nephropathy stage did not affect graft survival in this small sample study.

  15. SHAPING OF ACTION POTENTIALS BY TYPE I AND TYPE II BK CHANNELS

    PubMed Central

    Jaffe, David B.; Wang, Bin; Brenner, Robert

    2011-01-01

    The BK channel is a Ca2+ and voltage-gated conductance responsible for shaping action potential waveforms in many types of neurons. Type II BK channels are differentiated from type I channels by their pharmacology and slow gating kinetics. The β4 accessory subunit confers type II properties on BK α subunits. Empirically derived properties of BK channels, with and without the β4 accessory subunit, were obtained using a heterologous expression system under physiological ionic conditions. These data were then used to study how BK channels alone (type I) and with the accessory β4 subunit (type II) modulate action potential properties in biophysical neuron models. Overall, the models support the hypothesis that it is the slower kinetics provided by the β4 subunit that endows the BK channel with type II properties, which leads to broadening of action potentials and, secondarily, to greater recruitment of SK channels reducing neuronal excitability. Two regions of parameter space distinguished type II and type I effects; one where the range of BK-activating Ca2+ was high (>20 µM) and the other where BK-activating Ca2+ was low (~0.4–1.2 µM). The latter required an elevated BK channel density, possibly beyond a likely physiological range. BK-mediated sharpening of the spike waveform associated with the lack of the β4 subunit was sensitive to the properties of voltage-gated Ca2+ channels due to electrogenic effects on spike duration. We also found that depending on Ca2+ dynamics, type II BK channels may have the ability to contribute to the medium AHP, a property not generally ascribed to BK channels, influencing the frequency-current relationship. Finally, we show how the broadening of action potentials conferred by type II BK channels can also indirectly increase the recruitment of SK-type channels decreasing the excitability of the neuron. PMID:21723921

  16. BK channels modulate pre- and postsynaptic signaling at reciprocal synapses in retina.

    PubMed

    Grimes, William N; Li, Wei; Chávez, Andrés E; Diamond, Jeffrey S

    2009-05-01

    In the mammalian retina, A17 amacrine cells provide reciprocal inhibitory feedback to rod bipolar cells, thereby shaping the time course of visual signaling in vivo. Previous results have indicated that A17 feedback can be triggered by Ca(2+) influx through Ca(2+)-permeable AMPA receptors and can occur independently of voltage-gated Ca(2+) (Ca(v)) channels, whose presence and functional role in A17 dendrites have not yet been explored. We combined electrophysiology, calcium imaging and immunohistochemistry and found that L-type Ca(v) channels in rat A17 amacrine cells were located at the sites of reciprocal synaptic feedback and that their contribution to GABA release was diminished by large-conductance Ca(2+)-activated potassium (BK) channels, which suppress postsynaptic depolarization in A17s and limit Ca(v) channel activation. We also found that BK channels, by limiting GABA release from A17s, regulate the flow of excitatory synaptic transmission through the rod pathway.

  17. Epidemiology of polyomavirus BK (BKV) and the emergent African variant in kidney and bone marrow transplant recipients in the Fundacion Valle del Lili in Cali, Colombia.

    PubMed

    Ocampo, Luz Ángela; Rosso, Fernando; Pacheco, Robinson; Villegas, Adriana

    2017-07-01

    To describe the epidemiology of BKV and to assess the presence of the African variant in bone marrow and kidney transplant patients who have suspected BKV reactivation. A descriptive study was conducted, using institutional records, at the Fundación Valle del Lili, Cali-Colombia. The overall prevalence of BKV during the study period was 51%. The African variant was identified in 49.4% of samples that were positive for BKV. 50.6% of the samples were found to have the wild strain of BKV. Among BKV positive patients, 57% were kidney transplant recipients and 43% were bone marrow transplant recipients. This is the first epidemiological study describing the African variant of BKV in Colombia. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Detection of polyomavirus major capsid antigen (VP-1) in human pilomatricomas.

    PubMed

    Sanjuán, Norberto A; Símula, Silvina; Casas, José; Woscoff, Alberto

    2010-01-01

    The family Polyomaviridae is composed of small, non-enveloped, double-stranded DNA viruses widely used to study cell transformation in vitro and tumor induction in vivo. The development of pilomatricomas in mice experimentally infected with polyomavirus led us to detect the viral major capsid protein VP-1 in human pilomatricomas. This tumor, even uncommon, is one of the most frequent benign hair follicle tumors in humans and is composed of proliferating matrix cells that undergo keratinization, and form cystic neoplasms. The detection of VP-1 was performed using the peroxidase-antiperoxidase technique in paraffin-embedded slides with a specific primary serum. Adjacent slides treated with normal rabbit serum as a primary were employed as internal control. Positive and negative controls were also employed as well as slides of lesions caused by human papillomavirus to rule out any unspecific cross-reactivity. In 4 out of 10 cases polyomavirus VP-1 was clearly detected in nuclei of human pilomatricomas proliferating cells, in a patchy pattern of distribution. The controls confirmed the specificity of the immunocytochemical procedure. These results could indicate either an eventual infection of the virus in already developed tumors or alternatively, a direct involvement of polyomavirus in the pathogenesis of some pilomatricomas. The recent discovery of a new human polyomavirus associated with Merkel cell carcinomas has been a strong contribution to better understand the pathogenesis of some human uncommon skin cancers. Hopefully the results reported in this work will encourage further research on the role of polyomavirus in other human skin neoplasms.

  19. Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists.

    PubMed

    Kyle, Barry D; Bradley, Eamonn; Large, Roddy; Sergeant, Gerard P; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

    2013-09-15

    We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca(2+) was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd(2+) (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca(2+) release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca(2+) release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of

  20. Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists

    PubMed Central

    Kyle, Barry D.; Bradley, Eamonn; Large, Roddy; Sergeant, Gerard P.; McHale, Noel G.; Thornbury, Keith D.

    2013-01-01

    We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K+ (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca2+, resulting from ryanodine receptor (RyR) activation via Ca2+-induced Ca2+ release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca2+ was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd2+ (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on

  1. 11,12-EET Stimulates the Association of BK Channel α and β1 Subunits in Mitochondria to Induce Pulmonary Vasoconstriction

    PubMed Central

    Loot, Annemarieke E.; Oelze, Matthias; Syzonenko, Tetyana; Daiber, Andreas; Fleming, Ingrid

    2012-01-01

    In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca2+-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ1 subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ1−/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ1−/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β1 subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β1 subunits of mitochondrial BK channels. The 11

  2. Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation.

    PubMed

    Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar

    2012-07-15

    Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

  3. Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala.

    PubMed

    Faber, E S Louise; Sah, Pankaj

    2003-10-15

    In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K+ currents that contribute to action potential repolarisation. In different neuronal cell types these K+ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K+ currents. Recent findings show that inactivation of a Ca2+-dependent K+ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca2+ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or alpha-dendrotoxin. We conclude that while inactivation of BK-type Ca2+-activated K+ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.

  4. Mice with Deficient BK Channel Function Show Impaired Prepulse Inhibition and Spatial Learning, but Normal Working and Spatial Reference Memory

    PubMed Central

    Azzopardi, Erin; Ruettiger, Lukas; Ruth, Peter; Schmid, Susanne

    2013-01-01

    Genetic variations in the large-conductance, voltage- and calcium activated potassium channels (BK channels) have been recently implicated in mental retardation, autism and schizophrenia which all come along with severe cognitive impairments. In the present study we investigate the effects of functional BK channel deletion on cognition using a genetic mouse model with a knock-out of the gene for the pore forming α-subunit of the channel. We tested the F1 generation of a hybrid SV129/C57BL6 mouse line in which the slo1 gene was deleted in both parent strains. We first evaluated hearing and motor function to establish the suitability of this model for cognitive testing. Auditory brain stem responses to click stimuli showed no threshold differences between knockout mice and their wild-type littermates. Despite of muscular tremor, reduced grip force, and impaired gait, knockout mice exhibited normal locomotion. These findings allowed for testing of sensorimotor gating using the acoustic startle reflex, as well as of working memory, spatial learning and memory in the Y-maze and the Morris water maze, respectively. Prepulse inhibition on the first day of testing was normal, but the knockout mice did not improve over the days of testing as their wild-type littermates did. Spontaneous alternation in the y-maze was normal as well, suggesting that the BK channel knock-out does not impair working memory. In the Morris water maze knock-out mice showed significantly slower acquisition of the task, but normal memory once the task was learned. Thus, we propose a crucial role of the BK channels in learning, but not in memory storage or recollection. PMID:24303038

  5. Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels

    PubMed Central

    Ullrich, Florian; Xu, Rong; Heinemann, Stefan H.; Hou, Shangwei

    2015-01-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca2+- and voltage-dependent K+ (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2–19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation–dependent modulatory influences. PMID:25825171

  6. Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels.

    PubMed

    Tian, Yutao; Ullrich, Florian; Xu, Rong; Heinemann, Stefan H; Hou, Shangwei; Hoshi, Toshinori

    2015-04-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences. © 2015 Tian et al.

  7. Treatment of experimental asthma using a single small molecule with anti-inflammatory and BK channel-activating properties

    PubMed Central

    Goldklang, Monica P.; Perez-Zoghbi, Jose F.; Trischler, Jordis; Nkyimbeng, Takwi; Zakharov, Sergey I.; Shiomi, Takayuki; Zelonina, Tina; Marks, Andrew R.; D'Armiento, Jeanine M.; Marx, Steven O.

    2013-01-01

    Large conductance voltage- and calcium-activated potassium (BK) channels are highly expressed in airway smooth muscle (ASM). Utilizing the ovalbumin (OVA) and house dust mite (HDM) models of asthma in C57BL/6 mice, we demonstrate that systemic administration of the BK channel agonist rottlerin (5 μg/g) during the challenge period reduced methacholine-induced airway hyperreactivity (AHR) in OVA- and HDM-sensitized mice (47% decrease in peak airway resistance in OVA-asthma animals, P<0.01; 54% decrease in HDM-asthma animals, P<0.01) with a 35–40% reduction in inflammatory cells and 20–35% reduction in Th2 cytokines in bronchoalveolar lavage fluid. Intravenous rottlerin (5 μg/g) reduced AHR within 5 min in the OVA-asthma mice by 45% (P<0.01). With the use of an ex vivo lung slice technique, rottlerin relaxed acetylcholine-stimulated murine airway lumen area to 87 ± 4% of the precontracted area (P<0.01 vs. DMSO control). Rottlerin increased BK channel activity in human ASM cells (V50 shifted by 73.5±13.5 and 71.8±14.6 mV in control and asthmatic cells, respectively, both P<0.05 as compared with pretreatment) and reduced the frequency of acetylcholine-induced Ca2+ oscillations in murine ex vivo lung slices. These findings suggest that rottlerin, with both anti-inflammatory and ASM relaxation properties, may have benefit in treating asthma.—Goldklang, M. P., Perez-Zoghbi, J. F., Trischler, J., Nkyimbeng, T., Zakharov, S. I., Shiomi, T., Zelonina, T., Marks, A. R., D'Armiento, J. M., Marx, S. O. Treatment of experimental asthma using a single small molecule with anti-inflammatory and BK channel-activating properties. PMID:23995289

  8. Exposure to raccoon polyomavirus (RacPyV) in free-ranging North American raccoons (Procyon lotor).

    PubMed

    Church, M E; Dela Cruz, F N; Estrada, M; Leutenegger, C M; Pesavento, P A; Woolard, K D

    2016-02-01

    There is evidence that raccoon polyomavirus is causative for neuroglial brain tumors in the western United States. It is unknown if infection is limited to geographic locales where tumors have been reported or is widespread, like human polyomaviruses. We demonstrate raccoons in western, eastern and midwestern states have been exposed to RacPyV by detection of antibodies to capsid protein, VP1. While raccoons in eastern and midwestern states are seropositive, exposure is lower than in the western states. Additionally, across geographic areas seropositivity is higher in older as compared to younger raccoons, similar to polyomavirus exposure in humans. Serum titers are significantly higher in raccoons with tumors compared to raccoons without. Unlike polyomavirus-associated diseases in humans, we did not detect significant sequence variation between tumor and non-tumor tissue in raccoons with tumors compared to those without tumors. This warrants further investigation into co-morbid diseases or genetic susceptibility studies of the host.

  9. Exposure to raccoon polyomavirus (RacPyV) in free-ranging North American raccoons (Procyon lotor)

    PubMed Central

    Cruz Dela, FN; Estrada, M; Leutenegger, CM; Pesavento, PA; Woolard, KD

    2016-01-01

    There is evidence that raccoon polyomavirus is causative for neuroglial brain tumors in the western United States. It is unknown if infection is limited to geographic locales where tumors have been reported or is widespread, like human polyomaviruses. We demonstrate raccoons in western, eastern and midwestern states have been exposed to RacPyV by detection of antibodies to capsid protein, VP1. While raccoons in eastern and midwestern states are seropositive, exposure is lower than in the western states. Additionally, across geographic areas seropositivity is higher in older compared to younger raccoons, similar to polyomavirus exposure in humans. Serum titers are significantly higher in raccoons with tumors compared to raccoons without. Unlike polyomavirus-associated diseases in humans, we did not detect significant sequence variation between tumor and non-tumor tissue in raccoons with tumors compared to those without tumors. This warrants further investigation into co-morbid diseases or genetic susceptibility studies of the host. PMID:26802526

  10. The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

    SciTech Connect

    Shen, Peter S.; Enderlein, Dirk; Nelson, Christian D.S.; Carter, Weston S.; Kawano, Masaaki; Xing Li; Swenson, Robert D.; Olson, Norman H.; Baker, Timothy S.; Cheng, R. Holland; Atwood, Walter J.; Johne, Reimar; Belnap, David M.

    2011-03-01

    Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting {beta}-hairpin observed in other polyomaviruses. We postulate that the terminal {beta}-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

  11. Energy levels of neutral and singly ionized berkelium, /sup 249/Bk I and II

    SciTech Connect

    Worden, E.F.; Conway, J.G.; Blaise, J.

    1987-09-01

    Energy-level analyses of the observed emission spectrum of berkelium have yielded 179 odd and 186 even levels of neutral berkelium Bk I, and 42 odd and 117 even levels of singly ionized berkelium Bk II. The levels are tabulated with the J value, the g value, the configuration and hyperfine constants A and B, and the width given for many of the levels. The ground states of Bk I and Bk II are (Rn)5f/sup 9/7s/sup 2/ /sup 6/H/sup 0//sub 15/2/ and (Rn)5f/sup 9/7s /sup 7/H/sup 0//sub 8/, respectively. A table lists the lowest level of each identified electronic configuration of Bk I and Bk II.

  12. Two-quasiparticle states in {sup 250}Bk studied by decay scheme and transfer reaction spectroscopy

    SciTech Connect

    Ahmad, I.; Kondev, F. G.; Koenig, Z. M.; McHarris, Wm. C.; Yates, S. W.

    2008-05-15

    Two-quasiparticle states in {sup 250}Bk were investigated with decay scheme studies and the single-neutron transfer reaction {sup 249}Bk(d,p){sup 250}Bk. Mass-separated sources of {sup 254}Es were used for {alpha} singles and {alpha}-{gamma} coincidence measurements. These studies, plus previous studies of {sup 254}Es{sup m} {alpha} decay and the {sup 249}Bk(n,{gamma}) reaction, provide spins and parities of the observed levels. The transfer reaction {sup 249}Bk(d,p){sup 250}Bk was used to deduce neutron single-particle components of the observed bands. Six pairs of singlet and triplet states, formed by the coupling of proton and neutron one-quasiparticle states, were identified. The splitting energies between the triplet and singlet states were found to be in agreement with previous calculations.

  13. Complete Genome Sequence of a Novel WU Polyomavirus Isolate from Arkansas, USA, Associated with Acute Respiratory Infection

    PubMed Central

    Denson, J. L.; Schwalm, K. S.; Stoner, A. N.; Kincaid, J. C.; Abramo, T. J.; Thompson, T. M.; Ulloa, E. M.; Burchiel, S. W.; Dinwiddie, D. L.

    2017-01-01

    ABSTRACT We report here the complete genome sequence of a WU polyomavirus (WUPyV) isolate, also known as human polyomavirus 4, collected in 2016 from a patient in Arkansas with an acute respiratory infection. Isolate hPyV4/USA/AR001/2016 has a double-stranded DNA genome of 5,229 bp in length. PMID:28082496

  14. BIOPSY-PROVEN BK VIRUS NEPHROPATHY WITHOUT DETECTABLE BK VIREMIA IN A ONE-YEAR POST-KIDNEY TRANSPLANT RECIPIENT.

    PubMed

    Ruangkanchanasetr, Prajej; Pumchandh, Norawee; Satirapoj, Bancha; Termmathurapoj, Sumeth; Pongthanapisith, Viroj

    2015-07-01

    BK virus nephropathy (BKVN) is an important clinical problem in kidney transplant (KT) recipients. The sequence of disease is usually viruria, viremia and then nephropathy. Diagnosis of BK virus (BKV) infection includes checking BKV DNA in the urine, in the plasma and histology on renal biopsy. This last method is used to diagnose BKVN. We describe a KT patient with BKVN without detectable BK viremia. A 62-year-old female with hypertensive nephropathy underwent renal transplant from a living relative donor in December 2011. Fourteen months after transplantation, her serum creatinine(SCr) rose up from 1.2 to 1.6 mg/dl with biopsy-proven acute antibody-mediated and cellular rejection. After pulse methylprednisolone, plasmapheresis and intravenous immunoglobulin, her SCr decreased to baseline but she subsequently developed cytomegalovirus infection with pancytopenia and transaminitis. The SCr rose to 1.9 mg/dl despite ganciclovir treatment. Renal ultrasound and antegrade pyelogram showed partial obstruction of the proximal ureter with moderate hydronephrosis. A quantitative polymerase chain reaction (PCR) assay for BKV DNA was negative (less than 10 copies/ml). A renal biopsy was performed and the pathology revealed viral cytopathic changes in the tubular epithelium with interstitial inflammation. The renal biopsy also showed BKV nucleic acid sequences by in-situ hybridization confirming BKVN. Immunosuppression regimen was changed to cyclosporine, low-dose prednisolone and leflunomide. A temporary percutaneous nephrostomy was performed. Her renal function improved within one week. The diagnosis of BKVN should be considered in a KT recipient with a rising SCr with or without BK viremia and should be made by renal biopsy.

  15. Binding of cellular proteins to the regulatory region of BK virus DNA.

    PubMed Central

    Markowitz, R B; Dynan, W S

    1988-01-01

    The human papovavirus BK has a noncoding regulatory region located between the divergently transcribed early and late coding regions. Many strains of BK virus (BKV) have direct DNA sequence repeats in the regulatory region, although the number and extent of these repeats varies widely between independent isolates. Until recently, little was known about the individual functional elements within the BKV regulatory region, and the biological significance of the variable repeat structure has been unclear. To characterize the interaction between sequences in the BKV regulatory region and host cell transcription factors, we have carried out DNase I footprinting and competitive binding experiments on three strains of BKV, including one strain that does not contain direct sequence repeats. We have used relatively crude fractions from HeLa cell nuclear extracts, as well as DNA affinity-purified preparations of proteins. Our results demonstrate that BK(Dunlop), BK(WW), and BK(MM) each contain multiple binding sites for a factor, NF-BK, that is a member of the nuclear factor 1 family of transcription factors. We predict the presence of three to eight binding sites for NF-BK in the other strains of BKV for which a DNA sequence is available. This suggests that the binding of this protein is likely to be required for biological activity of the virus. In addition to NF-BK sites, BK(WW) and BK(MM) each contain a single binding site for transcription factor Sp1, and BK(Dunlop) contains two binding sites for transcription factor AP-1. The AP-1 sites in BK(Dunlop) span the junction of adjacent direct repeats, suggesting that repeat formation may be an important mechanism for de novo formation of binding sites not present in a parental strain. Images PMID:2841492

  16. An Exploratory Study of BK from NF = 2 Dynamical Clover-Improved Wilson Fermions

    NASA Astrophysics Data System (ADS)

    Flynn, J. M.; Mescia, F.; Tariq, A. S. B.

    2005-04-01

    We report calculations of BK using two flavours of dynamical clover-improved Wilson lattice fermions and look for dependence on the dynamical quark mass at fixed lattice spacing. We see some evidence for dynamical quark effects. In particular BK decreases as the sea quark masses are reduced towards the up/down quark mass. Our meson masses are quite heavy and a firm prediction of the BK value is a task for future simulations.

  17. BK virus associated nephropathy in renal transplantation: where do we stand.

    PubMed

    Gupta, A; Gupta, P

    2011-06-01

    BK virus is an increasingly identified complication in renal allograft recipients. During the last decade, the use of potent immunosuppressive medications has led to reemergence of this virus. Despite the paucity of randomized trials, we have come a long way in the knowledge of BK virus associated nephropathy. This review highlights the epidemiological, pathogenic, pathological, and clinical aspects of BK virus. It summarizes advances made in prophylaxis and treatment strategies to curtail this virus in an era of modern immunosuppression. The old word of wisdom- prevention is better than cure- might be relevant in context of BK virus prophylaxis with flouroquinolones in years to follow.

  18. Targeting BK (big potassium) Channels in Epilepsy

    PubMed Central

    N'Gouemo, Prosper

    2011-01-01

    Introduction Epilepsies are disorders of neuronal excitability characterized by spontaneous and recurrent seizures. Ion channels are critical for regulating neuronal excitability and, therefore, can contribute significantly to epilepsy pathophysiology. In particular, large conductance, Ca2+-activated K+ (BKCa) channels play an important role in seizure etiology. These channels are activated by both membrane depolarization and increased intracellular Ca2+. This unique coupling of Ca2+ signaling to membrane depolarization is important in controlling neuronal hyperexcitability, as outward K+ current through BKCa channels hyperpolarizes neurons. Areas covered This review focuses on BKCa channel structure-function and discusses the role of these channels in epilepsy pathophysiology. Expert opinion Loss-of-function BKCa channels contribute neuronal hyperexcitability that can lead to temporal lobe epilepsy, tonic-clonic seizures and alcohol withdrawal seizures. Similarly, BKCa channel blockade can trigger seizures and status epilepticus. Paradoxically, some mutations in BKCa channel subunit can give rise to the channel gain-of-function that leads to development of idiopathic epilepsy (primarily absence epilepsy). Seizures themselves also enhance BKCa channel currents associated with neuronal hyperexcitability, and blocking BKCa channels suppresses generalized tonic-clonic seizures. Thus, both loss-of-function and gain-of-function BKCa channels might serve as molecular targets for drugs to suppress certain seizure phenotypes including temporal lobe seizures and absence seizures, respectively. PMID:21923633

  19. Safety and efficacy of an inactivated Carbopol-adjuvanted goose haemorrhagic polyomavirus vaccine for domestic geese.

    PubMed

    Gelfi, Jacqueline; Pappalardo, Michael; Claverys, Carine; Peralta, Brigitte; Guerin, Jean-Luc

    2010-04-01

    Haemorrhagic nephritis enteritis of the goose (HNEG) is an epizootic viral disease in domestic geese. The causal agent is a polyomavirus, namely goose haemorrhagic polyomavirus. To help control the disease, an inactivated vaccine was developed, based on viral particles produced in goose kidney cells. Viral material was quantified using real-time quantitative polymerase chain reaction, inactivated with beta-propiolactone and adjuvanted with Carbopol, an acrylic acid polymer. Carbopol proved to be more immunogenic than aluminium hydroxide and was totally safe when administered to young goslings and breeders alike. Carbopol-adjuvanted vaccine induced a high serological response. Moreover, goslings hatched from vaccinated breeders were protected against viral challenge, indicating that maternally-derived neutralizing antibodies (MDA) were efficiently transferred. MDA were still detectable 15 days post-hatch. Clinical trials will be necessary to accurately evaluate a vaccine-based HNEG control strategy under field conditions.

  20. Merkel cell polyomavirus (MCPyV) in chronic lymphocytic leukemia/small lymphocytic lymphoma.

    PubMed

    Teman, Carolin J; Tripp, Sheryl R; Perkins, Sherrie L; Duncavage, Eric J

    2011-05-01

    Merkel cell polyomavirus (MCPyV) is a novel polyomavirus that shows a strong association with Merkel cell carcinoma (MCC). Recent studies have demonstrated MCPyV in some cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a malignancy with a similar demographic as MCC. We tested for the presence of MCPyV by PCR and immunohistochemistry in 18 cases of CLL/SLL. Very low-level MCPyV DNA was detected in 33% of CLL/SLL cases by real-time PCR, but only one case demonstrated immunohistochemical positivity for MCPyV. MCPyV was not identified in 17 cases of follicular lymphoma, suggesting either that MCPyV is involved in CLL/SLL pathogenesis or that the immunodeficiency state of CLL/SLL induces low-level MCPyV reactivation.

  1. In Vitro and In Vivo Models for the Study of Human Polyomavirus Infection

    PubMed Central

    Barth, Heidi; Solis, Morgane; Kack-Kack, Wallys; Soulier, Eric; Velay, Aurélie; Fafi-Kremer, Samira

    2016-01-01

    Developments of genome amplification techniques have rapidly expanded the family of human polyomaviruses (PyV). Following infection early in life, PyV persist in their hosts and are generally of no clinical consequence. High-level replication of PyV can occur in patients under immunosuppressive or immunomodulatory therapy and causes severe clinical entities, such as progressive multifocal leukoencephalopathy, polyomavirus-associated nephropathy or Merkel cell carcinoma. The characterization of known and newly-discovered human PyV, their relationship to human health, and the mechanisms underlying pathogenesis remain to be elucidated. Here, we summarize the most widely-used in vitro and in vivo models to study the PyV-host interaction, pathogenesis and anti-viral drug screening. We discuss the strengths and limitations of the different models and the lessons learned. PMID:27782080

  2. Production and biomedical applications of virus-like particles derived from polyomaviruses.

    PubMed

    Teunissen, Erik A; de Raad, Markus; Mastrobattista, Enrico

    2013-11-28

    Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargoes and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.

  3. Atomic Mass and Nuclear Binding Energy for Bk-282 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-282 (Berkelium, atomic number Z = 97, mass number A = 282).

  4. Atomic Mass and Nuclear Binding Energy for Bk-272 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-272 (Berkelium, atomic number Z = 97, mass number A = 272).

  5. Atomic Mass and Nuclear Binding Energy for Bk-297 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-297 (Berkelium, atomic number Z = 97, mass number A = 297).

  6. Atomic Mass and Nuclear Binding Energy for Bk-324 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-324 (Berkelium, atomic number Z = 97, mass number A = 324).

  7. Atomic Mass and Nuclear Binding Energy for Bk-247 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-247 (Berkelium, atomic number Z = 97, mass number A = 247).

  8. Atomic Mass and Nuclear Binding Energy for Bk-310 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-310 (Berkelium, atomic number Z = 97, mass number A = 310).

  9. Atomic Mass and Nuclear Binding Energy for Bk-260 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-260 (Berkelium, atomic number Z = 97, mass number A = 260).

  10. Atomic Mass and Nuclear Binding Energy for Bk-320 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-320 (Berkelium, atomic number Z = 97, mass number A = 320).

  11. Atomic Mass and Nuclear Binding Energy for Bk-301 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-301 (Berkelium, atomic number Z = 97, mass number A = 301).

  12. Atomic Mass and Nuclear Binding Energy for Bk-304 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-304 (Berkelium, atomic number Z = 97, mass number A = 304).

  13. Atomic Mass and Nuclear Binding Energy for Bk-309 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-309 (Berkelium, atomic number Z = 97, mass number A = 309).

  14. Atomic Mass and Nuclear Binding Energy for Bk-275 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-275 (Berkelium, atomic number Z = 97, mass number A = 275).

  15. Atomic Mass and Nuclear Binding Energy for Bk-298 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-298 (Berkelium, atomic number Z = 97, mass number A = 298).

  16. Atomic Mass and Nuclear Binding Energy for Bk-251 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-251 (Berkelium, atomic number Z = 97, mass number A = 251).

  17. Atomic Mass and Nuclear Binding Energy for Bk-318 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-318 (Berkelium, atomic number Z = 97, mass number A = 318).

  18. Atomic Mass and Nuclear Binding Energy for Bk-252 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-252 (Berkelium, atomic number Z = 97, mass number A = 252).

  19. Atomic Mass and Nuclear Binding Energy for Bk-261 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-261 (Berkelium, atomic number Z = 97, mass number A = 261).

  20. Atomic Mass and Nuclear Binding Energy for Bk-300 (Berkelium)

    NASA Astrophysics Data System (ADS)

    Sukhoruchkin, S. I.; Soroko, Z. N.

    This document is part of the Supplement containing the complete sets of data of Subvolume B `Nuclei with Z = 55 - 100' of Volume 22 `Nuclear Binding Energies and Atomic Masses' of Landolt-Börnstein - Group I `Elementary Particles, Nuclei and Atoms', and additionally including data for nuclei with Z = 101 - 130. It provides atomic mass, mass excess, nuclear binding energy, nucleon separation energies, Q-values, and nucleon residual interaction parameters for atomic nuclei of the isotope Bk-300 (Berkelium, atomic number Z = 97, mass number A = 300).