Science.gov

Sample records for activating kir genes

  1. Comparison of KIR gene content profiles revealed a difference between northern and southern Persians in the distribution of KIR2DS5 and its linked loci.

    PubMed

    Solgi, Ghasem; Ghafari, Hamidreza; Ashouri, Elham; Alimoghdam, Kamran; Rajalingam, Raja; Amirzargar, Aliakbar

    2011-11-01

    Killer cell immunoglobulin-like receptors (KIR) are the key receptors of human natural killer (NK) cells that mount an early immune response against infection and tumors. The number and type of KIR genes are substantially variable between individuals and populations. Recently we reported KIR gene content diversity in a Persian population living in the southern province of Fars, which is comparable to that of European Caucasians. These results are consistent with the ethnic ancestry and affinity between Persians and Caucasians. Herein we analyzed another Persian population living in the northern province of Tehran and discovered an unexpected increase in the distribution of KIR2DS5 and its linked loci KIR3DS1, -2DS1, and -2DL5 in northern Persians compared with that reported in the southern Persian population. Although the geographic barriers may have limited the gene flow, the impact of the local environment on the natural selection of KIR2DS5 and its linked loci in the northern Persians cannot be completely ruled out. The difference in northern and southern populations in activating KIR gene content creates an appealing hypothesis that KIR2DS5-enriched northern Persians are more resistant to developing clinical conditions demonstrated to be associated with KIR2DS5, such as psoriasis vulgaris, endometriosis, ankylosing spondylitis, and acute rejection of kidney grafts, compared with those living in the southern part of the country. PMID:21867738

  2. KIR Gene Content in Amerindians Indicates Influence of Demographic Factors

    PubMed Central

    Augusto, Danillo Gardenal; Piovezan, Bruno Zagonel; Tsuneto, Luiza Tamie; Callegari-Jacques, Sidia Maria; Petzl-Erler, Maria Luiza

    2013-01-01

    Although the KIR gene content polymorphism has been studied worldwide, only a few isolated or Amerindian populations have been analyzed. This extremely diverse gene family codifies receptors that are expressed mainly in NK cells and bind HLA class I molecules. KIR-HLA combinations have been associated to several diseases and population studies are important to comprehend their evolution and their role in immunity. Here we analyzed, by PCR-SSP (specific sequencing priming), 327 individuals from four isolated groups of two of the most important Brazilian Amerindian populations: Kaingang and Guarani. The pattern of KIR diversity among these and other ten Amerindian populations disclosed a wide range of variation for both KIR haplotypes and gene frequencies, indicating that demographic factors, such as bottleneck and founder effects, were the most important evolutionary factors in shaping the KIR polymorphism in these populations. PMID:23451080

  3. KIR genotypic diversity in Portuguese and analysis of KIR gene allocation after allogeneic hematopoietic stem cell transplantation.

    PubMed

    Ligeiro, D; Buhler, S; Abecasis, M; Abade, O; Sanchez-Mazas, A; da Silva, M Gomes; Trindade, H

    2016-05-01

    The diversity of killer-cell immunoglobulin-like receptors (KIR) genes was evaluated in Portuguese and the observed genotypic profiles were found related to the ones reported in European populations. The KIR repertoire after hematopoietic stem cell transplantation is determined by these gene frequencies and the KIR group B motifs are the less common. We estimated donor-KIR/recipient-ligand interactions in transplants with related donors and unrelated donors found in a local registry or from abroad. A large fraction of transplants had all three ligands of inhibitory receptors, and therefore, in theory were not prone to natural killer cell (NK) mediated alloreactivity. Furthermore, the distribution of KIR alloreactive interactions was found independent of the donor-recipient genetic proximity, probably because of different gene segregation and comparable KIR frequencies in the donor pools. PMID:27075774

  4. Definition of gene content for nine common group B haplotypes of the Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes.

    PubMed

    Uhrberg, Markus; Parham, Peter; Wernet, Peter

    2002-07-01

    The segregation of killer cell immunoglobulin-like receptor ( KIR) genes was determined for a panel of 21 Caucasoid families: 23 different KIR gene patterns were found and could be assigned to combinations of 16 different haplotypes. Four loci were held in common by all haplotypes: KIR2DL4, KIR3DL2, the putative pseudogene KIR3DL3 and KIR2DL2/KIR2DL3, the latter likely being alleles of one gene. Group A haplotypes, which have a unique combination of seven KIR genes, were found at 80% frequency in the family panel, the polygenic group B haplotypes at 65% frequency. KIR gene segregation was fully determined for the nine group B haplotypes, which occurred at highest frequencies in both the family panel and a panel of unrelated individuals. The group B haplotypes carried between seven and 11 KIR genes and encoded inhibitory KIR for one, two, or all three major HLA class I epitopes. Analysis of human leucocyte antigen (HLA) class I genotypes revealed that most, but not all, individuals possess an inhibitory KIR for a self HLA class I epitope. The number of stimulatory KIR genes in group B haplotypes varied considerably between one and five. The data show that group B haplotypes possess a broad spectrum of KIR gene patterns, which is largely complementary to the KIR gene set of group A haplotypes. The results suggest that rapid diversification of group B haplotypes is the result of pathogen-mediated selection for KIR genotypes that have more than the set of KIR genes provided by the group A haplotype.

  5. KIR and HLA haplotype analysis in a family lacking the KIR 2DL1-2DP1 genes

    PubMed Central

    Vojvodić, S; Ademović-Sazdanić, D

    2015-01-01

    The killer cell immunoglobulin-like receptor (KIR) gene cluster exhibits extensive allelic and haplotypic diversity that is observed as presence/absence of genes, resulting in expansion and contraction of KIR haplotypes and by allelic variation of individual KIR genes. We report a case of KIR pseudogene 2DP1 and 2DL1 gene absence in members of one family with the children suffering from acute myelogenous leukemia (AML). Killer cell immunoglobulin-like receptor low resolution genotyping was performed by the polymerase chain reaction (PCR)-sequence-specific primers (SSP)/sequence-specific oligonucleotide (SSO) method and haplotype assignment was done by gene content analysis. Both parents and the maternal grandfather, shared the same Cen-B2 KIR haplotype, containing KIR 3DL3, -2DS2, -2DL2 and -3DP1 genes. The second haplotype in the KIR genotype of the mother and grandfather was Tel-A1 with KIR 2DL4 (normal and deleted variant), -3DL1, -22 bp deletion variant of the 2DS4 gene and -3DL2, while the second haplotype in the KIR genotype of the father was Tel-B1 with 2DL4 (normal variant), -3DS1, -2DL5, -2DS5, -2DS1 and 3DL2 genes. Haplotype analysis in all three offsprings revealed that the children inherited the Cen-B2 haplotype with the same gene content but two of the children inherited a deleted variant of the 2DL4 gene, while the third child inherited a normal one. The second haplotype of all three offspring contained KIR 2DL4, -2DL5, -2DS1, -2DS4 (del 22bp variant), -2DS5, -3DL1 and -3DL2 genes, which was the basis of the assumption that there is a hybrid haplotype and that the present 3DL1 gene is a variant of the 3DS1 gene. Due to consanguinity among the ancestors, the results of KIR segregation analysis showed the existence of a very rare KIR genotype in the offspring. The family who is the subject of this case is even more interesting because the father was 10/10 human leukocyte antigen (HLA)-matched to his daughter, all members of the family have the

  6. KIR gene diversity in Mexican mestizos of San Luis Potosí.

    PubMed

    Alvarado-Hernández, Diana Lorena; Hernández-Ramírez, Daniel; Noyola, Daniel Ernesto; García-Sepúlveda, Christian Alberto

    2011-09-01

    Natural killer (NK) cell function is regulated by different types of membrane-bound receptors of which killer-cell immunoglobulin-like receptors (KIRs) are the most complex and diverse. KIRs are encoded by 17 different genes located within the leukocyte receptor complex (19q13.4). The frequency with which KIR gene features are present in different human populations differs. Here, we present our results on the KIR gene diversity observed in a large group of mestizos from the central Mexican city of San Luis Potosí. In total, 53 different KIR genotypes were observed, 47 with previously described gene profiles and six harboring novel KIR gene combinations. Group A homozygous haplotypes were seen in 102 individuals (34%), while group B homozygous haplotypes were present in 45 (15%). Heterozygous combinations of groups A and B haplotypes were seen in 153 individuals (51%). Haplotype frequency estimations based on a true content of 600 chromosomes showed a relatively balanced proportion of group A (59.5%) and group B (40.5%) haplotypes in our study population. A homozygous combination of the cA01|tA01 haplotype was present in 33% of the population with other frequent combinations being cA01|tA01, cB03|tB01 in 14.7% and cA01|tA01, cB02|tA01 in 12%. The dendrogram derived from activating KIR gene phylogenetic analysis revealed five clearly distinct clades corresponding to African, East Asian, Arab/Caucasoid, Mexican mestizo/Amerindian and South Asian populations. Our results illustrate the genetic contribution that Caucasoid and Amerindian populations have made toward present-day Mexicans and suggest an important Southeast Asian genetic contribution to native Amerindian populations.

  7. Conformational Dynamics of Kir3.1/Kir3.2 Channel Activation Via δ-Opioid Receptors

    PubMed Central

    Richard-Lalonde, Melissa; Nagi, Karim; Audet, Nicolas; Sleno, Rory; Amraei, Mohammad; Hogue, Mireille; Balboni, Gianfranco; Schiller, Peter W.; Bouvier, Michel; Hébert, Terence E.; Pineyro, Graciela

    2013-01-01

    This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gβγ, and Kir3 subunits constitutively interacted with one another. GαoA associated with DORs and Gβγ, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GαoA, DOR-Gβγ, GαoA-Gβγ, and Gβγ-Kir3 interfaces. Ligand-induced BRET changes assessing Gβγ-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GαoA-Gβγ interface, 2) displayed the same order of efficacy as those observed at the DOR-Gβγ interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gβγ/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gβγ-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gβγ with respect to DORs, GαoA, and channel subunits. Further, the fact that BRET changes at the Gβγ-Kir3 interface are predictive of a ligand’s ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation. PMID:23175530

  8. Common Genetic Variants Found in HLA and KIR Immune Genes in Autism Spectrum Disorder

    PubMed Central

    Torres, Anthony R.; Sweeten, Thayne L.; Johnson, Randall C.; Odell, Dennis; Westover, Jonna B.; Bray-Ward, Patricia; Ward, David C.; Davies, Christopher J.; Thomas, Aaron J.; Croen, Lisa A.; Benson, Michael

    2016-01-01

    The “common variant—common disease” hypothesis was proposed to explain diseases with strong inheritance. This model suggests that a genetic disease is the result of the combination of several common genetic variants. Common genetic variants are described as a 5% frequency differential between diseased vs. matched control populations. This theory was recently supported by an epidemiology paper stating that about 50% of genetic risk for autism resides in common variants. However, rare variants, rather than common variants, have been found in numerous genome wide genetic studies and many have concluded that the “common variant—common disease” hypothesis is incorrect. One interpretation is that rare variants are major contributors to genetic diseases and autism involves the interaction of many rare variants, especially in the brain. It is obvious there is much yet to be learned about autism genetics. Evidence has been mounting over the years indicating immune involvement in autism, particularly the HLA genes on chromosome 6 and KIR genes on chromosome 19. These two large multigene complexes have important immune functions and have been shown to interact to eliminate unwanted virally infected and malignant cells. HLA proteins have important functions in antigen presentation in adaptive immunity and specific epitopes on HLA class I proteins act as cognate ligands for KIR receptors in innate immunity. Data suggests that HLA alleles and KIR activating genes/haplotypes are common variants in different autism populations. For example, class I allele (HLA-A2 and HLA-G 14 bp-indel) frequencies are significantly increased by more than 5% over control populations (Table 2). The HLA-DR4 Class II and shared epitope frequencies are significantly above the control populations (Table 2). Three activating KIR genes: 3DS1, 2DS1, and 2DS2 have increased frequencies of 15, 22, and 14% in autism populations, respectively. There is a 6% increase in total activating KIR genes in

  9. Correlating gene-specific DNA methylation changes with expression and transcriptional activity of astrocytic KCNJ10 (Kir4.1)

    PubMed Central

    Nwaobi, Sinifunanya E.; Olsen, Michelle L.

    2016-01-01

    to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity. PMID:26436772

  10. KIR Genes and Patterns Given by the A Priori Algorithm: Immunity for Haematological Malignancies

    PubMed Central

    Rodríguez-Escobedo, J. Gilberto; García-Sepúlveda, Christian A.; Cuevas-Tello, Juan C.

    2015-01-01

    Killer-cell immunoglobulin-like receptors (KIRs) are membrane proteins expressed by cells of innate and adaptive immunity. The KIR system consists of 17 genes and 614 alleles arranged into different haplotypes. KIR genes modulate susceptibility to haematological malignancies, viral infections, and autoimmune diseases. Molecular epidemiology studies rely on traditional statistical methods to identify associations between KIR genes and disease. We have previously described our results by applying support vector machines to identify associations between KIR genes and disease. However, rules specifying which haplotypes are associated with greater susceptibility to malignancies are lacking. Here we present the results of our investigation into the rules governing haematological malignancy susceptibility. We have studied the different haplotypic combinations of 17 KIR genes in 300 healthy individuals and 43 patients with haematological malignancies (25 with leukaemia and 18 with lymphomas). We compare two machine learning algorithms against traditional statistical analysis and show that the “a priori” algorithm is capable of discovering patterns unrevealed by previous algorithms and statistical approaches. PMID:26495028

  11. Killer cell immunoglobulin-like receptor (KIR) gene diversity in a population naturally exposed to malaria in Porto Velho, Northern Brazil.

    PubMed

    Perce-da-Silva, D S; Silva, L A; Lima-Junior, J C; Cardoso-Oliveira, J; Ribeiro-Alves, M; Santos, F; Porto, L C M S; Oliveira-Ferreira, J; Banic, D M

    2015-03-01

    Killer cell immunoglobulin-like receptors (KIR) are expressed mainly in natural killer cells and specifically recognize human leukocyte antigen (HLA) class I molecules. The repertoire of KIR genes and KIR-HLA pairs is known to play a key role in the susceptibilities to and the outcomes of several diseases, including malaria. The aim of this study was to investigate the distribution of KIR genes, KIR genotypes and KIR-HLA pair combinations in a population naturally exposed to malaria from Brazilian Amazon. All 16 KIR genes investigated were present in the studied population. Overall, 46 KIR genotypes were defined. The two most common genotypes in the Porto Velho communities, genotypes 1 and 2, were present at similar frequencies as in the Americas. Principal component analysis based on the frequencies of the KIR genes placed the Porto Velho population closer to the Venezuela Mestizos, USA California hispanic and Brazil Paraná Mixed in terms of KIR gene frequencies. This analysis highlights the multi-ethnic profile of the Porto Velho population. Most of the individuals were found to have at least one inhibitory KIR-HLA pair. Seventy-five KIR-HLA pair combinations were identified. The KIR-2DL2/3_HLA-C1, KIR3DL1_HLA-Bw4 and KIR2DL1_HLA-C2 pairs were the most common. There was no association between KIR genes, KIR genotypes or KIR-HLA pair combinations and malaria susceptibility in the studied population. This is the first report on the distribution of KIR and known HLA ligands in the Porto Velho population. Taken together, these results should provide baseline information that will be relevant to population evolutionary history, malaria and other diseases studies in populations of the Brazilian Amazon.

  12. Protective KIR-HLA interactions for HCV infection in intravenous drug users

    PubMed Central

    Zúñiga, Joaquin; Romero, Viviana; Azocar, José; Terreros, Daniel; Vargas-Rojas, María Inés; Torres-García, Diana; Jimenez-Alvarez, Luis; Vargas-Alarcón, Gilberto; Granados-Montiel, Julio; Husain, Zaheed; Chung, Raymond T.; Alper, Chester A.; Yunis, Edmond J.

    2009-01-01

    Intravenous drug use has become the principal route of hepatitis C virus (HCV) transmission due to the sharing of infected needles. In this study, we analyzed the distribution of HLA-KIR genotypes among 160 Puerto Rican intravenous drug users (IDUs) with HCV infection and 92 HCV-negative Puerto Rican IDUs. We found a significant association between the presence of different combinations of KIR inhibitory receptor genes (KIR2DL2 and/or KIR2DL3, pC = 0.01, OR = 0.07; KIR2DL2 and/or KIR2DL3+KIR2DS4, pC = 0.01, OR = 0.39) and HLA-C1 homozygous genotypes (HLA-C1+KIR2DS4, pC = 0.02, OR = 0.43; HLA-C1+KIR2DL2+KIR2DS4, pC = 0.02, OR = 0.40) together with the activating receptor KIR2DS4 (HLA-C1+KIR2DS4+KIR2DL3 and/or KIR2DL2, pC = 0.004, OR = 0.38) with protection from HCV infection. Our findings in HCV-infected and non-infected IDUs suggest an important role for KIRs (KIR2DL2 and KIR2DL3) with group HLA-C1 molecules, in the presence of activating KIR2DS4, in protection from HCV infection. These results support the hypothesis that activator signaling, mediated by KIR2DS4, is a determinant in the regulation of NK cell antiviral-activity. PMID:19552960

  13. The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like receptors

    PubMed Central

    Guselnikov, Sergey V; Reshetnikova, Evdokiya S; Najakshin, Alexander M; Mechetina, Ludmila V; Robert, Jacques; Taranin, Alexander V

    2009-01-01

    In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3–4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcR subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory. PMID:19896971

  14. Functional advantage of educated KIR2DL1(+) natural killer cells for anti-HIV-1 antibody-dependent activation.

    PubMed

    Gooneratne, S L; Center, R J; Kent, S J; Parsons, M S

    2016-04-01

    Evidence from the RV144 HIV-1 vaccine trial implicates anti-HIV-1 antibody-dependent cellular cytotoxicity (ADCC) in vaccine-conferred protection from infection. Among effector cells that mediate ADCC are natural killer (NK) cells. The ability of NK cells to be activated in an antibody-dependent manner is reliant upon several factors. In general, NK cell-mediated antibody-dependent activation is most robust in terminally differentiated CD57(+) NK cells, as well as NK cells educated through ontological interactions between inhibitory killer immunoglobulin-like receptors (KIR) and their major histocompatibility complex class I [MHC-I or human leucocyte antigen (HLA-I)] ligands. With regard to anti-HIV-1 antibody-dependent NK cell activation, previous research has demonstrated that the epidemiologically relevant KIR3DL1/HLA-Bw4 receptor/ligand combination confers enhanced activation potential. In the present study we assessed the ability of the KIR2DL1/HLA-C2 receptor/ligand combination to confer enhanced activation upon direct stimulation with HLA-I-devoid target cells or antibody-dependent stimulation with HIV-1 gp140-pulsed CEM.NKr-CCR5 target cells in the presence of an anti-HIV-1 antibody source. Among donors carrying the HLA-C2 ligand for KIR2DL1, higher interferon (IFN)-γ production was observed within KIR2DL1(+) NK cells than in KIR2DL1(-) NK cells upon both direct and antibody-dependent stimulation. No differences in KIR2DL1(+) and KIR2DL1(-) NK cell activation were observed in HLA-C1 homozygous donors. Additionally, higher activation in KIR2DL1(+) than KIR2DL1(-) NK cells from HLA-C2 carrying donors was observed within less differentiated CD57(-) NK cells, demonstrating that the observed differences were due to education and not an overabundance of KIR2DL1(+) NK cells within differentiated CD57(+) NK cells. These observations are relevant for understanding the regulation of anti-HIV-1 antibody-dependent NK cell responses.

  15. An intersubunit salt bridge near the selectivity filter stabilizes the active state of Kir1.1.

    PubMed

    Sackin, Henry; Nanazashvili, Mikheil; Li, Hui; Palmer, Lawrence G; Walters, D Eric

    2009-08-19

    ROMK (Kir1.1) potassium channels are closed by internal acidification with a pKa of 6.7 +/- 0.01 in 100 mM external K and a pKa of 7.0 +/- 0.01 in 1 mM external K. Internal acidification in 1 mM K (but not 100 mM K) not only closed the pH gate but also inactivated Kir1.1, such that realkalization did not restore channel activity until high K was returned to the bath. We identified a new putative intersubunit salt bridge (R128-E132-Kir1.1b) in the P-loop of the channel near the selectivity filter that affected the K sensitivity of the inactivation process. Mutation of either R128-Kir1.1b or E132-Kir1.1b caused inactivation in both 1 mM and 100 mM external K during oocyte acidification. However, 300 mM external K (but not 200 mM Na + 100 mM K) protected both E132Q and R128Y from inactivation. External application of a modified honey-bee toxin, tertiapin Q (TPNQ), also protected Kir1.1 from inactivation in 1 mM K and protected E132Q and R128Y from inactivation in 100 mM K, which suggests that TPNQ binding to the outer mouth of the channel stabilizes the active state. Pretreatment of Kir1.1 with external Ba prevented Kir1.1 inactivation, similar to pretreatment with TPNQ. In addition, mutations that disrupted transmembrane helix H-bonding (K61M-Kir1.1b) or stabilized a selectivity filter to helix-pore linkage (V121T-Kir1.1b) also protected both E132Q and R128Y from inactivation in 1 mM K and 100 mM K. Our results are consistent with Kir inactivation arising from conformational changes near the selectivity filter, analogous to C-type inactivation. PMID:19686653

  16. Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine–induced temporal lobe epilepsy model

    PubMed Central

    Nagao, Yuki; Harada, Yuya; Mukai, Takahiro; Shimizu, Saki; Okuda, Aoi; Fujimoto, Megumi; Ono, Asuka; Sakagami, Yoshihisa; Ohno, Yukihiro

    2013-01-01

    The inwardly rectifying potassium (Kir) channel Kir4.1 in brain astrocytes mediates spatial K+ buffering and regulates neural activities. Recent studies have shown that loss-of-function mutations in the human gene KCNJ10 encoding Kir4.1 cause epileptic seizures, suggesting a close relationship between the Kir4.1 channel function and epileptogenesis. Here, we performed expressional analysis of Kir4.1 in a pilocarpine-induced rat model of temporal lobe epilepsy (TLE) to explore the role of Kir4.1 channels in modifying TLE epileptogenesis. Treatment of rats with pilocarpine (350 mg/kg, i.p.) induced acute status epilepticus, which subsequently caused spontaneous seizures 7–8 weeks after the pilocarpine treatment. Western blot analysis revealed that TLE rats (interictal condition) showed significantly higher levels of Kir4.1 than the control animals in the cerebral cortex, striatum, and hypothalamus. However, the expression of other Kir subunits, Kir5.1 and Kir2.1, remained unaltered. Immunohistochemical analysis illustrated that Kir4.1-immunoreactivity-positive astrocytes in the pilocarpine-induced TLE model were markedly increased in most of the brain regions examined, concomitant with an increase in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes. In addition, Kir4.1 expression ratios relative to the number of astrocytes (Kir4.1-positive cells/GFAP-positive cells) were region-specifically elevated in the amygdala (i.e., medial and cortical amygdaloid nuclei) and sensory cortex. The present study demonstrated for the first time that the expression of astrocytic Kir4.1 channels was elevated in a pilocarpine-induced TLE model, especially in the amygdala, suggesting that astrocytic Kir4.1 channels play a role in modifying TLE epileptogenesis, possibly by acting as an inhibitory compensatory mechanism. PMID:23922547

  17. Expressional analysis of the astrocytic Kir4.1 channel in a pilocarpine-induced temporal lobe epilepsy model.

    PubMed

    Nagao, Yuki; Harada, Yuya; Mukai, Takahiro; Shimizu, Saki; Okuda, Aoi; Fujimoto, Megumi; Ono, Asuka; Sakagami, Yoshihisa; Ohno, Yukihiro

    2013-01-01

    The inwardly rectifying potassium (Kir) channel Kir4.1 in brain astrocytes mediates spatial K(+) buffering and regulates neural activities. Recent studies have shown that loss-of-function mutations in the human gene KCNJ10 encoding Kir4.1 cause epileptic seizures, suggesting a close relationship between the Kir4.1 channel function and epileptogenesis. Here, we performed expressional analysis of Kir4.1 in a pilocarpine-induced rat model of temporal lobe epilepsy (TLE) to explore the role of Kir4.1 channels in modifying TLE epileptogenesis. Treatment of rats with pilocarpine (350 mg/kg, i.p.) induced acute status epilepticus, which subsequently caused spontaneous seizures 7-8 weeks after the pilocarpine treatment. Western blot analysis revealed that TLE rats (interictal condition) showed significantly higher levels of Kir4.1 than the control animals in the cerebral cortex, striatum, and hypothalamus. However, the expression of other Kir subunits, Kir5.1 and Kir2.1, remained unaltered. Immunohistochemical analysis illustrated that Kir4.1-immunoreactivity-positive astrocytes in the pilocarpine-induced TLE model were markedly increased in most of the brain regions examined, concomitant with an increase in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes. In addition, Kir4.1 expression ratios relative to the number of astrocytes (Kir4.1-positive cells/GFAP-positive cells) were region-specifically elevated in the amygdala (i.e., medial and cortical amygdaloid nuclei) and sensory cortex. The present study demonstrated for the first time that the expression of astrocytic Kir4.1 channels was elevated in a pilocarpine-induced TLE model, especially in the amygdala, suggesting that astrocytic Kir4.1 channels play a role in modifying TLE epileptogenesis, possibly by acting as an inhibitory compensatory mechanism.

  18. Killer Cell Immunoglobulin-Like Receptor (KIR) Genotype Distribution in Familial Mediterranean Fever (FMF) Patients

    PubMed Central

    Erken, Ertugrul; Ozturk, Ozlem Goruroglu; Kudas, Ozlem; Tas, Didem Arslan; Demirtas, Ahmet; Kibar, Filiz; Dinkci, Suzan; Erken, Eren

    2015-01-01

    Background Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disease predominantly affecting Mediterranean populations. The gene associated with FMF is the MEFV gene, which encodes for a protein called pyrin. Mutations of pyrin lead to uncontrolled attacks of inflammation, and subclinical inflammation continues during attack-free intervals. Killer cell immunoglobulin-like receptor (KIR) genes encode HLA class I receptors expressed by NK cells. The aim this study was to look for immunogenetic determinants in the pathogenesis of FMF and find out if KIR are related to susceptibility to disease or complications like renal amyloidosis. Material/Methods One hundred and five patients with FMF and 100 healthy individuals were involved in the study. Isolated DNA from peripheral blood was amplified by sequence specific PCR probes and analyzed by Luminex for KIR genotypes. Fisher Exact test was used to evaluate the variation of KIR gene distribution. Results All patients and healthy controls expressed the framework genes. An activator KIR gene, KIR2DS2, was significantly more frequent in FMF patients (p=0.036). Renal amyloidosis and presence of arthritis were not associated with KIR genes and genotype. KIR3DL1 gene was more common in patients with high serum CRP (p=0.016). Conclusions According to our findings, we suggest that presence of KIR2DS2, which is an activator gene for NK cell functions, might be related to the autoinflammation in FMF. The potential effect of KIR genes on amyloidosis and other clinical features requires studies with larger sample sizes. PMID:26574972

  19. Large spectrum of HLA-C recognition by killer Ig-like receptor (KIR)2DL2 and KIR2DL3 and restricted C1 SPECIFICITY of KIR2DS2: dominant impact of KIR2DL2/KIR2DS2 on KIR2D NK cell repertoire formation.

    PubMed

    David, Gaëlle; Djaoud, Zakia; Willem, Catherine; Legrand, Nolwenn; Rettman, Pauline; Gagne, Katia; Cesbron, Anne; Retière, Christelle

    2013-11-01

    The interactions of killer Ig-like receptor 2D (KIR2D) with HLA-C ligands contribute to functional NK cell education and regulate NK cell functions. Although simple alloreactive rules have been established for inhibitory KIR2DL, those governing activating KIR2DS function are still undefined, and those governing the formation of the KIR2D repertoire are still debated. In this study, we investigated the specificity of KIR2DL1/2/3 and KIR2DS1/2, dissected each KIR2D function, and assessed the impact of revisited specificities on the KIR2D NK cell repertoire formation from a large cohort of 159 KIR and HLA genotyped individuals. We report that KIR2DL2(+) and KIR2DL3(+) NK cells reacted similarly against HLA-C(+) target cells, irrespective of C1 or C2 allele expression. In contrast, KIR2DL1(+) NK cells specifically reacted against C2 alleles, suggesting a larger spectrum of HLA-C recognition by KIR2DL2 and KIR2DL3 than KIR2DL1. KIR2DS2(+) KIR2DL2(-) NK cell clones were C1-reactive irrespective of their HLA-C environment. However, when KIR2DS2 and KIR2DL2 were coexpressed, NK cell inhibition via KIR2DL2 overrode NK cell activation via KIR2DS2. In contrast, KIR2DL1 and KIR2DS2 had an additive enhancing effect on NK cell responses against C1C1 target cells. KIR2DL2/3/S2 NK cells predominated within the KIR repertoire in KIR2DL2/S2(+) individuals. In contrast, the KIR2DL1/S1 NK cell compartment is dominant in C2C2 KIR2DL2/S2(-) individuals. Moreover, our results suggest that together with KIR2DL2, activating KIR2DS1 and KIR2DS2 expression limits KIR2DL1 acquisition on NK cells. Altogether, our results suggest that the NK cell repertoire is remolded by the activating and inhibitory KIR2D and their cognate ligands.

  20. Definition of the Cattle Killer Cell Ig–like Receptor Gene Family: Comparison with Aurochs and Human Counterparts

    PubMed Central

    Sanderson, Nicholas D.; Norman, Paul J.; Guethlein, Lisbeth A.; Ellis, Shirley A.; Williams, Christina; Breen, Matthew; Park, Steven D. E.; Magee, David A.; Babrzadeh, Farbod; Warry, Andrew; Watson, Mick; Bradley, Daniel G.; MacHugh, David E.; Parham, Peter

    2014-01-01

    Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle. PMID:25398326

  1. Definition of the cattle killer cell Ig-like receptor gene family: comparison with aurochs and human counterparts.

    PubMed

    Sanderson, Nicholas D; Norman, Paul J; Guethlein, Lisbeth A; Ellis, Shirley A; Williams, Christina; Breen, Matthew; Park, Steven D E; Magee, David A; Babrzadeh, Farbod; Warry, Andrew; Watson, Mick; Bradley, Daniel G; MacHugh, David E; Parham, Peter; Hammond, John A

    2014-12-15

    Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig-like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle.

  2. Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1.

    PubMed

    Garcia-Beltran, Wilfredo F; Hölzemer, Angelique; Martrus, Gloria; Chung, Amy W; Pacheco, Yovana; Simoneau, Camille R; Rucevic, Marijana; Lamothe-Molina, Pedro A; Pertel, Thomas; Kim, Tae-Eun; Dugan, Haley; Alter, Galit; Dechanet-Merville, Julie; Jost, Stephanie; Carrington, Mary; Altfeld, Marcus

    2016-09-01

    The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1(+) NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4(+) T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1(+) NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease.

  3. Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1.

    PubMed

    Garcia-Beltran, Wilfredo F; Hölzemer, Angelique; Martrus, Gloria; Chung, Amy W; Pacheco, Yovana; Simoneau, Camille R; Rucevic, Marijana; Lamothe-Molina, Pedro A; Pertel, Thomas; Kim, Tae-Eun; Dugan, Haley; Alter, Galit; Dechanet-Merville, Julie; Jost, Stephanie; Carrington, Mary; Altfeld, Marcus

    2016-09-01

    The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1(+) NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4(+) T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1(+) NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease. PMID:27455421

  4. The distribution of KIR-HLA functional blocks is different from north to south of Italy.

    PubMed

    Fasano, M E; Rendine, S; Pasi, A; Bontadini, A; Cosentini, E; Carcassi, C; Capittini, C; Cornacchini, G; Espadas de Arias, A; Garbarino, L; Carella, G; Mariotti, M L; Mele, L; Miotti, V; Moscetti, A; Nesci, S; Ozzella, G; Piancatelli, D; Porfirio, B; Riva, M R; Romeo, G; Tagliaferri, C; Lombardo, C; Testi, M; Amoroso, A; Martinetti, M

    2014-03-01

    The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells. Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival. We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification. We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A. We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy. By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0.012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 0.0003) and the Bw4 Ile80 epitope (P = 0.0005). The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -0.1211), possibly as a consequence of their similar function in inhibiting NK cells. The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 0.0067) and with HLA-B Bw4 Ile80 (P = 0.0027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 0.0044). Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections. Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate

  5. High effectiveness of triptolide, an active diterpenoid triepoxide, in suppressing Kir-channel currents from human glioma cells.

    PubMed

    So, Edmund Cheung; Lo, Yi-Ching; Chen, Li-Tzong; Kao, Chin-An; Wu, Sheng-Nan

    2014-09-01

    Triptolide (Trip), a diterpene triepoxide isolated from medicinal vine Trypterygium wilfordii Hook. F. possessed multiple biological activities including antineoplastic actions. However, no report concerning its effects on ion currents has been published. In this study, we attempted to determine whether this compound has any effects on ion currents in malignant glioma cells. The mRNA expression of KCNJ10 (Kir4.1) was detected in U373 glioma cells. The inwardly rectifying K(+) currents (IK(IR)) in U373 cells were almost fully blocked by BaCl2 (1mM). Trip (30 nM-10 μM) effectively decreased the amplitude of IK(IR) in a concentration-dependent manner with an IC50 value of 0.72 μM. In chlorotoxin-treated U373 cells, Trip-mediated block of IK(IR) remained effective. Addition of Trip (3 μM) slightly inhibited the amplitude of Ca(2+)-activated K(+) current and sustained K(+) outward current in U373 cells. In cell-attached configuration, when Trip was added to the bath, the activity of inwardly rectifying K(+) (Kir) channels diminished with no change in single-channel conductance. Its suppression of Kir channels was accompanied by a reduction in the slow component of mean open time. Under current-clamp conditions, addition of Trip depolarized the membrane along with changes in frequency histogram of resting potential. Block by this component of Kir4.1 channels may be an important mechanism underlying its actions on the functional activity of glioma cells. Targeting at Kir4.1 channels may be clinically useful as an adjunctive regimen to anti-cancer drugs.

  6. ATP Sensitive Potassium Channels in the Skeletal Muscle Function: Involvement of the KCNJ11(Kir6.2) Gene in the Determination of Mechanical Warner Bratzer Shear Force

    PubMed Central

    Tricarico, Domenico; Selvaggi, Maria; Passantino, Giuseppe; De Palo, Pasquale; Dario, Cataldo; Centoducati, Pasquale; Tateo, Alessandra; Curci, Angela; Maqoud, Fatima; Mele, Antonietta; Camerino, Giulia M.; Liantonio, Antonella; Imbrici, Paola; Zizzo, Nicola

    2016-01-01

    The ATP-sensitive K+-channels (KATP) are distributed in the tissues coupling metabolism with K+ ions efflux. KATP subunits are encoded by KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1), and ABCC9 (SUR2) genes, alternative RNA splicing give rise to SUR variants that confer distinct physiological properties on the channel. An high expression/activity of the sarco-KATP channel is observed in various rat fast-twitch muscles, characterized by elevated muscle strength, while a low expression/activity is observed in the slow-twitch muscles characterized by reduced strength and frailty. Down-regulation of the KATP subunits of fast-twitch fibers is found in conditions characterized by weakness and frailty. KCNJ11 gene knockout mice have reduced glycogen, lean phenotype, lower body fat, and weakness. KATP channel is also a sensor of muscle atrophy. The KCNJ11 gene is located on BTA15, close to a QTL for meat tenderness, it has also a role in glycogen storage, a key mechanism of the postmortem transformation of muscle into meat. The role of KCNJ11 gene in muscle function may underlie an effect of KCNJ11 genotypes on meat tenderness, as recently reported. The fiber phenotype and genotype are important in livestock production science. Quantitative traits including meat production and quality are influenced both by environment and genes. Molecular markers can play an important role in the genetic improvement of animals through breeding strategies. Many factors influence the muscle Warner-Bratzler shear force including breed, age, feeding, the biochemical, and functional parameters. The role of KCNJ11gene and related genes on muscle tenderness will be discussed in the present review. PMID:27242541

  7. Unconventional role of the inwardly rectifying potassium channel Kir2.2 as a constitutive activator of RelA in cancer.

    PubMed

    Lee, Inkyoung; Lee, Sook-Ja; Kang, Tong Mook; Kang, Won Ki; Park, Chaehwa

    2013-02-01

    The constitutive activation of NF-κB is a major event leading to the initiation, development, and progression of cancer. Recently, we showed that the size of preestablished tumors was reduced after the depletion of Kir2.2, an inwardly rectifying potassium channel. To determine the precise mechanism of action of Kir2.2 in the control of tumor growth, we searched for interacting proteins. Notably, NF-κB p65/RelA was identified as a binding partner of Kir2.2 in a yeast two-hybrid analysis. Further analyses revealed that Kir2.2 directly interacted with RelA in vitro and coimmunoprecipitated with RelA from cell lysates. Kir2.2 increased RelA phosphorylation at S536 and facilitated its translocation from the cytoplasm to the nucleus, thereby activating the transcription factor and increasing the expression level of NF-κB targets, including cyclin D1, matrix metalloproteinase (MMP)9, and VEGF. Kir2.2 was overexpressed in human cancer and the expression level was correlated with increased colony formation and tumor growth in mouse tumor models. On the basis of these findings, we propose an unconventional role for Kir2.2 as a constitutive RelA-activating protein, which is likely to contribute to tumor progression in vivo. PMID:23269273

  8. Genotypic diversity of the Killer Cell Immunoglobulin-like Receptors (KIR) and their HLA class I Ligands in a Saudi population

    PubMed Central

    Omar, Suliman Y. Al; Alkuriji, Afrah; Alwasel, Saleh; Dar, javid Ahmed; Alhammad, Alwaleed; Christmas, Stephen; Mansour, Lamjed

    2016-01-01

    Abstract Killer Cell Immunoglobulin-like Receptors (KIR) have been used as good markers for the study of genetic predisposition in many diseases and in human genetic population dynamics. In this context, we have investigated the genetic diversity of KIR genes and their main HLA class I ligands in Saudi population and compared the data with other studies of neighboring populations. One hundred and fourteen randomly selected healthy Saudi subjects were genotyped for the presence or absence of 16 KIR genes and their HLA-C1, -C2, -Bw4Thr80 and Bw4Ile80 groups, using a PCR-SSP technique. The results show the occurrence of the framework genes (3DL2, 3DL3 and 2DL4) and the pseudogenes (2DP1 and 3DP1) at highest frequencies. All inhibitory KIR (iKIR) genes appeared at higher frequencies than activating genes (aKIR), except for 2DS4 with a frequency of 90.35%. A total of 55 different genotypes were observed appearing at different frequencies, where 12 are considered novel. Two haplotypes were characterized, AA and Bx (BB and AB), which were observed in 24.5% and 75.5% respectively of the studied group. The frequencies of iKIR + HLA associations were found to be much higher than aKIR + HLA. KIR genes frequencies in the Saudi population are comparable with other Middle Eastern and North African populations. PMID:27007893

  9. Genotypic diversity of the Killer Cell Immunoglobulin-like Receptors (KIR) and their HLA class I Ligands in a Saudi population.

    PubMed

    Omar, Suliman Y Al; Alkuriji, Afrah; Alwasel, Saleh; Dar, Javid Ahmed; Alhammad, Alwaleed; Christmas, Stephen; Mansour, Lamjed

    2016-03-01

    Killer Cell Immunoglobulin-like Receptors (KIR) have been used as good markers for the study of genetic predisposition in many diseases and in human genetic population dynamics. In this context, we have investigated the genetic diversity of KIR genes and their main HLA class I ligands in Saudi population and compared the data with other studies of neighboring populations. One hundred and fourteen randomly selected healthy Saudi subjects were genotyped for the presence or absence of 16 KIR genes and their HLA-C1, -C2, -Bw4Thr80 and Bw4Ile80 groups, using a PCR-SSP technique. The results show the occurrence of the framework genes (3DL2, 3DL3 and 2DL4) and the pseudogenes (2DP1 and 3DP1) at highest frequencies. All inhibitory KIR (iKIR) genes appeared at higher frequencies than activating genes (aKIR), except for 2DS4 with a frequency of 90.35%. A total of 55 different genotypes were observed appearing at different frequencies, where 12 are considered novel. Two haplotypes were characterized, AA and Bx (BB and AB), which were observed in 24.5% and 75.5% respectively of the studied group. The frequencies of iKIR + HLA associations were found to be much higher than aKIR + HLA. KIR genes frequencies in the Saudi population are comparable with other Middle Eastern and North African populations.

  10. HLA Class I and KIR Genes Do Not Protect Against HIV Type 1 Infection in Highly Exposed Uninfected Individuals With Hemophilia A

    PubMed Central

    Vince, Nicolas; Bashirova, Arman A.; Lied, Alexandra; Gao, Xiaojiang; Dorrell, Lucy; McLaren, Paul J.; Fellay, Jacques; Carrington, Mary

    2014-01-01

    A recent genome-wide association study (GWAS) involving patients with hemophilia A who were exposed to but uninfected with human immunodeficiency virus type 1 (HIV-1) did not reveal genetic variants associated with resistance to HIV-1 infection, beyond homozygosity for CCR5-Δ32. Since variation in HLA class I and KIR genes is not well interrogated by standard GWAS techniques, we tested whether these 2 loci were involved in protection from HIV-1 infection in the same hemophilia cohort, using controls from the general population. Our data indicate that HLA class I alleles, presence or absence of KIR genes, and functionally relevant combinations of the HLA/KIR genotypes are not involved in resistance to parenterally transmitted HIV-1 infection. PMID:24719475

  11. Interaction of KIR genes and G1M immunoglobulin allotypes confer susceptibility to type 2 diabetes in Puerto Rican Americans.

    PubMed

    Zuniga, Joaquin; Romero, Viviana; Azocar, Jose; Stern, Joel N H; Clavijo, Olga; Almeciga, Ingrid; Encinales, Liliana; Avendano, Angel; Fridkis-Hareli, Masha; Pandey, Janardan P; Yunis, Edmond J

    2006-11-01

    The susceptibility to type 2 diabetes (T2D) involves genetic factors. We studied the distribution of KIR and MHC class I ligands phenotype and genotype frequencies, as well as immunoglobulin KM and GM allotype frequencies in a group of patients (N = 95) with T2D and ethnically matched healthy controls (N = 74) with Puerto Rican ethnic background. We found a slight increase of the 2DL3/2DL3 homozygous genotype in T2D. Moreover, the association between 2DL3/2DL3 genotype was significant in the presence of 2DS4 (pC = 0.01). Also, we observed an epistatic effect of the interaction of 2DL3/2DL3, 2DS4 with allele z of G1M in T2D (pC = 0.004, OR = 3.60, 95% CI, 1.62-8.10). This genetic interaction between KIR and G1M allotypes, associated with T2D, was also significant by multiple logistic regression analysis (p < 0.0001, OR = 4.90, 95% CI, 2.12-11.3). We did not detect population stratification using unlinked short tandem repeat (STR) markers, demonstrating that the patients and controls were ethnically matched. Hence, we have demonstrated in this study an epistatic interaction between KIR genes and the G1M allotype that influences the susceptibility to T2D in Puerto Rican Americans. Our findings are important for understanding the autoimmune or innate immune inflammatory-mediated mechanisms involved in the pathogenesis of T2D.

  12. KIR and HLA Genotypes Implicated in Reduced Killer Lymphocytes Immunity Are Associated with Vogt-Koyanagi-Harada Disease

    PubMed Central

    Levinson, Ralph D.; Yung, Madeline; Meguro, Akira; Ashouri, Elham; Yu, Fei; Mizuki, Nobuhisa; Ohno, Shigeaki

    2016-01-01

    Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are killer lymphocytes that provide defense against viral infections and tumor transformation. Analogous to that of CTL, interactions of killer-cell immunoglobulin-like receptors (KIR) with specific human leukocyte antigen (HLA) class I ligands calibrate NK cell education and response. Gene families encoding KIRs and HLA ligands are located on different chromosomes, and feature variation in the number and type of genes. The independent segregation of KIR and HLA genes results in variable KIR-HLA interactions in individuals, which may impact disease susceptibility. We tested whether KIR-HLA combinations are associated with Vogt-Koyanagi-Harada (VKH) disease, a bilateral granulomatous panuveitis that has strong association with HLA-DR4. We present a case control study of 196 VKH patients and 209 controls from a highly homogeneous native population of Japan. KIR and HLA class I genes were typed using oligonucleotide hybridization method and analyzed using two-tailed Fisher’s exact probabilities. The incidence of Bx-KIR genotypes was decreased in VKH patients (odds ratio [OR] 0.58, P = 0.007), due primarily to a decrease in centromeric B-KIR motif and its associated KIRs 2DS2, 2DL2, 2DS3, and 2DL5B. HLA-B22, implicated in poor immune response, was increased in VKH (OR = 4.25, P = 0.0001). HLA-Bw4, the ligand for KIR3DL1, was decreased in VKH (OR = 0.59, P = 0.01). The KIR-HLA combinations 2DL2+C1/C2 and 3DL1+Bw4, which function in NK education, were also decreased in VKH (OR = 0.49, P = 0.012; OR = 0.59, P = 0.013). Genotypes missing these two inhibitory KIR-HLA combinations in addition to missing activating KIRs 2DS2 and 2DS3 were more common in VKH (OR = 1.90, P = 0.002). These results suggest that synergistic hyporesponsiveness of NK cells (due to poor NK education along with missing of activating KIRs) and CTL (due to HLA-B22 restriction) fail to mount an effective immune response against viral

  13. NK Cells Expressing the Inhibitory Killer Immunoglobulin-Like Receptors (iKIR) KIR2DL1, KIR2DL3 and KIR3DL1 Are Less Likely to Be CD16+ than Their iKIR Negative Counterparts

    PubMed Central

    Lisovsky, Irene; Bruneau, Julie; Lebouché, Bertrand; Routy, Jean-Pierre; Bernard, Nicole F.

    2016-01-01

    Natural Killer (NK) cell education, which requires the engagement of inhibitory NK cell receptors (iNKRs) by their ligands, is important for generating self-tolerant functional NK cells. While the potency of NK cell education is directly related to their functional potential upon stimulation with HLA null cells, the influence of NK cell education on the potency of the antibody dependent cellular cytotoxicity (ADCC) function of NK cells is unclear. ADCC occurs when the Fc portion of an immunoglobulin G antibody bridges the CD16 Fc receptor on NK cells and antigen on target cells, resulting in NK cell activation, cytotoxic granule release, and target cell lysis. We previously reported that education via the KIR3DL1/HLA-Bw4 iNKR/HLA ligand combination supported higher KIR3DL1+ than KIR3DL1- NK cell activation levels but had no impact on ADCC potency measured as the frequency of granzyme B positive (%GrB+) targets generated in an ADCC GranToxiLux assay. A lower frequency of KIR3DL1+ compared to KIR3DL1- NK cells were CD16+, which may in part explain the discrepancy between NK cell activation and target cell effects. Here, we investigated the frequency of CD16+ cells among NK cells expressing other iNKRs. We found that CD16+ cells were significantly more frequent among NK cells negative for the inhibitory KIR (iKIR) KIR2DL1, KIR2DL3, and KIR3DL1 than those positive for any one of these iKIR to the exclusion of the others, making iKIR+ NK cells poorer ADCC effectors than iKIR- NK cells. The education status of these iKIR+ populations had no effect on the frequency of CD16+ cells. PMID:27732638

  14. Kir6.2 activation by sulfonylurea receptors: a different mechanism of action for SUR1 and SUR2A subunits via the same residues

    PubMed Central

    Principalli, Maria A; Dupuis, Julien P; Moreau, Christophe J; Vivaudou, Michel; Revilloud, Jean

    2015-01-01

    ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K+ channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels. PMID:26416970

  15. Cloning and characterization of a novel dehydrin gene, SiDhn2, from Saussurea involucrata Kar. et Kir.

    PubMed

    Qiu, Honglin; Zhang, Linhua; Liu, Chao; He, Li; Wang, Aiying; Liu, Hai-Liang; Zhu, Jian-Bo

    2014-04-01

    Saussurea involucrata Kar. et Kir. is a hardy dicotyledonous plant capable of tolerating severe abiotic stress conditions. In a previous study, we created a cDNA library to determine what factors are associated with the cold acclimation response in S. involucrata. From this, a full-length cDNA of a dehydrin-like gene (SiDhn2) was obtained by RT-PCR. The SiDhn2 gene was characterized in this study. The full-length SiDhn2 cDNA comprised 693 bp containing an open reading frame of 345 bp specifying a protein of 115 amino acids. An alignment of the deduced amino acid sequence showed that SiDhn2 shared 55 % identity with two Brassica dehydrins. Agrobacterium tumefaciens was used to transform RD29A:SiDhn2 and 35S:SiDhn2 constructs into tobacco to investigate the germination and resistance to freezing and drought stress of transgenic plants. The RD29A:SiDhn2 transgenic plants showed greater resistance to freezing and drought stress than 35S:SiDhn2 transgenic plants or the wild-type. This study demonstrates that SiDhn2 confers cold hardiness and drought resistance, and may be a candidate resistance gene for genetic improvement of crops to increase stress resistance. PMID:24337866

  16. Polymorphic HLA-C receptors balance the functional characteristics of KIR haplotypes

    PubMed Central

    Hilton, Hugo G.; Guethlein, Lisbeth A.; Goyos, Ana; Nemat-Gorgani, Neda; Bushnell, David A.; Norman, Paul J.; Parham, Peter

    2015-01-01

    The human killer cell immunoglobulin-like receptor (KIR) locus comprises two groups of KIR haplotypes, termed A and B. These are present in all human populations but with different relative frequencies, suggesting they have different functional properties that underlie their balancing selection. We studied the genomic organization and functional properties of the alleles of the inhibitory and activating HLA-C receptors encoded by KIR haplotypes. Because every HLA-C allotype functions as a ligand for KIR, the interactions between KIR and HLA-C dominate the HLA class I mediated regulation of human NK cells. The C2 epitope is recognized by inhibitory KIR2DL1 and activating KIR2DS1, whereas the C1 epitope is recognized by inhibitory KIR2DL2 and KIR2DL3. This study shows that the KIR2DL1 and 2DS1 and KIR2DL2/3 alleles form distinctive phylogenetic clades that associate with specific KIR haplotypes. KIR A haplotypes are characterized by KIR2DL1 alleles that encode strong inhibitory C2 receptors and KIR2DL3 alleles encoding weak inhibitory C1 receptors. In striking contrast, KIR B haplotypes are characterized by KIR2DL1 alleles that encode weak inhibitory C2 receptors and KIR2DL2 alleles encoding strong inhibitory C1 receptors. The wide-ranging properties of KIR allotypes arise from substitutions throughout the KIR molecule. Such substitutions can influence cell-surface expression, as well as the avidity and specificity for HLA-C ligands. Consistent with the crucial role of inhibitory HLA-C receptors in self-recognition, and natural killer cell education and response, most KIR haplotypes have both a functional C1 and C2 receptor, despite the considerable variation that occurs in ligand recognition and surface expression. PMID:26311903

  17. Susceptibility to Crohn's disease is mediated by KIR2DL2/KIR2DL3 heterozygosity and the HLA-C ligand.

    PubMed

    Hollenbach, Jill A; Ladner, Martha B; Saeteurn, Koy; Taylor, Kent D; Mei, Ling; Haritunians, Talin; McGovern, Dermot P B; Erlich, Henry A; Rotter, Jerome I; Trachtenberg, Elizabeth A

    2009-10-01

    In the present study, we investigated the relationship between the KIR loci and the genes encoding their HLA ligands and genetic susceptibility to Crohn's disease (CD). Analyses of the interactions between KIR3DL1, KIR2DL1, KIR2DL2, and KIR2DL3 with their respective HLA ligands indicate that there is a protective effect for KIR2DL2 in the absence of its HLA ligand C1. Given that KIR2DL2 and KIR2DL3 segregate as alleles, we compared their genotypic distributions to expectations under Hardy-Weinberg Equilibrium (HWE) with regard to the HLA ligand C1 status. While all the genotypic distributions conform to expectations under HWE in controls, in C2 ligand homozygous cases there is significant deviation from HWE, with a reduction of KIR2DL2, KIR2DL3 heterozygotes. KIR2DL2, KIR2DL3 heterozygosity is the only genotypic combination that confers protection from CD. In addition to the protective effect (OR = 0.44, CI = 0.22-0.87; p = 0.018) observed in C2 ligand homozygotes, the KIR2DL2, KIR2DL3 genotype is predisposing (OR = 1.34, CI = 1.03-4.53; p = 0.031) in the presence of C1 ligand. A test for trend of HLA class I C ligand group genotypes with KIR2DL2, KIR2DL3 heterozygosity in cases and controls indicates that C1, C2 ligand group heterozygotes have an intermediate effect on predisposition. These results show for the first time that disease susceptibility may be related to heterozygosity at a specific KIR locus, and that HLA ligand genotype influences the relative effect of the KIR genotype.

  18. Both the nature of KIR3DL1 alleles and the KIR3DL1/S1 allele combination affect the KIR3DL1 NK-cell repertoire in the French population.

    PubMed

    Gagne, Katia; Willem, Catherine; Legrand, Nolwenn; Djaoud, Zakia; David, Gaëlle; Rettman, Pauline; Bressollette-Bodin, Céline; Senitzer, David; Esbelin, Julie; Cesbron-Gautier, Anne; Schneider, Thierry; Retière, Christelle

    2013-04-01

    NK-cell functions are regulated by many activating and inhibitory receptors including KIR3DL1. Extensive allelic polymorphism and variability in expression can directly alter NK-cell phenotype and functions. Here we investigated the KIR3DL1(+) NK-cell repertoire, taking into account the allelic KIR3DL1/S1 polymorphism, KIR3DL1 phenotype, and function. All 109 studied individuals possessed at least one KIR3DL1 allele, with weak KIR3DL1*054, or null alleles being frequently present. In KIR3DL1(high/null) individuals, we observed a bimodal distribution of KIR3DL1(+) NK cells identified by a different KIR3DL1 expression level and cell frequency regardless of a similar amount of both KIR3DL1 transcripts, HLA background, or KIR2D expression. However, this bimodal distribution can be explained by a functional selection following a hierarchy of KIR3DL1 receptors. The higher expression of KIR3DL1 observed on cord blood NK cells suggests the expression of the functional KIR3DL1*004 receptors. Thus, the low amplification of KIR3DL1(high) , KIR3DL1*004 NK-cell subsets during development may be due to extensive signaling via these two receptors. Albeit in a nonexclusive manner, individual immunological experience may contribute to shaping the KIR3DL1 NK-cell repertoire. Together, this study provides new insight into the mechanisms regulating the KIR3DL1 NK-cell repertoire.

  19. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse.

    PubMed

    Hong, Seung Hwa; Kyeong, Kyu-Sang; Kim, Chan Hyung; Kim, Young Chul; Choi, Woong; Yoo, Ra Young; Kim, Hun Sik; Park, Yeon Jin; Ji, Il Woon; Jeong, Eun-Hwan; Kim, Hak Soon; Xu, Wen-Xie; Lee, Sang Jin

    2016-08-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2.

  20. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse

    PubMed Central

    HONG, Seung Hwa; KYEONG, Kyu-Sang; KIM, Chan Hyung; KIM, Young Chul; CHOI, Woong; YOO, Ra Young; KIM, Hun Sik; PARK, Yeon Jin; JI, Il Woon; JEONG, Eun-Hwan; KIM, Hak Soon; XU, Wen-Xie; LEE, Sang Jin

    2016-01-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2. PMID:27086859

  1. Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse.

    PubMed

    Hong, Seung Hwa; Kyeong, Kyu-Sang; Kim, Chan Hyung; Kim, Young Chul; Choi, Woong; Yoo, Ra Young; Kim, Hun Sik; Park, Yeon Jin; Ji, Il Woon; Jeong, Eun-Hwan; Kim, Hak Soon; Xu, Wen-Xie; Lee, Sang Jin

    2016-08-01

    ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2. PMID:27086859

  2. Killer cell immunoglobulin-like receptor (KIR) locus profiles in the Tunisian population.

    PubMed

    Meriem, Bani; Jihen, Seket; Houda, Kaabi; Ghaya, Cherif; Manel, Chaabane; Hedi, Bellali; Slama, Hmida

    2015-05-01

    Killer cell immunoglobulin-like receptors (KIRs) are a family of inhibitory and activatory receptors that are expressed by most natural killer (NK) cells. The KIR gene family is polymorphic: genomic diversity is achieved through differences in gene content and allelic polymorphism. The number of KIR loci has been reported to vary among individuals, resulting in different KIR haplotypes. In this study we report the genotypic structure of KIRs in 267 unrelated and healthy Tunisian subjects by polymerase chain reaction-sequence-specific primer (PCR-SSP) method. All 16 KIR genes were observed in the population with different frequencies; framework genes KIR3DP1 and KIR3DL2 and the nonframework genes KIR2DL1 and KIR2DP1 were present in all individuals. A total of 26 different KIR gene profiles and 54 subgenotypes were observed in the tested population samples. Genotype 1, with a frequency of 36.6%, is the most commonly observed in the Tunisian population. Our results showed that the Tunisian population possesses the previously reported general features of the Caucasian as well as African populations, with some additional interesting differences. Such knowledge of the KIR gene distribution in populations is very useful in the study of associations with diseases and in selection of donors for haploidentical bone marrow transplantation.

  3. Direct Regulation of Prokaryotic Kir Channel by Cholesterol*

    PubMed Central

    Singh, Dev K.; Rosenhouse-Dantsker, Avia; Nichols, Colin G.; Enkvetchakul, Decha; Levitan, Irena

    2009-01-01

    Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by 86Rb+ uptake. Our results show that 86Rb+ flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses 86Rb+ flux by >50%, and activity is completely inhibited at 12–15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated 86Rb+ uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 μg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific. PMID:19740741

  4. A High-Throughput Electrophysiology Assay Identifies Inhibitors of the Inwardly Rectifying Potassium Channel Kir7.1.

    PubMed

    Wright, Paul D; Kanumilli, Srinivasan; Tickle, David; Cartland, Jamie; Bouloc, Nathalie; Dale, Timothy; Tresize, Derek J; McCloskey, Conor; McCavera, Samantha; Blanks, Andrew M; Kettleborough, Catherine; Jerman, Jeffrey C

    2015-07-01

    Kir7.1 is an inwardly rectifying potassium channel that has been implicated in controlling the resting membrane potential of the myometrium. Abnormal uterine activity in pregnancy plays an important role in postpartum hemorrhage, and novel therapies for this condition may lie in manipulation of membrane potential. This work presents an assay development and screening strategy for identifying novel inhibitors of Kir7.1. A cell-based automated patch-clamp electrophysiology assay was developed using the IonWorks Quattro (Molecular Devices, Sunnyvale, CA) system, and the iterative optimization is described. In total, 7087 compounds were tested, with a hit rate (>40% inhibition) of 3.09%. During screening, average Z' values of 0.63 ± 0.09 were observed. After chemistry triage, lead compounds were resynthesized and activity confirmed by IC50 determinations. The most potent compound identified (MRT00200769) gave rise to an IC50 of 1.3 µM at Kir7.1. Compounds were assessed for selectivity using the inwardly rectifying potassium channel Kir1.1 (ROMK) and hERG (human Ether-à-go-go Related Gene). Pharmacological characterization of known Kir7.1 inhibitors was also carried out and analogues of VU590 tested to assess selectivity at Kir7.1.

  5. Inhibition of Cardiac Kir Current (IK1) by Protein Kinase C Critically Depends on PKCβ and Kir2.2

    PubMed Central

    Scherer, Daniel; Seyler, Claudia; Xynogalos, Panagiotis; Scholz, Eberhard P.; Thomas, Dierk; Backs, Johannes; Andrassy, Martin; Völkers, Mirko; Karle, Christoph A.; Katus, Hugo A.; Zitron, Edgar

    2016-01-01

    Background Cardiac inwardly rectifying Kir current (IK1) mediates terminal repolarisation and is critical for the stabilization of the diastolic membrane potential. Its predominant molecular basis in mammalian ventricle is heterotetrameric assembly of Kir2.1 and Kir2.2 channel subunits. It has been shown that PKC inhibition of IK1 promotes focal ventricular ectopy. However, the underlying molecular mechanism has not been fully elucidated to date. Methods and Results In the Xenopus oocyte expression system, we observed a pronounced PKC-induced inhibition of Kir2.2 but not Kir2.1 currents. The PKC regulation of Kir2.2 could be reproduced by an activator of conventional PKC isoforms and antagonized by pharmacological inhibition of PKCβ. In isolated ventricular cardiomyocytes (rat, mouse), pharmacological activation of conventional PKC isoforms induced a pronounced inhibition of IK1. The PKC effect in rat ventricular cardiomyocytes was markedly attenuated following co-application of a small molecule inhibitor of PKCβ. Underlining the critical role of PKCβ, the PKC-induced inhibition of IK1 was absent in homozygous PKCβ knockout-mice. After heterologous expression of Kir2.1-Kir2.2 concatemers in Xenopus oocytes, heteromeric Kir2.1/Kir2.2 currents were also inhibited following activation of PKC. Conclusion We conclude that inhibition of cardiac IK1 by PKC critically depends on the PKCβ isoform and Kir2.2 subunits. This regulation represents a potential novel target for the antiarrhythmic therapy of focal ventricular arrhythmias. PMID:27214373

  6. The Structure of the Atypical Killer Cell Immunoglobulin-like Receptor, KIR2DL4*

    PubMed Central

    Moradi, Shoeib; Berry, Richard; Pymm, Phillip; Hitchen, Corinne; Beckham, Simone A.; Wilce, Matthew C. J.; Walpole, Nicholas G.; Clements, Craig S.; Reid, Hugh H.; Perugini, Matthew A.; Brooks, Andrew G.; Rossjohn, Jamie; Vivian, Julian P.

    2015-01-01

    The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor. PMID:25759384

  7. Regulation of Aldosterone Biosynthesis by the Kir3.4 (KCNJ5) Potassium Channel

    PubMed Central

    Velarde-Miranda, Carolina; Gomez-Sanchez, Elise P.; Gomez-Sanchez, Celso E.

    2013-01-01

    Summary The G-protein-activated inwardly rectifying potassium channel Kir3.4 is expressed in the zona glomerulosa cell membrane and transports potassium out of the cell. Angiotensin II stimulation of aldosterone secretion is mediated in part by suppression of the transcription of KCNJ5, the gene coding for Kir3.4, and blocking channel activity. This results in membrane depolarization, mobilization of intracellular calcium, activation of the calcium-calmodulin pathway, and increasing gene transcription of steroidogenic enzymes required for aldosterone secretion. In 40–60% of aldosterone-producing adenomas there is a somatic mutation in the region of the KCNJ5 gene that codes for the selectivity filter that decreases potassium selectivity, allowing sodium to leak into the cells, thus depolarizing the membrane and initiating events that result in increased aldosterone synthesis. The mechanism by which mutated KCNJ5 induces cell proliferation and adenoma formation remains unclear. PMID:23829355

  8. Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: A novel role in egg production

    PubMed Central

    Raphemot, Rene; Estévez-Lao, Tania Y.; Rouhier, Matthew F.; Piermarini, Peter M.; Denton, Jerod S.; Hillyer, Julián F.

    2014-01-01

    Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical RIP features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood in mosquito fecundity, and further validates them as promising molecular targets for the meal, but decreased their egg output. These data provide evidence for a new role of Kir channels development of a new class of mosquitocides to be used in vector control. PMID:24855023

  9. 5-Hydroxytryptamine1A receptor-activation hyperpolarizes pyramidal cells and suppresses hippocampal gamma oscillations via Kir3 channel activation

    PubMed Central

    Johnston, April; McBain, Chris J; Fisahn, André

    2014-01-01

    Rhythmic cortical neuronal oscillations in the gamma frequency band (30–80 Hz, gamma oscillations) have been associated with cognitive processes such as sensory perception and integration, attention, learning, and memory. Gamma oscillations are disrupted in disorders for which cognitive deficits are hallmark symptoms such as schizophrenia and Alzheimer's disease. In vitro, various neurotransmitters have been found to modulate gamma oscillations. Serotonin (5-HT) has long been known to be important for both behavioural and cognitive functions such as learning and memory. Multiple 5-HT receptor subtypes are expressed in the CA3 region of the hippocampus and high doses of 5-HT reduce the power of induced gamma oscillations. Hypothesizing that 5-HT may have cell- and receptor subtype-specific modulatory effects, we investigated the receptor subtypes, cell types and cellular mechanisms engaged by 5-HT in the modulation of gamma oscillations in mice and rats. We found that 5-HT decreases the power of kainate-induced hippocampal gamma oscillations in both species via the 5-HT1A receptor subtype. Whole-cell patch clamp recordings demonstrated that this decrease was caused by a hyperpolarization of CA3 pyramidal cells and a reduction of their firing frequency, but not by alteration of inhibitory neurotransmission. Finally, our results show that the effect on pyramidal cells is mediated via the G protein-coupled receptor inwardly rectifying potassium channel Kir3. Our findings suggest this novel cellular mechanism as a potential target for therapies that are aimed at alleviating cognitive decline by helping the brain to maintain or re-establish normal gamma oscillation levels in neuropsychiatric and neurodegenerative disorders. PMID:25107925

  10. Human NK cells maintain licensing status and are subject to killer immunoglobulin-like receptor (KIR) and KIR-ligand inhibition following ex vivo expansion.

    PubMed

    Wang, Wei; Erbe, Amy K; Alderson, Kory A; Phillips, Emily; Gallenberger, Mikayla; Gan, Jacek; Campana, Dario; Hank, Jacquelyn A; Sondel, Paul M

    2016-09-01

    Infusion of allogeneic NK cells is a potential immunotherapy for both hematopoietic malignancies and solid tumors. Interactions between killer immunoglobulin-like receptors (KIR) on human NK cells and KIR-ligands on tumor cells influence the magnitude of NK function. To obtain sufficient numbers of activated NK cells for infusion, one potent method uses cells from the K562 human erythroleukemia line that have been transfected to express activating 41BB ligand (41BBL) and membrane-bound interleukin 15 (mbIL15). The functional importance of KIRs on ex vivo expanded NK cells has not been studied in detail. We found that after a 12-day co-culture with K562-mbIL15-41BBL cells, expanded NK cells maintained inhibition specificity and prior in vivo licensing status determined by KIR/KIR-ligand interactions. Addition of an anti-CD20 antibody (rituximab) induced NK-mediated antibody-dependent cellular cytotoxicity and augmented killing of CD20+ target cells. However, partial inhibition induced by KIR/KIR-ligand interactions persisted. Finally, we found that extended co-cultures of NK cells with stimulatory cells transduced to express various KIR-ligands modified both the inhibitory and activating KIR repertoires of the expanded NK cell product. These studies demonstrate that the licensing interactions known to occur during NK ontogeny also influence NK cell function following NK expansion ex vivo with HLA-null stimulatory cells. PMID:27392940

  11. Killer-cell immunoglobulin-like receptor genes and ligands and their role in hematologic malignancies.

    PubMed

    Varbanova, Viktoria; Naumova, Elissaveta; Mihaylova, Anastasiya

    2016-04-01

    Natural killer (NK) cells are considered crucial for the elimination of emerging tumor cells. Effector NK-cell functions are controlled by interactions of inhibitory and activating killer-cell immunoglobulin-like receptors (KIRs) on NK cells with human leukocyte antigen (HLA) class I ligands on target cells. KIR and HLA are highly polymorphic genetic systems segregating independently, creating a great diversity in KIR/HLA gene profiles in different individuals. There is an increasing evidence supporting the relevance of KIR and HLA ligand gene background for the occurrence and outcome of certain cancers. However, the data are still controversial and the mechanisms of receptor-ligand mediated NK-cell action remain unclear. Here, the main characteristics and functions of KIRs and their HLA class I ligands are reviewed. In addition, we review the HLA and KIR correlations with different hematological malignancies and discuss our current understanding of the biological significance and mechanisms underlying these associations.

  12. Amazonian Amerindians exhibit high variability of KIR profiles.

    PubMed

    Ewerton, Paloma Daguer; Leite, Mauro de Meira; Magalhães, Milena; Sena, Leonardo; Melo dos Santos, Eduardo José

    2007-08-01

    Natural killer cell immunoglobulin-like receptors (KIRs) mediate cell lysis through the recognition of human leukocyte antigen class I complexes in target cells, playing an important role in innate immune response. In this context, disease-based selective pressures could be relevant, leaving signatures detected by population studies. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania, and Africa remain poorly studied. The aim of this study was to analyze the variability of KIR genes in Amerindian tribes from the Amazon region to infer about their evolutionary history. KIR profiles were estimated in 40 individuals from six Amazonian Amerindian tribes using single specific primer polymerase chain reaction. Twenty-five different profiles were identified, and surprisingly, the haplogroup A frequency was the lowest observed in human populations (16%). Results showed also that KIR variability was higher in this group in contrast to Venezuelan Amerindians. Principal components analysis evidenced that Amerindians formed a separated group from other worldwide populations and showed a higher intraethnic differentiation in comparison to other ethnic groups. Such pattern may reflect small effective size and intense genetic drift. However, because of the role of KIR in immune response, selective pressures cannot be entirely ruled out.

  13. Interaction of Galphaq and Kir3, G protein-coupled inwardly rectifying potassium channels.

    PubMed

    Kawano, Takeharu; Zhao, Peng; Floreani, Christina V; Nakajima, Yasuko; Kozasa, Tohru; Nakajima, Shigehiro

    2007-04-01

    Activation of substance P receptors, which are coupled to Galpha(q), inhibits the Kir3.1/3.2 channels, resulting in neuronal excitation. We have shown previously that this channel inactivation is not caused by reduction of the phosphatidylinositol 4,5-bisphosphate level in membrane. Moreover, Galpha(q) immunoprecipitates with Kir3.2 (J Physiol 564:489-500, 2005), suggesting that Galpha(q) interacts with Kir3.2. Positive immunoprecipitation, however, does not necessarily indicate direct interaction between the two proteins. Here, the glutathione transferase pull-down assay was used to investigate interaction between Galpha(q) and the K(+) channels. We found that Galpha(q) interacted with N termini of Kir3.1, Kir3.2, and Kir3.4. However, Galpha(q) did not interact with the C terminus of any Kir3 or with the C or N terminus of Kir2.1. TRPC6 is regulated by the signal initiated by Galpha(q). Immunoprecipitation, however, showed that Galpha(q) did not interact with TRPC6. Thus, the interaction between Galpha(q) and the Kir3 N terminus is quite specific. This interaction occurred in the presence of GDP or GDP-AlF(-)(4). The Galpha(q) binding could take place somewhere between residues 51 to 90 of Kir3.2; perhaps the segment between 81 to 90 residues is crucial. Gbetagamma, which is known to bind to N terminus of Kir3, did not compete with Galpha(q) for the binding, suggesting that these two binding regions are different. These findings agree with the hypothesis (J Physiol 564:489-500, 2005) that the signal to inactivate the Kir3 channel could be mainly transmitted directly from Galpha(q) to Kir3. PMID:17296805

  14. The potential influence of KIR cluster profiles on disease patterns of Canadian Aboriginals and other indigenous peoples of the Americas

    PubMed Central

    Rempel, Julia D; Hawkins, Kim; Lande, Erin; Nickerson, Peter

    2011-01-01

    Genetic differences in immune regulators influence disease resistance and susceptibility patterns. There are major health discrepancies in immune-mediated diseases between Caucasians and Canadian Aboriginal people, as well as with other indigenous people of the Americas. Environmental factors offer a limited explanation as Aboriginal people also demonstrate a rare resistance to chronic hepatitis C virus infection. Killer immunoglobulin-like receptors (KIRs) are known modulators of viral responses and autoimmune diseases. The possibility that variation in KIR cluster profiles contribute to the health outcomes of Aboriginal people was evaluated with Canadian Caucasian (n=93, population controls) and Aboriginal (n=86) individuals. Relative to Caucasians, the Aboriginal KIR cluster displayed a greater immune activating phenotype associated with genes of the B haplotype situated within the telomeric region. In conjunction, there was a decrease in the genes of the B haplotype from the centromeric region. Caucasian and Aboriginal cohorts further demonstrated distinct genotype and haplotype relationships enforcing the disconnect between the B haplotype centromeric and telomeric regions within the Aboriginal population. Moreover, Caucasian KIR cluster patterns reflected studies of Caucasians globally, as well as Asians. In contrast, the unique pattern of the Canadian Aboriginal cohort mirrored the phenotype of other indigenous peoples of the Americas, but not that of Caucasians or Asians. Taken together, these data suggest that historically indigenous peoples of the Americas were subject to immune selection processes that could be influencing the current disease resistance and susceptibility patterns of their descendents. PMID:21731058

  15. Defining KIR and HLA Class I Genotypes at Highest Resolution via High-Throughput Sequencing.

    PubMed

    Norman, Paul J; Hollenbach, Jill A; Nemat-Gorgani, Neda; Marin, Wesley M; Norberg, Steven J; Ashouri, Elham; Jayaraman, Jyothi; Wroblewski, Emily E; Trowsdale, John; Rajalingam, Raja; Oksenberg, Jorge R; Chiaroni, Jacques; Guethlein, Lisbeth A; Traherne, James A; Ronaghi, Mostafa; Parham, Peter

    2016-08-01

    The physiological functions of natural killer (NK) cells in human immunity and reproduction depend upon diverse interactions between killer cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands: HLA-A, HLA-B, and HLA-C. The genomic regions containing the KIR and HLA class I genes are unlinked, structurally complex, and highly polymorphic. They are also strongly associated with a wide spectrum of diseases, including infections, autoimmune disorders, cancers, and pregnancy disorders, as well as the efficacy of transplantation and other immunotherapies. To facilitate study of these extraordinary genes, we developed a method that captures, sequences, and analyzes the 13 KIR genes and HLA-A, HLA-B, and HLA-C from genomic DNA. We also devised a bioinformatics pipeline that attributes sequencing reads to specific KIR genes, determines copy number by read depth, and calls high-resolution genotypes for each KIR gene. We validated this method by using DNA from well-characterized cell lines, comparing it to established methods of HLA and KIR genotyping, and determining KIR genotypes from 1000 Genomes sequence data. This identified 116 previously uncharacterized KIR alleles, which were all demonstrated to be authentic by sequencing from source DNA via standard methods. Analysis of just two KIR genes showed that 22% of the 1000 Genomes individuals have a previously uncharacterized allele or a structural variant. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations and defining the precise HLA and KIR factors associated with disease. The methods are applicable to other highly polymorphic genes. PMID:27486779

  16. Kiwifruit Information Resource (KIR): a comparative platform for kiwifruit genomics.

    PubMed

    Yue, Junyang; Liu, Jian; Ban, Rongjun; Tang, Wei; Deng, Lin; Fei, Zhangjun; Liu, Yongsheng

    2015-01-01

    The Kiwifruit Information Resource (KIR) is dedicated to maintain and integrate comprehensive datasets on genomics, functional genomics and transcriptomics of kiwifruit (Actinidiaceae). KIR serves as a central access point for existing/new genomic and genetic data. KIR also provides researchers with a variety of visualization and analysis tools. Current developments include the updated genome structure of Actinidia chinensis cv. Hongyang and its newest genome annotation, putative transcripts, gene expression, physical markers of genetic traits as well as relevant publications based on the latest genome assembly. Nine thousand five hundred and forty-seven new transcripts are detected and 21 132 old transcripts are changed. At the present release, the next-generation transcriptome sequencing data has been incorporated into gene models and splice variants. Protein-protein interactions are also identified based on experimentally determined orthologous interactions. Furthermore, the experimental results reported in peer-reviewed literature are manually extracted and integrated within a well-developed query page. In total, 122 identifications are currently associated, including commonly used gene names and symbols. All KIR datasets are helpful to facilitate a broad range of kiwifruit research topics and freely available to the research community. Database URL: http://bdg.hfut.edu.cn/kir/index.html. PMID:26656885

  17. Kiwifruit Information Resource (KIR): a comparative platform for kiwifruit genomics.

    PubMed

    Yue, Junyang; Liu, Jian; Ban, Rongjun; Tang, Wei; Deng, Lin; Fei, Zhangjun; Liu, Yongsheng

    2015-01-01

    The Kiwifruit Information Resource (KIR) is dedicated to maintain and integrate comprehensive datasets on genomics, functional genomics and transcriptomics of kiwifruit (Actinidiaceae). KIR serves as a central access point for existing/new genomic and genetic data. KIR also provides researchers with a variety of visualization and analysis tools. Current developments include the updated genome structure of Actinidia chinensis cv. Hongyang and its newest genome annotation, putative transcripts, gene expression, physical markers of genetic traits as well as relevant publications based on the latest genome assembly. Nine thousand five hundred and forty-seven new transcripts are detected and 21 132 old transcripts are changed. At the present release, the next-generation transcriptome sequencing data has been incorporated into gene models and splice variants. Protein-protein interactions are also identified based on experimentally determined orthologous interactions. Furthermore, the experimental results reported in peer-reviewed literature are manually extracted and integrated within a well-developed query page. In total, 122 identifications are currently associated, including commonly used gene names and symbols. All KIR datasets are helpful to facilitate a broad range of kiwifruit research topics and freely available to the research community. Database URL: http://bdg.hfut.edu.cn/kir/index.html.

  18. A KIR B centromeric region present in Africans but not Europeans protects pregnant women from pre-eclampsia.

    PubMed

    Nakimuli, Annettee; Chazara, Olympe; Hiby, Susan E; Farrell, Lydia; Tukwasibwe, Stephen; Jayaraman, Jyothi; Traherne, James A; Trowsdale, John; Colucci, Francesco; Lougee, Emma; Vaughan, Robert W; Elliott, Alison M; Byamugisha, Josaphat; Kaleebu, Pontiano; Mirembe, Florence; Nemat-Gorgani, Neda; Parham, Peter; Norman, Paul J; Moffett, Ashley

    2015-01-20

    In sub-Saharan Africans, maternal mortality is unacceptably high, with >400 deaths per 100,000 births compared with <10 deaths per 100,000 births in Europeans. One-third of the deaths are caused by pre-eclampsia, a syndrome arising from defective placentation. Controlling placentation are maternal natural killer (NK) cells that use killer-cell immunoglobulin-like receptor (KIR) to recognize the fetal HLA-C molecules on invading trophoblast. We analyzed genetic polymorphisms of maternal KIR and fetal HLA-C in 484 normal and 254 pre-eclamptic pregnancies at Mulago Hospital, Kampala, Uganda. The combination of maternal KIR AA genotypes and fetal HLA-C alleles encoding the C2 epitope associates with pre-eclampsia [P = 0.0318, odds ratio (OR) = 1.49]. The KIR genes associated with protection are located in centromeric KIR B regions that are unique to sub-Saharan African populations and contain the KIR2DS5 and KIR2DL1 genes (P = 0.0095, OR = 0.59). By contrast, telomeric KIR B genes protect Europeans against pre-eclampsia. Thus, different KIR B regions protect sub-Saharan Africans and Europeans from pre-eclampsia, whereas in both populations, the KIR AA genotype is a risk factor for the syndrome. These results emphasize the importance of undertaking genetic studies of pregnancy disorders in African populations with the potential to provide biological insights not available from studies restricted to European populations.

  19. ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels.

    PubMed

    Swale, Daniel R; Kurata, Haruto; Kharade, Sujay V; Sheehan, Jonathan; Raphemot, Rene; Voigtritter, Karl R; Figueroa, Eric E; Meiler, Jens; Blobaum, Anna L; Lindsley, Craig W; Hopkins, Corey R; Denton, Jerod S

    2016-07-20

    The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 μM and Tmax = 3 h) and favorable CNS distribution (mouse brain/plasma Kp of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo. PMID:27184474

  20. Identification of a Cholesterol-Binding Pocket in Inward Rectifier K+ (Kir) Channels

    PubMed Central

    Fürst, Oliver; Nichols, Colin G.; Lamoureux, Guillaume; D’Avanzo, Nazzareno

    2014-01-01

    Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket. PMID:25517146

  1. KIR haplotypes are associated with late-onset type 1 diabetes in European–American families

    PubMed Central

    Traherne, J A; Jiang, W; Valdes, A M; Hollenbach, J A; Jayaraman, J; Lane, J A; Johnson, C; Trowsdale, J; Noble, J A

    2016-01-01

    Classical human leukocyte antigens (HLA) genes confer the strongest, but not the only, genetic susceptibility to type 1 diabetes. Killer cell immunoglobulin-like receptors (KIR), on natural killer (NK) cells, bind ligands including class I HLA. We examined presence or absence, with copy number, of KIR loci in 1698 individuals, from 339 multiplex type 1 diabetes families, from the Human Biological Data Interchange, previously genotyped for HLA. Combining family data with KIR copy number information allowed assignment of haplotypes using identity by descent. This is the first disease study to use KIR copy number typing and unambiguously define haplotypes by gene transmission. KIR A1 haplotypes were positively associated with T1D in the subset of patients without the high T1D risk HLA genotype, DR3/DR4 (odds ratio=1.29, P=0.0096). The data point to a role for KIR in type 1 diabetes risk in late-onset patients. In the top quartile (age of onset>14), KIR A2 haplotype was overtransmitted (63.4%, odds ratio=1.73, P=0.024) and KIR B haplotypes were undertransmitted (41.1%, odds ratio=0.70, P=0.0052) to patients. The data suggest that inhibitory ‘A' haplotypes are predisposing and stimulatory ‘B' haplotypes confer protection in both DR3/DR4-negative and late-onset patient groups. PMID:26492518

  2. Diversity of killer cell immunoglobulin-like receptor (KIR) genotypes and KIR2DL2/3 variants in HCV treatment outcome.

    PubMed

    Vidal-Castiñeira, Jose Ramón; López-Vázquez, Antonio; Martínez-Borra, Jesús; Martínez-Camblor, Pablo; Prieto, Jesús; López-Rodríguez, Rosario; Sanz-Cameno, Paloma; de la Vega, Juan; Rodrigo, Luis; Pérez-López, Rosa; Pérez-Álvarez, Ramón; López-Larrea, Carlos

    2014-01-01

    The aim of this study was to analyse the distribution of KIR haplotypes and the KIR2DL2/3 alleles in chronic HCV-infected patients in order to establish the influence on the response to pegylated interferon plus ribavirin classical treatment. The alleles study of previously associated KIR2DL2/3 showed that KIR2DL2*001 was more frequent in non-SVR (NSVR) (42.2% vs. 27.5%, p<0.05) and KIR2DL3*001 was associated with sustained viral response (SVR) (41.6% vs. 61.2%, p<0.005). The KIR2DL3*001-HLA-C1 association was also significant (24.5% vs. 45.7%, p<0.001). From the frequencies of KIR obtained, 35 genotypes were assigned on the basis of previous studies. The centromeric A/A genotype was more frequent in SVR (44.1% vs. 34.5%, p<0.005) and the centromeric B/B genotype was found to be significantly more frequent in NSVR (20.9% vs. 11.2%, p<0.001). The logic regression model showed the importance of KIR genes in predicting the response to combined treatment, since the positive predictive value (PPV) was improved (from 55.9% to 75.3%) when the analysis of KIR was included in addition to the IFNL3 rs12979860 polymorphism. The study of KIR receptors may be a powerful tool for predicting the combined treatment response in patients with chronic HCV infection in association with the determination of IFNL3 polymorphism. PMID:24927414

  3. Cdo Regulates Surface Expression of Kir2.1 K+ Channel in Myoblast Differentiation

    PubMed Central

    Koh, Jewoo; Kang, KyeongJin; Bae, Gyu-Un; Cho, Hana; Kang, Jong-Sun

    2016-01-01

    A potassium channel Kir2.1-associated membrane hyperpolarization is required for myogenic differentiation. However the molecular regulatory mechanisms modulating Kir2.1 channel activities in early stage of myogenesis are largely unknown. A cell surface protein, Cdo functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that Cdo forms a complex with Kir2.1 during myogenic differentiation, and is required for the channel activity by enhancing the surface expression of Kir2.1 in the early stage of differentiation. The expression of a constitutively active form of the upstream kinase for p38MAPK, MKK6(EE) can restore Kir2.1 activities in Cdo-depleted C2C12 cells, while the treatment with a p38MAPK inhibitor, SB203580 exhibits a similar effect of Cdo depletion on Kir2.1 surface expression. Furthermore, Cdo-/- primary myoblasts, which display a defective differentiation program, exhibit a defective Kir2.1 activity. Taken together, our results suggest that a promyogenic Cdo signaling is critical for Kir2.1 activities in the induction of myogenic differentiation. PMID:27380411

  4. KIR2DL2/2DL3-E35 alleles are functionally stronger than -Q35 alleles

    NASA Astrophysics Data System (ADS)

    Bari, Rafijul; Thapa, Rajoo; Bao, Ju; Li, Ying; Zheng, Jie; Leung, Wing

    2016-03-01

    KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E35) are functionally stronger than those with glutamine at the same position (Q35). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E35 could kill more target cells lacking their ligands than NK cells with the weaker -Q35 alleles, indicating better licensing of KIR2DL2/L3+ NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation.

  5. KIR2DL2/2DL3-E35 alleles are functionally stronger than -Q35 alleles

    PubMed Central

    Bari, Rafijul; Thapa, Rajoo; Bao, Ju; Li, Ying; Zheng, Jie; Leung, Wing

    2016-01-01

    KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E35) are functionally stronger than those with glutamine at the same position (Q35). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E35 could kill more target cells lacking their ligands than NK cells with the weaker -Q35 alleles, indicating better licensing of KIR2DL2/L3+ NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation. PMID:27030405

  6. KIR/HLA interactions negatively affect rituximab- but not GA101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity.

    PubMed

    Terszowski, Grzegorz; Klein, Christian; Stern, Martin

    2014-06-15

    Ab-dependent cellular cytotoxicity (ADCC) mediated by NK cells is regulated by inhibitory killer cell Ig-like receptors (KIRs), which interact with target cell HLA class I. We analyzed how KIR/HLA interactions influence ADCC induced by rituximab and by GA101, a novel type II CD20 Ab glycoengineered for increased FcgRIII binding and ADCC capacity. We found that KIR/HLA interactions strongly and selectively inhibit rituximab-induced in vitro ADCC toward target cells expressing cognate HLA KIR ligands. NK cells of donors carrying all three ligands to inhibitory KIR showed weak activation and target cell depletion capacity when incubated with rituximab and KIR-ligand matched target B cells. In contrast, NK cells from individuals missing one or more KIR ligands activated more strongly and depleted KIR ligand-matched target B cells more efficiently in the presence of rituximab. NK cells expressing a KIR for which the ligand was absent were the main effectors of ADCC in these donors. Notably, the influence of KIR/HLA interactions on NK cell activation was synergistic with the effect of the V158F FCGR3A single nucleotide polymorphism. In contrast, GA101 induced activation of NK cells irrespective of inhibitory KIR expression, and efficiency of target cell depletion was not negatively affected by KIR/HLA interactions. These data show that modification of the Fc fragment to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic mAbs by recruiting NK cells irrespective of their inhibitory KIR expression.

  7. Conjunctival Scarring in Trachoma Is Associated with the HLA-C Ligand of KIR and Is Exacerbated by Heterozygosity at KIR2DL2/KIR2DL3

    PubMed Central

    Roberts, Chrissy h.; Molina, Sandra; Makalo, Pateh; Joof, Hassan; Harding-Esch, Emma M.; Burr, Sarah E.; Mabey, David C. W.; Bailey, Robin L.; Burton, Matthew J.; Holland, Martin J.

    2014-01-01

    Background Chlamydia trachomatis is globally the predominant infectious cause of blindness and one of the most common bacterial causes of sexually transmitted infection. Infections of the conjunctiva cause the blinding disease trachoma, an immuno-pathological disease that is characterised by chronic conjunctival inflammation and fibrosis. The polymorphic Killer-cell Immunoglobulin-like Receptors (KIR) are found on Natural Killer cells and have co-evolved with the Human Leucocyte Antigen (HLA) class I system. Certain genetic constellations of KIR and HLA class I polymorphisms are associated with a number of diseases in which modulation of the innate responses to viral and intracellular bacterial pathogens is central. Methodology A sample of 134 Gambian pedigrees selected to contain at least one individual with conjunctival scarring in the F1 generation was used. Individuals (n = 830) were genotyped for HLA class I and KIR gene families. Family Based Association Tests and Case Pseudo-control tests were used to extend tests for transmission disequilibrium to take full advantage of the family design, genetic model and phenotype. Principle findings We found that the odds of trachomatous scarring increased with the number of genome copies of HLA-C2 (C1/C2 OR = 2.29 BHP-value = 0.006; C2/C2 OR = 3.97 BHP-value = 0.0004) and further increased when both KIR2DL2 and KIR2DL3 (C2/C2 OR = 5.95 BHP-value = 0.006) were present. Conclusions To explain the observations in the context of chlamydial infection and trachoma we propose a two-stage model of response and disease that balances the cytolytic response of KIR expressing NK cells with the ability to secrete interferon gamma, a combination that may cause pathology. The data presented indicate that HLA-C genotypes are important determinants of conjunctival scarring in trachoma and that KIR2DL2/KIR2DL3 heterozygosity further increases risk of conjunctival scarring in individuals carrying HLA-C2. PMID

  8. Selective inhibition of the Kir2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR and pharmacological characterization of ML133

    PubMed Central

    Wang, Hao-Ran; Wu, Meng; Yu, Haibo; Long, Shunyou; Stevens, Amy; Engers, Darren W.; Sackin, Henry; Daniels, J. Scott; Dawson, Eric S.; Hopkins, Corey R.; Lindsley, Craig W.; Li, Min; McManus, Owen B

    2011-01-01

    The Kir inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (Kir1 – Kir7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective Kir2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of Kir2.1 function. Here we report one potent Kir2.1 inhibitor, ML133, which inhibits Kir2.1 with IC50 of 1.8 μM at pH 7.4 and 290 nM at pH 8.5, but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on Kir1.1 (IC50 > 300 μM), and displays weak activity for Kir4.1 (76 μM) and Kir7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the Kir family reported to date. Due to the high homology within the Kir family, the channels share a common design of a pore region flanked by two transmembrane domains, identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent Kir inhibitors. Using chimeric channels between Kir2.1 and Kir1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of Kir2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of Kir1.1 to those of Kir2.1 (N171D and C175I) transplants ML133 inhibition to Kir1.1. Together, the combination of a potent, Kir2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of Kir2 inward rectifier potassium channels. PMID:21615117

  9. Different KIRs confer susceptibility and protection to adults with latent autoimmune diabetes in Latvian and Asian Indian populations.

    PubMed

    Shastry, Arun; Sedimbi, Saikiran K; Rajalingam, Raja; Rumba, Ingrida; Kanungo, Alok; Sanjeevi, C B

    2008-12-01

    KIRs (killer Ig-like receptors) expressed on natural killer (NK) cells are an important component of innate (and adaptive) immunity. They are either activatory or inhibitory, and certain KIRs are known to interact with specific motifs of HLA Class I molecules, which is very crucial in determining whether a cell is targeted to lysis or otherwise. Latent autoimmune diabetes in adults (LADA) is a slowly progressive form of autoimmune diabetes, with an adult onset (>30 years). Because autoantibodies and autoimmunity involved are involved in the etiology of LADA, KIRs might play an important role in conferring susceptibility to or protection against the disease. The purpose of this study was to identify killer immunoglobulin-like receptor (KIR) genes, which are associated with susceptibility to and protection against type 1 diabetes in Latvian and Asian Indian patients with LADA. KIR and HLA-C ligand genotyping was performed using PCR-SSP in LADA patients from Latvia (n= 45) with age- and sex-matched controls (n= 92) and from India (n= 86) with controls (n= 98). Results showed that in Latvian patients with LADA, KIRs 2DL1, 2DS2, and 2DS4 were associated with susceptibility and KIR 2DS5 with protection. In Asian Indian LADA patients, KIRs 2DL5 and 3DL1 were associated with susceptibility and KIRs 2DS1 and 2DS3 with protection. Stratification analyses for KIRs that bind to HLA-C1 and C2 were performed. We concluded that KIRs are important in conferring susceptibility (or protection) to adult patients with LADA in both our study populations. However the KIR genes (and their HLA-C ligands) conferring susceptibility or protection in these two populations differ, showing a role of ethnicity in disease susceptibility.

  10. Potential role of killer immunoglobulin receptor genes among individuals vaccinated against hepatitis B virus in Lebanon

    PubMed Central

    Melhem, Nada M; Mahfouz, Rami A; Kreidieh, Khalil; Abdul-Khalik, Rabab; El-Khatib, Rolla; Talhouk, Reem; Musharrafieh, Umayya; Hamadeh, Ghassan

    2016-01-01

    AIM To explore the role of killer immunoglobulin receptor (KIR) genes in responsiveness or non-responsiveness to vaccination against hepatitis B virus. METHODS We recruited 101 voluntary participants between March 2010 and December 2011. Sera samples from vaccinated and non-vaccinated participants were tested for the presence of anti-HBs antibodies as a measure of protection against hepatitis B, hepatitis B surface antigen and hepatitis B core antibody as indicators of infection by enzyme-linked immunosorbent assay. KIR gene frequencies were determined by polymerase chain reaction. RESULTS Sera samples from 99 participants were tested for the levels of anti-HBs as an indicator of protection (≥ 10 mIU/mL) following vaccination as defined by the World Health Organization international reference standard. Among the vaccinated participants, 47% (35/74) had anti-HBs titers above 100 mIU/mL, 22% (16/74) had anti-HBs ranging between 10-100 mIU/mL, and 20% (15/74) had values of less than 10 mIU/mL. We report the lack of significant association between the number of vaccine dosages and the titer of antibodies among our vaccinated participants. The inhibitory KIR2DL1, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL were detected in more than 95%, whereas KIR2DL2, KIR2DL3, KIR2DL5 (KR2DL5A and KIR2DL5B) were expressed in 56%, 84% and 42% (25% and 29%) of participants, respectively. The observed frequency of the activating KIR genes ranged between 35% and 55% except for KIR2DS4, detected in 95% of the study participants (40.6% 2DS4*001/002; 82.2% 2DS4*003/007). KIR2DP1 pseudogene was detected in 99% of our participants, whereas KIR3DP*001/02/04 and KIR3DP1*003 had frequencies of 17% and 100%, respectively. No association between the frequency of KIR genes and anti-HBs antibodies was detected. When we compared the frequency of KIR genes between vaccinated individuals with protective antibodies titers and those who lost their protective antibody levels, we did not detect a significant

  11. The role of glial-specific Kir4.1 in normal and pathological states of the CNS.

    PubMed

    Nwaobi, Sinifunanya E; Cuddapah, Vishnu A; Patterson, Kelsey C; Randolph, Anita C; Olsen, Michelle L

    2016-07-01

    Kir4.1 is an inwardly rectifying K(+) channel expressed exclusively in glial cells in the central nervous system. In glia, Kir4.1 is implicated in several functions including extracellular K(+) homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and facilitation of glutamate uptake. Knockout of Kir4.1 in rodent models leads to severe neurological deficits, including ataxia, seizures, sensorineural deafness, and early postnatal death. Accumulating evidence indicates that Kir4.1 plays an integral role in the central nervous system, prompting many laboratories to study the potential role that Kir4.1 plays in human disease. In this article, we review the growing evidence implicating Kir4.1 in a wide array of neurological disease. Recent literature suggests Kir4.1 dysfunction facilitates neuronal hyperexcitability and may contribute to epilepsy. Genetic screens demonstrate that mutations of KCNJ10, the gene encoding Kir4.1, causes SeSAME/EAST syndrome, which is characterized by early onset seizures, compromised verbal and motor skills, profound cognitive deficits, and salt-wasting. KCNJ10 has also been linked to developmental disorders including autism. Cerebral trauma, ischemia, and inflammation are all associated with decreased astrocytic Kir4.1 current amplitude and astrocytic dysfunction. Additionally, neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis demonstrate loss of Kir4.1. This is particularly exciting in the context of Huntington disease, another neurodegenerative disorder in which restoration of Kir4.1 ameliorated motor deficits, decreased medium spiny neuron hyperexcitability, and extended survival in mouse models. Understanding the expression and regulation of Kir4.1 will be critical in determining if this channel can be exploited for therapeutic benefit. PMID:26961251

  12. The role of glial-specific Kir4.1 in normal and pathological states of the CNS.

    PubMed

    Nwaobi, Sinifunanya E; Cuddapah, Vishnu A; Patterson, Kelsey C; Randolph, Anita C; Olsen, Michelle L

    2016-07-01

    Kir4.1 is an inwardly rectifying K(+) channel expressed exclusively in glial cells in the central nervous system. In glia, Kir4.1 is implicated in several functions including extracellular K(+) homeostasis, maintenance of astrocyte resting membrane potential, cell volume regulation, and facilitation of glutamate uptake. Knockout of Kir4.1 in rodent models leads to severe neurological deficits, including ataxia, seizures, sensorineural deafness, and early postnatal death. Accumulating evidence indicates that Kir4.1 plays an integral role in the central nervous system, prompting many laboratories to study the potential role that Kir4.1 plays in human disease. In this article, we review the growing evidence implicating Kir4.1 in a wide array of neurological disease. Recent literature suggests Kir4.1 dysfunction facilitates neuronal hyperexcitability and may contribute to epilepsy. Genetic screens demonstrate that mutations of KCNJ10, the gene encoding Kir4.1, causes SeSAME/EAST syndrome, which is characterized by early onset seizures, compromised verbal and motor skills, profound cognitive deficits, and salt-wasting. KCNJ10 has also been linked to developmental disorders including autism. Cerebral trauma, ischemia, and inflammation are all associated with decreased astrocytic Kir4.1 current amplitude and astrocytic dysfunction. Additionally, neurodegenerative diseases such as Alzheimer disease and amyotrophic lateral sclerosis demonstrate loss of Kir4.1. This is particularly exciting in the context of Huntington disease, another neurodegenerative disorder in which restoration of Kir4.1 ameliorated motor deficits, decreased medium spiny neuron hyperexcitability, and extended survival in mouse models. Understanding the expression and regulation of Kir4.1 will be critical in determining if this channel can be exploited for therapeutic benefit.

  13. Donor KIR B Genotype Improves Progression Free Survival of Non-Hodgkin lymphoma Patients Receiving Unrelated Donor Transplantation

    PubMed Central

    Bachanova, Veronika; Weisdorf, Daniel J.; Wang, Tao; Marsh, Steven G.E.; Trachtenberg, Elizabeth; Haagenson, Michael D; Spellman, Stephen R.; Ladner, Martha; Guethlein, Lisbeth A.; Parham, Peter; Miller, Jeffrey S.; Cooley, Sarah A.

    2016-01-01

    Donor killer immunoglobulin-like receptor (KIR) genotypes associate with relapse protection and survival after allotransplantation for acute myelogenous leukemia. We examined the possibility of a similar effect in a cohort of 614 non-Hodgkin lymphoma (NHL) patients receiving unrelated donor (URD) T-cell replete marrow or peripheral blood grafts. Sixty four percent (n=396) of donor-recipient pairs were 10/10 allele HLA-matched; 26% were 9/10 allele matched. Seventy percent of donors had KIR B/x genotype; the others had KIR A/A genotype. NHL patients receiving 10/10 HLA-matched URD grafts with KIR B/x donors experienced significantly lower relapse at 5 years (26%; CI 21–32% vs. 37%; CI 27–46%, p=0.05) compared with KIR A/A donors, resulting in improved 5 year progression-free survival (PFS) (35%; CI 26–44% vs. 22%; CI 11–35%; p=0.007). In multivariate analysis, use of KIR B/x donors associated with significantly reduced relapse risk (RR 0.63, p=0.02) and improved PFS (RR 0.71, p=0.008). The relapse protection afforded by KIR B/x donors was not observed in HLA-mismatched transplants, and was not specific to any particular KIR-B gene. Selecting 10/10 HLA-matched and KIR B/x donors should benefit patients with NHL receiving URD allogeneic transplantation. PMID:27220262

  14. The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy.

    PubMed

    McCloskey, Conor; Rada, Cara; Bailey, Elizabeth; McCavera, Samantha; van den Berg, Hugo A; Atia, Jolene; Rand, David A; Shmygol, Anatoly; Chan, Yi-Wah; Quenby, Siobhan; Brosens, Jan J; Vatish, Manu; Zhang, Jie; Denton, Jerod S; Taggart, Michael J; Kettleborough, Catherine; Tickle, David; Jerman, Jeff; Wright, Paul; Dale, Timothy; Kanumilli, Srinivasan; Trezise, Derek J; Thornton, Steve; Brown, Pamela; Catalano, Roberto; Lin, Nan; England, Sarah K; Blanks, Andrew M

    2014-09-01

    Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility.

  15. Differential transcription factor use by the KIR2DL4 promoter under constitutive and IL-2/15-treated conditions.

    PubMed

    Presnell, Steven R; Zhang, Lei; Chlebowy, Corrin N; Al-Attar, Ahmad; Lutz, Charles T

    2012-05-01

    KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, EMSA, and cotransfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a cyclic AMP response element (CRE) and initiator elements. IL-2- and IL-15-stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells.

  16. Impact of KIR and HLA Genotypes on Outcomes after Reduced-Intensity Conditioning Hematopoietic Cell Transplantation.

    PubMed

    Sobecks, Ronald M; Wang, Tao; Askar, Medhat; Gallagher, Meighan M; Haagenson, Michael; Spellman, Stephen; Fernandez-Vina, Marcelo; Malmberg, Karl-Johan; Müller, Carlheinz; Battiwalla, Minoo; Gajewski, James; Verneris, Michael R; Ringdén, Olle; Marino, Susana; Davies, Stella; Dehn, Jason; Bornhäuser, Martin; Inamoto, Yoshihiro; Woolfrey, Ann; Shaw, Peter; Pollack, Marilyn; Weisdorf, Daniel; Milller, Jeffrey; Hurley, Carolyn; Lee, Stephanie J; Hsu, Katharine

    2015-09-01

    Natural killer cells are regulated by killer cell immunoglobulin-like receptor (KIR) interactions with HLA class I ligands. Several models of natural killer cell reactivity have been associated with improved outcomes after myeloablative allogeneic hematopoietic cell transplantation (HCT), but this issue has not been rigorously addressed in reduced-intensity conditioning (RIC) unrelated donor (URD) HCT. We studied 909 patients undergoing RIC-URD HCT. Patients with acute myeloid leukemia (AML, n = 612) lacking ≥ 1 KIR ligands experienced higher grade III to IV acute graft-versus-host disease (GVHD) (HR, 1.6; 95% CI, 1.16 to 2.28; P = .005) compared to those with all ligands present. Absence of HLA-C2 for donor KIR2DL1 was associated with higher grade II to IV (HR, 1.4; P = .002) and III to IV acute GVHD (HR, 1.5; P = .01) compared with HLA-C2(+) patients. AML patients with KIR2DS1(+), HLA-C2 homozygous donors had greater treatment-related mortality compared with others (HR, 2.4; 95% CI, 1.4 to 4.2; P = .002) but did not experience lower relapse. There were no significant associations with outcomes for AML when assessing donor-activating KIRs or centromeric KIR content or for any donor-recipient KIR-HLA assessments in patients with myelodysplastic syndrome (n = 297). KIR-HLA combinations in RIC-URD HCT recapitulate some but not all KIR-HLA effects observed in myeloablative HCT. PMID:25960307

  17. Impact of KIR and HLA Genotypes on Outcomes after Reduced-Intensity Conditioning Hematopoietic Cell Transplantation

    PubMed Central

    Sobecks Ronald, M; Tao, Wang; Medhat, Askar; Gallagher Meighan, M; Michael, Haagenson; Stephen, Spellman; Marcelo, Fernandez-Vina; Karl-Johan, Malmberg; Carlheinz, Muller; Minoo, Battiwalla; James, Gajewski; Verneris Michael, R; Olle, Ringden; Marino Susana, R; Stella, Davies; Jason, Dehn; Martin, Bornhäuser; Yoshihiro, Inamoto; Ann, Woolfrey; Peter, Shaw; Marilyn, Pollack; Daniel, Weisdorf; Jeffrey, Miller; Hurley Carolyn, K; Lee Stephanie, J; Hsu Katharine, C

    2015-01-01

    Natural killer (NK) cells are regulated killer immunoglobulin-like receptor (KIR) interactions with HLA class I ligands. Several models of NK reactivity have been associated with improved outcomes following myeloablative allogeneic hematopoietic cell transplantation (HCT), but this issue has not been rigorously addressed in reduced-intensity conditioning (RIC) unrelated donor (URD) HCT. We studied 909 patients undergoing RIC-URD HCT. Patients with acute myeloid leukemia (AML, n=612) lacking ≥1 KIR ligands experienced higher grade III–IV acute graft-vs.-host disease (GvHD) (HR 1.6, 95%CI 1.16–2.28, p=0.005) compared to those with all ligands present. Absence of HLA-C2 for donor KIR2DL1 was associated with higher grade II–IV (HR 1.4, p=0.002) and III–IV acute GvHD (HR 1.5, p=0.01) compared to HLA-C2+patients. AML patients with KIR2DS1+, HLA-C2 homozygous donors had greater treatment-related mortality compared to others (HR 2.4, 95%CI 1.4–4.2, p=0.002), but did not experience lower relapse. There were no significant associations with outcomes for AML when assessing donor activating KIRs or centromeric KIR content, nor for any donor-recipient KIR-HLA assessments in patients with myelodysplastic syndrome (n=297). KIR-HLA combinations in RIC-URD HCT recapitulate some but not all KIR-HLA effects observed in myeloablative HCT. PMID:25960307

  18. HLA-F and MHC-I Open Conformers Bind Natural Killer Cell Ig-Like Receptor KIR3DS1.

    PubMed

    Burian, Aura; Wang, Kevin L; Finton, Kathryn A K; Lee, Ni; Ishitani, Akiko; Strong, Roland K; Geraghty, Daniel E

    2016-01-01

    Based on previous findings supporting HLA-F as a ligand for KIR3DL2 and KIR2DS4, we investigated the potential for MHC-I open conformers (OCs) as ligands for KIR3DS1 and KIR3DL1 through interactions measured by surface plasmon resonance. These measurements showed physical binding of KIR3DS1 but not KIR3DL1 with HLA-F and other MHC-I OC while also confirming the allotype specific binding of KIR3DL1 with MHC-I peptide complex. Concordant results were obtained with biochemical pull-down from cell lines and biochemical heterodimerization experiments with recombinant proteins. In addition, surface binding of HLA-F and KIR3DS1 to native and activated NK and T cells was coincident with specific expression of the putative ligand or receptor. A functional response of KIR3DS1 was indicated by increased granule exocytosis in activated cells incubated with HLA-F bound to surfaces. The data extend a model for interaction between MHC-I open conformers and activating KIR receptors expressed during an inflammatory response, potentially contributing to communication between the innate and adaptive immune response. PMID:27649529

  19. HLA-F and MHC-I Open Conformers Bind Natural Killer Cell Ig-Like Receptor KIR3DS1

    PubMed Central

    Burian, Aura; Wang, Kevin L.; Finton, Kathryn A. K.; Lee, Ni; Ishitani, Akiko; Strong, Roland K.; Geraghty, Daniel E.

    2016-01-01

    Based on previous findings supporting HLA-F as a ligand for KIR3DL2 and KIR2DS4, we investigated the potential for MHC-I open conformers (OCs) as ligands for KIR3DS1 and KIR3DL1 through interactions measured by surface plasmon resonance. These measurements showed physical binding of KIR3DS1 but not KIR3DL1 with HLA-F and other MHC-I OC while also confirming the allotype specific binding of KIR3DL1 with MHC-I peptide complex. Concordant results were obtained with biochemical pull-down from cell lines and biochemical heterodimerization experiments with recombinant proteins. In addition, surface binding of HLA-F and KIR3DS1 to native and activated NK and T cells was coincident with specific expression of the putative ligand or receptor. A functional response of KIR3DS1 was indicated by increased granule exocytosis in activated cells incubated with HLA-F bound to surfaces. The data extend a model for interaction between MHC-I open conformers and activating KIR receptors expressed during an inflammatory response, potentially contributing to communication between the innate and adaptive immune response. PMID:27649529

  20. Donor-Recipient Matching for KIR Genotypes Reduces Chronic GVHD and Missing Inhibitory KIR Ligands Protect against Relapse after Myeloablative, HLA Matched Hematopoietic Cell Transplantation

    PubMed Central

    Faridi, Rehan Mujeeb; Kemp, Taylor J.; Dharmani-Khan, Poonam; Lewis, Victor; Rajalingam, Raja; Berka, Noureddine; Storek, Jan; Masood Khan, Faisal

    2016-01-01

    Background Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT Methods and Findings The study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into ‘discovery’ (135 pairs) and ‘validation’ (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50–5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33–5.11). High incidence of cGVHD associated

  1. KIR3DL1 and HLA-B Density and Binding Calibrate NK Education and Response to HIV.

    PubMed

    Boudreau, Jeanette E; Mulrooney, Tiernan J; Le Luduec, Jean-Benoît; Barker, Edward; Hsu, Katharine C

    2016-04-15

    NK cells recognize self-HLA via killer Ig-like receptors (KIR). Homeostatic HLA expression signals for inhibition via KIR, and downregulation of HLA, a common consequence of viral infection, allows NK activation. Like HLA, KIR are highly polymorphic, and allele combinations of the most diverse receptor-ligand pair, KIR3DL1 and HLA-B, correspond to hierarchical HIV control. We used primary cells from healthy human donors to demonstrate how subtype combinations of KIR3DL1 and HLA-B calibrate NK education and their consequent capacity to eliminate HIV-infected cells. High-density KIR3DL1 and Bw4-80I partnerships endow NK cells with the greatest reactivity against HLA-negative targets; NK cells exhibiting the remaining KIR3DL1/HLA-Bw4 combinations demonstrate intermediate responsiveness; and Bw4-negative KIR3DL1(+) NK cells are poorly responsive. Cytotoxicity against HIV-infected autologous CD4(+) T cells strikingly correlated with reactivity to HLA-negative targets. These findings suggest that the programming of NK effector function results from defined features of receptor and ligand subtypes. KIR3DL1 and HLA-B subtypes exhibit an array of binding strengths. Like KIR3DL1, subtypes of HLA-Bw4 are expressed at distinct, predictable membrane densities. Combinatorial permutations of common receptor and ligand subtypes reveal binding strength, receptor density, and ligand density to be functionally important. These findings have immediate implications for prognosis in patients with HIV infection. Furthermore, they demonstrate how features of KIR and HLA modified by allelic variation calibrate NK cell reactive potential. PMID:26962229

  2. Pharmacological validation of an inward-rectifier potassium (Kir) channel as an insecticide target in the yellow fever mosquito Aedes aegypti.

    PubMed

    Rouhier, Matthew F; Raphemot, Rene; Denton, Jerod S; Piermarini, Peter M

    2014-01-01

    Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, for insecticide development. Here we provide pharmacological validation of a specific mosquito Kir channel (AeKir1) in the yellow fever mosquito Aedes aegypti. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits AeKir1 but not another mosquito Kir channel (AeKir2B) in vitro. Moreover, we show that a previously identified inhibitor of AeKir1 (VU573) elicits an unexpected agonistic effect on AeKir2B in vitro. Injection of VU590 into the hemolymph of adult female mosquitoes significantly inhibits their capacity to excrete urine and kills them within 24 h, suggesting a mechanism of action on the excretory system. Importantly, a structurally-related VU590 analog (VU608), which weakly blocks AeKir1 in vitro, has no significant effects on their excretory capacity and does not kill mosquitoes. These observations suggest that the toxic effects of VU590 are associated with its inhibition of AeKir1.

  3. Kir3 channel signaling complexes: focus on opioid receptor signaling

    PubMed Central

    Nagi, Karim; Pineyro, Graciela

    2014-01-01

    Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels. Kir3 channels control cellular excitability in the central nervous system and in the heart and, because of their ubiquitous distribution, they mediate the effects of a large range of hormones and neurotransmitters which, upon activation of corresponding G protein-coupled receptors (GPCRs) lead to channel opening. Here we analyze GPCR signaling via these effectors in reference to precoupling and collision models. Existing knowledge on signaling bias is discussed in relation to these models as a means of developing strategies to produce novel opioid analgesics with an improved side effects profile. PMID:25071446

  4. The inwardly rectifying potassium channel Kir1.1: development of functional assays to identify and characterize channel inhibitors.

    PubMed

    Felix, John P; Priest, Birgit T; Solly, Kelli; Bailey, Timothy; Brochu, Richard M; Liu, Chou J; Kohler, Martin G; Kiss, Laszlo; Alonso-Galicia, Magdalena; Tang, Haifeng; Pasternak, Alexander; Kaczorowski, Gregory J; Garcia, Maria L

    2012-10-01

    The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartter's syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization. PMID:22881347

  5. Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

    PubMed

    Yang, Yuqin; Chen, Fangyi; Karasawa, Takatoshi; Ma, Ke-Tao; Guan, Bing-Cai; Shi, Xiao-Rui; Li, Hongzhe; Steyger, Peter S; Nuttall, Alfred L; Jiang, Zhi-Gen

    2015-01-01

    The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba(2+) 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K(+), while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K(+) typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba(2+)-sensitive inward rectifier K(+) (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir

  6. Protein kinase C modulation of recombinant ATP-sensitive K(+) channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B.

    PubMed

    Thorneloe, Kevin S; Maruyama, Yoshiaki; Malcolm, A Todd; Light, Peter E; Walsh, Michael P; Cole, William C

    2002-05-15

    The molecular identity of smooth muscle ATP-sensitive K(+) channels (K(ATP)) is not established with certainty. Patch clamp methods were employed to determine if recombinant K(ATP) channels composed of Kir6.1 and SUR2B subunits expressed by human embryonic kidney (HEK293) cells share an identical modulation by protein kinase C (PKC) with the vascular K(NDP) subtype of K(ATP) channel. The open probability of Kir6.1/SUR2B channels was determined before and after sequential exposure to pinacidil (50 microM) and the combination of pinacidil and phorbol 12,13-dibutyrate (PdBu; 50 nM). Treatment with PdBu caused a decline in channel activity, but this was not seen with an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (PdDe; 50 nM). Angiotensin II (0.1 microM) induced a similar inhibition of Kir6.1/SUR2B channels in cells expressing angiotensin AT(1) receptors. The effects of PdBu and angiotensin II were blocked by the PKC inhibitor, chelerythrine (3 microM). Purified PKC inhibited Kir6.1/SUR2B activity (in 0.5 mM ATP/ 0.5 mM ADP), and the inhibition was blocked by a specific peptide inhibitor of PKC, PKC(19-31). In contrast, PdBu increased the activity of recombinant K(ATP) channels composed of Kir6.2 and SUR2B, or the combination of Kir6.1, Kir6.2 and SUR2B subunits. The results indicate that the modulation by PKC of Kir6.1/SUR2B, but not Kir6.2/SUR2B or Kir6.1-Kir6.2/SUR2B channel gating mimics that of native vascular K(NDP) channels. Physiological inhibition of vascular K(ATP) current by vasoconstrictors which utilize intracellular signalling cascades involving PKC is concluded to involve the modulation of K(NDP) channel complexes composed of four Kir6.1 and their associated SUR2B subunits.

  7. Genetic polymorphism of KIR2DL4 in the Polish population.

    PubMed

    Nowak, I; Barcz, E; Majorczyk, E; Malinowski, A; Wilczyński, J R; Banasik, M; Motak-Pochrzęst, H; Kuśnierczyk, P

    2015-06-01

    The KIR2DL4 gene is characterized by alleles with either 9 or 10 consecutive adenines in exon 7, which encodes the transmembrane domain. The 9A variant produces either a protein with a truncated cytoplasmic tail or one lacking the transmembrane region. This causes a lack of KIR2DL4 expression. In contrast, 10A alleles encode receptors that may be expressed at the cell surface. We tested 438 healthy individuals for polymorphism of the KIR2DL4 gene. KIR2DL4 9A/10A alleles were distinguished by the high resolution melting (HRM) method, and restriction fragment length polymorphism (RFLP) was used for genotyping of three other single nucleotide polymorphisms (SNPs) spanning the near vicinity of the poly-adenine fragment. We found a weak difference between males and females in 9769 C/A genotypes and alleles. In addition, we observed complete linkage disequilibrium (LD) between 9A insertion/deletion in the 9620 position and the 9571T/C position of the gene (r(2)  = 1) both in females and males and almost complete LD with the 9797G/A position (r(2)  = 0.963 for females and r(2)  = 0.892 for males). Most importantly, we detected, in a group of fertile women, a high frequency (30.2%) of homozygosity for the defective 9A variant, which suggests that KIR2DL4 as a functional cell surface receptor is not absolutely necessary for reproduction. On the other hand, lower representation of 10A/10A homozygotes and high frequency of 10A/9A heterozygotes indicates a need for both cell membrane-anchored and soluble KIR2DL4 molecules. Finally, cost-reducing RFLP instead of HRM is proposed for typing 9A and 10A variants. PMID:25818657

  8. Inwardly rectifying Kir3.1 subunit knockdown impairs learning and memory in an olfactory associative task in rat.

    PubMed

    Kourrich, Saïd; Masmejean, Frédérique; Martin-Eauclaire, Marie France; Soumireu-Mourat, Bernard; Mourre, Christiane

    2003-05-12

    Inward-rectifier potassium channels gated by the direct action of G proteins are activated or inhibited by numerous neurotransmitters and they modulate neuronal excitability. Using an olfactory associative task, the effect of Kir3.1 subunit knockdown was tested on learning and memory. Repeated intracerebroventricular injections of antisense oligodeoxyribonucleotide to the Kir3.1 subunit significantly reduced hippocampal expression of its mRNA target determined by Western blotting. The antisense knockdown had no effect on locomotor and drinking activity or on attention processes. The reduction in Kir3.1 subunit impaired the learning of the odor associations and the procedural side of the task. This reduction correlated with the performance impairment. The results suggest that Kir3.1 channel activity is implicated in the memory processes. PMID:12750011

  9. KIR3DL01 Recognition of Bw4 Ligands in the Rhesus Macaque: Maintenance of Bw4 Specificity since the Divergence of Apes and Old World Monkeys

    PubMed Central

    Schafer, Jamie L.; Colantonio, Arnaud D.; Neidermyer, William J.; Dudley, Dawn M.; Connole, Michelle; O’Connor, David H.; Evans, David T.

    2014-01-01

    The identification of MHC class I ligands for rhesus macaque KIRs is fundamental to our basic understanding of KIR and MHC class I co-evolution and to the study of NK cell responses in this non-human primate model for AIDS and other viral diseases. Here we show that Mamu-KIR3DL01, which is expressed by approximately 90% of rhesus macaques, recognizes MHC class I molecules with a Bw4 motif. Primary NK cells expressing Mamu-KIR3DL01 were identified by staining with a mAb herein shown to bind Mamu-KIR3DL01 allotypes with an aspartic acid at position 233. The cytolytic activity of Mamu-KIR3DL01+ NK cells was suppressed by cell lines expressing the Bw4 molecules Mamu-B*007:01, -B*041:01, -B*058:02, and -B*065:01. The Bw4 motif was necessary for Mamu-KIR3DL01 recognition, since substitutions in this region abrogated Mamu-KIR3DL01+ NK cell inhibition. However, the presence of a Bw4 motif was not sufficient for recognition, since another Bw4 molecule, Mamu-B*017:01, failed to suppress the cytolytic activity of these NK cells. Replacement of three residues in Mamu-B*017:01, predicted to be KIR-contacts based on the 3-dimensional structure of the human KIR3DL1-HLA-Bw4 complex, with the corresponding residues at these positions for the other Mamu-Bw4 ligands restored Mamu-KIR3DL01+ NK cell inhibition. These results define the ligand specificity of one of the most polymorphic and commonly expressed KIRs in the rhesus macaque, and reveal similarities in Bw4 recognition by Mamu-KIR3DL01 and human KIR3DL1, despite the absence of an orthologous relationship between these two KIRs or conservation of surface residues predicted to interact with MHC class I ligands. PMID:24453246

  10. Persistence of decidual NK cells and KIR genotypes in healthy pregnant and preeclamptic women: a case-control study in the third trimester of gestation

    PubMed Central

    2011-01-01

    Background Natural Killer (NK) cells are the most abundant lymphocytes in the decidua during early gestation. The interactions of NK cells with the extravillous cytotrophoblast have been associated with a normal spiral artery remodeling process, an essential event for a successful pregnancy. Recent data indicate that alterations in the amount of decidual NK (dNK) cells contribute to the development of preeclampsia (PE). Moreover, genetic studies suggest that Killer Immunoglobulin-like Receptors (KIR) expressed in dNK cells influence the susceptibility to PE. Although dNK cells have been well characterized during early pregnancy, they have been scarcely studied in the third trimester of gestation. The aim of this work was to characterize dNK cells at the last trimester of gestation and to analyze the KIR genotype of healthy and PE women. Methods Decidual samples were obtained during Caesarean section from control (n = 10) and PE (n = 9) women. Flow cytometric analysis of CD3, CD56, CD16 and CD9 was used to characterize and quantify dNK cells in both groups. Cell surface markers from decidual leukocytes were compared with PBMC from healthy donors. KIR genotyping was performed in genomic DNA (control, n = 86; PE, n = 90) using PCR-SSP. Results The results indicate that dNK cells persist throughout pregnancy. They represented 20% of total leukocytes in control and PE groups, and they expressed the same cell surface markers (CD3-, CD56+, CD16- and CD9+) as dNK in the first trimester of gestation. There were no significant differences in the percentage of dNK cells between control and PE groups. The analysis of KIR gene frequencies and genotypes was not statistically different between control and PE groups. The ratio of activating to inhibitory genes indicated that the overall inhibitory balance (0.2-0.5) was more frequent in the PE group (control, 31.3% vs PE, 45.5%), and the activating balance (0.6-1.1) was more frequent in the control group (control, 68.6% vs PE, 54

  11. Specific amplification of cDNA ends (SPACE): a new tool for the analysis of rare transcripts and its application for the promoter analysis of killer cell receptor genes.

    PubMed

    Radeloff, Britta; Nagler, Lydia; Zirra, Maja; Ziegler, Andreas; Volz, Armin

    2005-02-01

    The expression control of activating and inhibitory killer cell Ig-like receptors (KIR) on natural killer (NK) cells is highly relevant for the initiation of NK cell mediated cytolysis and cytokine secretion. Transcription start points of nine human KIR genes from two Caucasian donors and the NK cell line NK3.3 were investigated. To overcome sensitivity problems due to the low abundance of the respective transcripts, a novel protocol, specific amplification of cDNA ends (SPACE) with superior specificity and sensitivity was applied. A total of 235 individual SPACE clones resulting from different KIR genes were analysed and revealed a series of transcription start sites tightly clustered between 10 and 60 bp upstream of the start codon. The comparison of the adjacent putative promoter region of the human, chimpanzee and macaque KIR genes revealed a very high conservation for almost all of the KIR family members. An inter-gene and inter-species comparative approach revealed transcription factor binding sites at regions of maximal homology for all primate KIR genes analysed.

  12. Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 β

    PubMed Central

    2012-01-01

    Objective Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K+ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β) on Kir4.1 mRNA and protein expression. Methods We investigated Kir4.1 (Kcnj10) and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64), comparing the expression in tumor patients with (n = 38) and without epilepsy (n = 26). Results Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1). Conclusions Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the

  13. Parametrisation of the free energy of ATP binding to wild-type and mutant Kir6.2 potassium channels.

    PubMed

    Moran, Oscar; Grottesi, Alessandro; Chadburn, Andrew J; Tammaro, Paolo

    2013-01-01

    ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions; because of their sensitivity to inhibition by intracellular ATP, K(ATP) channels provide a link between cell metabolism and membrane electrical activity. We constructed structural homology models of Kir6.2 and a series of Kir6.2 channels carrying mutations within the putative ATP-binding site. Computational docking was carried out to determine the conformation of ATP in its binding site. The Linear Interaction Energy (LIE) method was used to estimate the free-energy of ATP binding to wild-type and mutant Kir6.2 channels. Comparisons of the theoretical binding free energies for ATP with those determined from mutational experiments enabled the identification of the most probable conformation of ATP bound to the Kir6.2 channel. A set of LIE parameters was defined that may enable prediction of the effects of additional Kir6.2 mutations within the ATP binding site on the affinity for ATP.

  14. Mutational and structural analysis of KIR3DL1 reveals a lineage-defining allotypic dimorphism that impacts both HLA and peptide sensitivity.

    PubMed

    O'Connor, Geraldine M; Vivian, Julian P; Widjaja, Jacqueline M; Bridgeman, John S; Gostick, Emma; Lafont, Bernard A P; Anderson, Stephen K; Price, David A; Brooks, Andrew G; Rossjohn, Jamie; McVicar, Daniel W

    2014-03-15

    Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01-restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation.

  15. Interaction between mutations in the slide helix of Kir6.2 associated with neonatal diabetes and neurological symptoms

    PubMed Central

    Männikkö, Roope; Jefferies, Craig; Flanagan, Sarah E.; Hattersley, Andrew; Ellard, Sian; Ashcroft, Frances M.

    2010-01-01

    ATP-sensitive potassium (KATP) channels regulate insulin secretion from pancreatic beta-cells. Gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause neonatal diabetes. We report two novel mutations on the same haplotype (cis), F60Y and V64L, in the slide helix of Kir6.2 in a patient with neonatal diabetes, developmental delay and epilepsy. Functional analysis revealed the F60Y mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly producing a marked decrease in channel inhibition by ATP and an increase in whole-cell KATP currents. When expressed alone, the V64L mutation caused a small reduction in apparent ATP inhibition, by enhancing the ability of MgATP to stimulate channel activity. The V64L mutation also ameliorated the deleterious effects on the F60Y mutation when it was expressed on the same (but not a different) subunit. These data indicate that F60Y is the pathogenic mutation and reveal that interactions between slide helix residues can influence KATP channel gating. PMID:20022885

  16. Neonatal diabetes mellitus: description of two Puerto Rican children with KCNJ11 activating gene mutation.

    PubMed

    Nieves-Rivera, Francisco; González-Pijem, Lilliam

    2011-06-01

    Neonatal diabetes mellitus (NDM) is a rare disorder. A one-month-old boy presented with vomiting, hyperglycemia (968 mg/dl [53.8 mmol/L]), severe acetonemia, and metabolic acidosis (pH 6.95, HCO3-4.2 mmol/L). A second child (three months of age) presented with upper respiratory tract symptoms and a plasma glucose level of 835 mg/dl, without acetonemia or acidosis. Both were hospitalized and managed with intravenous fluids and then discharged on insulin. Genetic testing identified the presence of the de nova V59M and E322K activating mutations in the KCNJ11 gene encoding the sulphonylurea/potassium channel (Kir6.2 subunit) of the insulin beta cell. Both patients were switched to glibenclamide and remain off insulin. To our knowledge, these are the first children in Puerto Rico identified with NDM secondary to a KCNJ11 activating mutation. We conclude that NDM secondary to KCNJ11/Kir6.2 activating mutations, although unusual, should be considered in similar cases since patients with these mutations could come off insulin.

  17. Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids.

    PubMed

    Lee, Sun-Joo; Ren, Feifei; Zangerl-Plessl, Eva-Maria; Heyman, Sarah; Stary-Weinzinger, Anna; Yuan, Peng; Nichols, Colin G

    2016-09-01

    Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(-)) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL(-) Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(-) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL(-) binding and PIP2 sensitivity. PMID:27527100

  18. KIR-HLA-A and B alleles of the Bw4 epitope against HIV infection in discordant heterosexual couples in Chaco Argentina

    PubMed Central

    Habegger de Sorrentino, Alicia; Sinchi, Jessica L; Marinic, Karina; López, Rosana; Iliovich, Ernesto

    2013-01-01

    Activating and inhibitory killer immunoglobulin-like receptors (KIR) and their ligands HLA-Bw4 (loci A and B) were studied by way of establishing whether they can contribute to protection against HIV-1 infection in highly exposed and persistently seronegative (HESN) patients. Twenty-three HIV-1 serodiscordant heterosexual couples, 100 HIV-1+ patients and 200 healthy individuals were included in this retrospective case–control study. HLA typing was performed by means of PCR followed by sequence-specific oligonucleotide probe reverse hybridization. KIR3DL1 and KIR3DS1 were studied by PCR sequence-specific primers. The frequency of KIR3DS1(3DS1/3DL1)-Bw4 combination was significantly higher in HESN patients versus the discordant couples (P = 0·0003) and HIV-1+ patients (P = 0·0001). Conversely, the KIR3DL1/KIR3DL1 homozygosity was significantly decreased in HESN patients versus the discordant couples (P = 0·00003), and HIV-1+ patients (P = 0·00066). The frequency of HLA-A*32 and HLA-B*44 was higher in HESN versus their discordant couples (P = 0·009; P = 0·049), and HIV-1+ patients (P = 0·00002; P = 0·0001). This had greater significance in combination with KIR3DS1 (3DS1/3DL1). KIR3DS1(3DS1/3DL1) could have a greater effect on protection against HIV-1 infection in HESN patients when bound to a specific HLA allele, in this case HLA-A*32 and HLA-B*44, both Bw4 alleles. The differences probably arise both in the HLA alleles and in the subtypes of KIR receptors depending on the ethnic group studied. PMID:23789883

  19. Diverse roles of K(ATP) channels learned from Kir6.2 genetically engineered mice.

    PubMed

    Seino, S; Iwanaga, T; Nagashima, K; Miki, T

    2000-03-01

    The regulation of insulin secretion from pancreatic beta-cells depends critically on the activities of their plasma membrane ion channels. ATP-sensitive K+ channels (K(ATP) channels) are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. The activity of the K(ATP) channels in pancreatic beta-cells is regulated by changes in the ATP and ADP concentrations (ATP/ADP ratio) caused by glucose metabolism. Thus, the K(ATP) channels are the ATP and ADP sensors in the regulation of glucose-induced insulin secretion. K(ATP) channels are also the target of sulfonylureas, which are widely used in the treatment of type 2 diabetes. Molecular cloning of the two subunits of the pancreatic beta-cell K(ATP) channel, Kir6.2 (an inward rectifier K+ channel member) and SUR1 (a receptor for sulfonylureas), has provided great insight into its structure and function. Kir6.2 subunits form the K+ ion-permeable pore and primarily confer inhibition of the channels by ATP, while SUR1 subunits confer activation of the channels by MgADP and K+ channel openers, such as diazoxide, as well as inhibition by sulfonylureas. The SUR1 subunits also enhance the sensitivity of the channels to ATP. To determine the physiological roles of K(ATP) channels directly, we have generated two kinds of genetically engineered mice: mice expressing a dominant-negative form of Kir6.2 specifically in the pancreatic beta-cells (Kir6.2G132S Tg mice) and mice lacking Kir6.2 (Kir6.2 knockout mice). Studies of these mice elucidated various roles of the K(ATP) channels in endocrine pancreatic function: 1) the K(ATP) channels are the major determinant of the resting membrane potential of pancreatic beta-cells, 2) both glucose- and sulfonylurea-induced membrane depolarization of beta-cells require closure of the K(ATP) channels, 3) both glucose- and sulfonylurea-induced rises in intracellular calcium concentration in beta-cells require closure of the K

  20. Combination of KIR 2DL2 and HLA-C1 (Asn 80) confers susceptibility to type 1 diabetes in Latvians.

    PubMed

    Shastry, A; Sedimbi, S K; Rajalingam, R; Nikitina-Zake, L; Rumba, I; Wigzell, H; Sanjeevi, C B

    2008-12-01

    Killer immunoglobulin-like receptors (KIRs) are known to modulate natural killer (NK) and NK T-cell function by interacting with human leucocyte antigen (HLA) class I ligands on target cells. The aim of our study was to investigate the influence of KIR2D genes with their HLA-C ligands in susceptibility to type 1 diabetes. A total of 98 type 1 diabetes patients and 70 healthy subjects from Latvia were typed for KIR genes and HLA-C ligands using polymerase chain reaction-based genotyping. The HLA C1+/C2+ combination was positively, and C1-/C2+ combination was negatively, associated with type 1 diabetes. Stratification analysis of KIR/HLA-C ligand combinations showed 2DL2+/C1+, 2DL3+/C1+, and 2DS2+ /C1+ to be positively, and 2DL2-/C1- and 2DS2-/ C1- to be negatively, associated. The presence of 2DL2-HLA-C1 in the absence of 2DS1, 2DS2 confers maximum susceptibility. Absence of 2DL2 and HLA-C1 along with absence of 2DS1 and 2DS2 confer maximum protection. A hypothetical model of KIR/ligand combinations on immune responses and type 1 diabetes susceptibility is proposed. Our results suggest that a combination KIR2DL2- HLA-C1 plays a critical role in susceptibility or protection in Latvians against type 1 diabetes.

  1. Overexpression of KIR inhibitory ligands (HLA-I) determines that immunosurveillance of myeloma depends on diverse and strong NK cell licensing

    PubMed Central

    Martínez-Sánchez, María V.; Periago, Adela; Legaz, Isabel; Gimeno, Lourdes; Mrowiec, Anna; Montes-Barqueros, Natividad R.; Campillo, José A.; Bolarin, José M.; Bernardo, María V.; López-Álvarez, María R.; González, Consuelo; García-Garay, María C.; Muro, Manuel; Cabañas-Perianes, Valentin; Fuster, Jose L.; García-Alonso, Ana M.; Moraleda, José M.; Álvarez-Lopez, María R.; Minguela, Alfredo

    2016-01-01

    ABSTRACT Missing self recognition makes cancer sensitive to natural killer cell (NKc) reactivity. However, this model disregards the NKc licensing effect, which highly increases NKc reactivity through interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) with their cognate HLA-I ligands. The influence of iKIR/HLA-ligand (HLA-C1/C2) licensing interactions on the susceptibility to and progression of plasma cell (PC) dyscrasias was evaluated in 164 Caucasian patients and 286 controls. Compared to controls, myeloma accumulates KIR2DL1−L2+L3− genotypes (2.8% vs. 13.2%, p < 0.01, OR = 5.29) and less diverse peripheral repertoires of NKc clones. Less diverse and weaker-affinity repertoires of iKIR2D/HLA-C licensing interactions increased myeloma susceptibility. Thus, the complete absence of conventional iKIR2D/HLA-C licensing interactions (KIR2DL1−L2+L3−/C2C2, 2.56% vs. 0.35%; p < 0.05; OR = 15.014), single-KIR2DL3+/C1+ (20.51% vs. 10.84%; p < 0.05; OR = 2.795) and single-KIR2DL2+/C1+ (12.82% vs. 4.9%; p < 0.01; OR = 5.18) interactions were over-represented in myeloma, compared to controls. Additionally, KIR2DL1−L2+L3− (20% vs. 83%, p < 0.00001) as well as KIR3DL1− (23% vs. 82%, p < 0.00001) genotypes had a dramatic negative impact on the 3-y progression-free survival (PFS), particularly in patients with low-tumor burden. Remarkably, myeloma-PCs, compared to K562 and other hematological cancers, showed substantial over-expression of HLA-I (“increasing-self” instead of missing-self), including HLA-C, and mild expression of ligands for NKc activating receptors (aRec) CD112, CD155, ULBP-1 and MICA/B, which apparently renders myeloma-PCs susceptible to lysis mainly by licensed NKc. KIR2DL1−L2+L3−/C2C2 patients (with no conventional iKIR2D/HLA-C licensing interactions) lyse K562 but barely lyse myeloma-PCs (4% vs. 15%; p < 0.05, compared to controls). These results support a model where immunosurveillance of no

  2. HLA-C and KIR combined genotype as new response marker for HBeAg-positive chronic hepatitis B patients treated with interferon-based combination therapy.

    PubMed

    Stelma, F; Jansen, L; Sinnige, M J; van Dort, K A; Takkenberg, R B; Janssen, H L A; Reesink, H W; Kootstra, N A

    2016-08-01

    Current treatment for chronic hepatitis B infection (CHB) consists of interferon-based therapy. However, for unknown reasons, a large proportion of patients with CHB do not respond to this treatment. Hence, there is a pressing need to establish response markers to select patients who will benefit from therapy and to spare potential nonresponders from unnecessary side effects of antiviral therapy. Here, we assessed whether HLA-C and KIR genotypes were associated with treatment outcome for CHB. Twelve SNPs in or near the HLA-C gene were genotyped in 86 CHB patients (41 HBeAg positive; 45 HBeAg negative) treated with peginterferon alfa-2a + adefovir. Genotyping of killer immunoglobin-like receptors (KIRs) was performed by SSP-PCR. One SNP in HLA-C (rs2308557) was significantly associated with combined response in HBeAg-positive CHB patients (P = 0.003). This SNP is linked to the HLA-C group C1 or C2 classification, which controls KIR binding. The combination of KIR2DL1 with its ligand HLA-C2 was observed significantly more often in HBeAg-positive patients with a combined response (13/14) than in nonresponders (11/27, P = 0.001). Patients with the KIR2DL1/C2 genotype had significantly higher baseline ALT levels (136 vs 50 U/L, P = 0.002) than patients without this combination. Furthermore, KIR2DL1-C2 predicted response independent of HBV genotype and ALT at baseline. HLA-C and KIR genotype is strongly associated with response in HBeAg-positive CHB patients treated with interferon-based therapy. In combination with other known response markers, HLA-C/KIR genotype could enable the selection of patients more likely to respond to interferon-based therapy. PMID:26945896

  3. Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

    PubMed

    Townsley, E; O'Connor, G; Cosgrove, C; Woda, M; Co, M; Thomas, S J; Kalayanarooj, S; Yoon, I-K; Nisalak, A; Srikiatkhachorn, A; Green, S; Stephens, H A F; Gostick, E; Price, D A; Carrington, M; Alter, G; McVicar, D W; Rothman, A L; Mathew, A

    2016-03-01

    Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

  4. Potassium inhibits nitric oxide and adenosine arteriolar vasodilatation via K(IR) and Na(+)/K(+) ATPase: implications for redundancy in active hyperaemia.

    PubMed

    Lamb, Iain R; Murrant, Coral L

    2015-12-01

    Redundancy, in active hyperaemia, where one vasodilator can compensate for another if the first is missing, would require that one vasodilator inhibits the effects of another; therefore, if the first vasodilator is inhibited, its inhibitory influence on the second vasodilator is removed and the second vasodilator exerts a greater vasodilatory effect. We aimed to determine whether vasodilators relevant to skeletal muscle contraction [potassium chloride (KCl), adenosine (ADO) and nitric oxide] inhibit one another and, in addition, to investigate the mechanisms for this interaction. We used the hamster cremaster muscle and intravital microscopy to directly visualize 2A arterioles when exposed to a range of concentrations of one vasodilator [10(-8) to 10(-5) M S-nitroso-N-acetyl penicillamine (SNAP), 10(-8) to 10(-5) M ADO, 10 and 20 mM KCl] in the absence and then in the presence of a second vasodilator (10(-7) M ADO, 10(-7) M SNAP, 10 mM KCl). We found that KCl significantly attenuated SNAP-induced vasodilatations by ∼65.8% and vasodilatations induced by 10(-8) to 10(-6) M ADO by ∼72.8%. Furthermore, we observed that inhibition of KCl vasodilatation, by antagonizing either Na(+)/K(+) ATPase using ouabain or inward rectifying potassium channels using barium chloride, could restore the SNAP-induced vasodilatation by up to ∼53.9% and 30.6%, respectively, and also restore the ADO-induced vasodilatations by up to ∼107% and 76.7%, respectively. Our data show that vasodilators relevant to muscle contraction can interact in a way that alters the effectiveness of other vasodilators. These data suggest that active hyperaemia may be the result of complex interactions between multiple vasodilators via a redundant control paradigm.

  5. KCNJ15/Kir4.2 couples with polyamines to sense weak extracellular electric fields in galvanotaxis

    PubMed Central

    Nakajima, Ken-ichi; Zhu, Kan; Sun, Yao-Hui; Hegyi, Bence; Zeng, Qunli; Murphy, Christopher J.; Small, J. Victor; Chen-Izu, Ye; Izumiya, Yoshihiro; Penninger, Josef M.; Zhao, Min

    2015-01-01

    Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K+ channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields. PMID:26449415

  6. RNA-mediated gene activation

    PubMed Central

    Jiao, Alan L; Slack, Frank J

    2014-01-01

    The regulation of gene expression by non-coding RNAs (ncRNAs) has become a new paradigm in biology. RNA-mediated gene silencing pathways have been studied extensively, revealing diverse epigenetic and posttranscriptional mechanisms. In contrast, the roles of ncRNAs in activating gene expression remains poorly understood. In this review, we summarize the current knowledge of gene activation by small RNAs, long non-coding RNAs, and enhancer-derived RNAs, with an emphasis on epigenetic mechanisms. PMID:24185374

  7. Cystic fibrosis transmembrane conductance regulator mediates sulphonylurea block of the inwardly rectifying K+ channel Kir6.1

    PubMed Central

    Ishida-Takahashi, Ayako; Otani, Hideo; Takahashi, Chiaki; Washizuka, Takashi; Tsuji, Keiko; Noda, Makoto; Horie, Minoru; Sasayama, Shigetake

    1998-01-01

    Recombinant ATP-sensitive K+ channels (KATP channels) were heterologously expressed in the NIH3T3 mouse cell line, and the electrophysiological properties were studied using patch-clamp techniques. The NIH3T3 cell lines transfected with the inwardly rectifying K+ channel Kir6.1 alone or with both Kir6.1 and cystic fibrosis transmembrane conductance regulator (CFTR) exhibited time-independent K+ currents with weak inward rectification. In contrast, no measurable K+ conductance was observed in mock-transfected cells or in cells transfected with CFTR alone. Regardless of co-transfection with Kir6.1, the transfection with CFTR produced a Cl− conductance that was activated by cell dialysis with cAMP (1 mm). The conductance was reversibly suppressed by glibenclamide (30 μm). Whole-cell currents at +60 mV were blocked in a concentration-dependent manner by Ba2+ ions with similar IC50 values: 89.3 ± 23.3 μm (Kir6.1 alone) and 67.3 ± 24.9 μm (Kir6.1-CFTR). The currents recorded from Kir6.1-transfected cells were not affected by glibenclamide, whereas glibenclamide did inhibit the conductance expressed in cells co-transfected with CFTR (IC50= 35.9 ± 6.6 μm). In the cell-attached mode with a 150 mm K+ pipette solution, both Kir6.1- and Kir6.1-CFTR-transfected cells displayed a class of K+ channels showing weak inward rectification and a slope conductance of 50.7 ± 1.0 and 52.4 ± 4.9 pS, respectively. In the inside-out mode, the single-channel currents recorded from both types of cells were not inhibited by intracellular ATP (1 mm). However, glibenclamide was found to block the single-channel activities in the co-transfected cells. PMID:9490811

  8. Crystal Structure of a Kir3.1-Prokaryotic Kir Channel Chimera

    SciTech Connect

    Nishida,M.; Cadene, M.; Chait, B.; MacKinnon, R.

    2007-01-01

    The Kir3.1 K+ channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2 Angstroms. The selectivity filter is identical to the Streptomyces lividans K+ channel within error of measurement (r.m.s.d.<0.2 Angstroms), suggesting that K+ selectivity requires extreme conservation of three-dimensional structure. Multiple K+ ions reside within the pore and help to explain voltage-dependent Mg2+ and polyamine blockade and strong rectification. Two constrictions, at the inner helix bundle and at the apex of the cytoplasmic pore, may function as gates: in one structure the apex is open and in the other, it is closed. Gating of the apex is mediated by rigid-body movements of the cytoplasmic pore subunits. Phosphatidylinositol 4, 5-biphosphate-interacting residues suggest a possible mechanism by which the signaling lipid regulates the cytoplasmic pore.

  9. Secondary anionic phospholipid binding site and gating mechanism in Kir2.1 inward rectifier channels

    PubMed Central

    Lee, Sun-Joo; Wang, Shizhen; Borschel, William; Heyman, Sarah; Gyore, Jacob; Nichols, Colin G.

    2013-01-01

    Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a crystallographically identified binding site, but an additional nonspecific secondary anionic phospholipid (PL(−)) is required to generate high PIP2 sensitivity of Kir2 channel gating. The PL(−)-binding site and mechanism are yet to be elucidated. Here we report docking simulations that identify a putative PL(−)-binding site, adjacent to the PIP2-binding site, generated by two lysine residues from neighbouring subunits. When either lysine is mutated to cysteine (K64C and K219C), channel activity is significantly decreased in cells and in reconstituted liposomes. Directly tethering K64C to the membrane by modification with decyl-MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(−)s. The results provide a coherent molecular mechanism whereby PL(−) interaction with a discrete binding site results in a conformational change that stabilizes the high-affinity PIP2 activatory site. PMID:24270915

  10. Estimating KIR Haplotype Frequencies on a Cohort of 10,000 Individuals: A Comprehensive Study on Population Variations, Typing Resolutions, and Reference Haplotypes

    PubMed Central

    Jayaraman, Jyothi; Trowsdale, John; Traherne, James; Kuang, Rui; Spellman, Stephen; Maiers, Martin

    2016-01-01

    The killer cell immunoglobulin-like receptors (KIR) mediate human natural killer (NK) cell cytotoxicity via activating or inhibiting signals. Although informative and functional haplotype patterns have been reported, most genotyping has been performed at resolutions that are structurally ambiguous. In order to leverage structural information given low-resolution genotypes, we performed experiments to quantify the effects of population variations, reference haplotypes, and genotyping resolutions on population-level haplotype frequency estimations as well as predictions of individual haplotypes. We genotyped 10,157 unrelated individuals in 5 populations (518 African American[AFA], 258 Asian or Pacific Islander[API], 8,245 European[EUR], 1,073 Hispanic[HIS], and 63 Native American[NAM]) for KIR gene presence/absence (PA), and additionally half of the AFA samples for KIR gene copy number variation (CNV). A custom EM algorithm was used to estimate haplotype frequencies for each population by interpretation in the context of three sets of reference haplotypes. The algorithm also assigns each individual the haplotype pairs of maximum likelihood. Generally, our haplotype frequency estimates agree with similar previous publications to within <5% difference for all haplotypes. The exception is that estimates for NAM from the U.S. showed higher frequency association of cB02 with tA01 (+14%) instead of tB01 (-8.5%) compared to a previous study of NAM from south of the U.S. The higher-resolution CNV genotyping on the AFA samples allowed unambiguous haplotype-pair assignments for the majority of individuals, resulting in a 22% higher median typing resolution score (TRS), which measures likelihood of self-match in the context of population-specific haplo- and geno-types. The use of TRS to quantify reduced ambiguity with CNV data clearly revealed the few individuals with ambiguous genotypes as outliers. It is observed that typing resolution and reference haplotype set influence

  11. Protective genotypes in HIV infection reflect superior function of KIR3DS1+ over KIR3DL1+ CD8+ T cells.

    PubMed

    Zipperlen, Katrin; Gallant, Maureen; Stapleton, Staci; Heath, John; Barrett, Lisa; Grant, Michael

    2015-01-01

    Certain human class I histocompatibility-linked leukocyte antigen (HLA)/killer cell immunoglobulin-like receptor (KIR) genotypic combinations confer more favourable prognoses upon exposure to human immunodeficiency virus (HIV). These combinations influence natural killer (NK) cell function, thereby implicating NK cells in protection from HIV infection or disease progression. Because CD8(+) T cells restrict HIV replication, depend upon HLA class I antigen presentation and can also express KIR molecules, we investigated how these HLA/KIR combinations relate to the phenotype and function of CD8(+) T cells from uninfected controls and individuals with chronic HIV infection. CD8(+) T cells from KIR3DL1 and KIR3DS1 homozygous individuals, and expressing the corresponding KIR, were enumerated and phenotyped for CD127, CD57 and CD45RA expression. Ex vivo and in vitro responsiveness to antigen-specific and polyclonal stimulation was compared between KIR-expressing and non-expressing CD8(+) T cells by interferon-γ production. There were higher numbers and fractions of KIR3DL1-expressing CD8(+) T cells in HIV-infected individuals independent of HLA-Bw4 co-expression, whereas expansion of KIR3DS1-expressing CD8(+) T cells reflected HLA-Bw4*80I co-expression. KIR3DL1(+) and S1(+) CD8(+) T cells were predominantly CD127(-)CD57(+)CD45RA(+). KIR3DL1-expressing CD8(+) T cells were insensitive to ex vivo stimulation with peptides from HIV or common viruses, but responded to anti-CD3 and recovered responsiveness to common viruses in vitro. Ex vivo non-responsiveness of KIR3DL1-expressing CD8(+) T cells was also independent of HLA-Bw4. KIR3DS1-expressing T cells responded normally to ex vivo antigenic stimulation, illustrating functional superiority over KIR3DL1(+) CD8(+) T cells.

  12. A hyperprostaglandin E syndrome mutation in Kir1.1 (renal outer medullary potassium) channels reveals a crucial residue for channel function in Kir1.3 channels.

    PubMed

    Derst, C; Wischmeyer, E; Preisig-Müller, R; Spauschus, A; Konrad, M; Hensen, P; Jeck, N; Seyberth, H W; Daut, J; Karschin, A

    1998-09-11

    Loss of function mutations in kidney Kir1.1 (renal outer medullary potassium channel, KCNJ1) inwardly rectifying potassium channels can be found in patients suffering from hyperprostaglandin E syndrome (HPS), the antenatal form of Bartter syndrome. A novel mutation found in a sporadic case substitutes an asparagine by a positively charged lysine residue at amino acid position 124 in the extracellular M1-H5 linker region. When heterologously expressed in Xenopus oocytes and mammalian cells, current amplitudes from mutant Kir1.1a[N124K] channels were reduced by a factor of approximately 12 as compared with wild type. A lysine at the equivalent position is present in only one of the known Kir subunits, the newly identified Kir1.3, which is also poorly expressed in the recombinant system. When the lysine residue in guinea pig Kir1.3 (gpKir1.3) isolated from a genomic library was changed to an asparagine (reverse HPS mutation), mutant channels yielded macroscopic currents with amplitudes increased 6-fold. From single channel analysis it became apparent that the decrease in mutant Kir1.1 channels and the increase in mutant gpKir1.3 macroscopic currents were mainly due to the number of expressed functional channels. Coexpression experiments revealed a dominant-negative effect of Kir1.1a[N124K] and gpKir1.3 on macroscopic current amplitudes when coexpressed with wild type Kir1.1a and gpKir[K110N], respectively. Thus we postulate that in Kir1.3 channels the extracellular positively charged lysine is of crucial functional importance. The HPS phenotype in man can be explained by the lower expression of functional channels by the Kir1. 1a[N124K] mutant. PMID:9727001

  13. Identification of the ancestral killer immunoglobulin-like receptor gene in primates

    PubMed Central

    Sambrook, Jennifer G; Bashirova, Arman; Andersen, Hanne; Piatak, Mike; Vernikos, George S; Coggill, Penny; Lifson, Jeff D; Carrington, Mary; Beck, Stephan

    2006-01-01

    Background Killer Immunoglobulin-like Receptors (KIR) are essential immuno-surveillance molecules. They are expressed on natural killer and T cells, and interact with human leukocyte antigens. KIR genes are highly polymorphic and contribute vital variability to our immune system. Numerous KIR genes, belonging to five distinct lineages, have been identified in all primates examined thus far and shown to be rapidly evolving. Since few KIR remain orthologous between species, with only one of them, KIR2DL4, shown to be common to human, apes and monkeys, the evolution of the KIR gene family in primates remains unclear. Results Using comparative analyses, we have identified the ancestral KIR lineage (provisionally named KIR3DL0) in primates. We show KIR3DL0 to be highly conserved with the identification of orthologues in human (Homo sapiens), common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), rhesus monkey (Macaca mulatta) and common marmoset (Callithrix jacchus). We predict KIR3DL0 to encode a functional molecule in all primates by demonstrating expression in human, chimpanzee and rhesus monkey. Using the rhesus monkey as a model, we further show the expression profile to be typical of KIR by quantitative measurement of KIR3DL0 from an enriched population of natural killer cells. Conclusion One reason why KIR3DL0 may have escaped discovery for so long is that, in human, it maps in between two related leukocyte immunoglobulin-like receptor clusters outside the known KIR gene cluster on Chromosome 19. Based on genomic, cDNA, expression and phylogenetic data, we report a novel lineage of immunoglobulin receptors belonging to the KIR family, which is highly conserved throughout 50 million years of primate evolution. PMID:16911775

  14. Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2: A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection.

    PubMed

    Jovanović, Sofija; Ballantyne, Thomas; Du, Qingyou; Blagojević, Miloš; Jovanović, Aleksandar

    2016-01-01

    ATP-sensitive K(+) (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100nM) for 24h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K(+) current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10mM), which was accompanied by increased in K(+) current in response to DNP (10mM). Cytoprotection afforded by phenylephrine (100nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K(+) conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes.

  15. Co-evolution of Human Leukocyte Antigen (HLA) Class I Ligands with Killer-Cell Immunoglobulin-Like Receptors (KIR) in a Genetically Diverse Population of Sub-Saharan Africans

    PubMed Central

    Norman, Paul J.; Hollenbach, Jill A.; Nemat-Gorgani, Neda; Guethlein, Lisbeth A.; Hilton, Hugo G.; Pando, Marcelo J.; Koram, Kwadwo A.; Riley, Eleanor M.; Abi-Rached, Laurent; Parham, Peter

    2013-01-01

    Interactions between HLA class I molecules and killer-cell immunoglobulin-like receptors (KIR) control natural killer cell (NK) functions in immunity and reproduction. Encoded by genes on different chromosomes, these polymorphic ligands and receptors correlate highly with disease resistance and susceptibility. Although studied at low-resolution in many populations, high-resolution analysis of combinatorial diversity of HLA class I and KIR is limited to Asian and Amerindian populations with low genetic diversity. At the other end of the spectrum is the West African population investigated here: we studied 235 individuals, including 104 mother-child pairs, from the Ga-Adangbe of Ghana. This population has a rich diversity of 175 KIR variants forming 208 KIR haplotypes, and 81 HLA-A, -B and -C variants forming 190 HLA class I haplotypes. Each individual we studied has a unique compound genotype of HLA class I and KIR, forming 1–14 functional ligand-receptor interactions. Maintaining this exceptionally high polymorphism is balancing selection. The centromeric region of the KIR locus, encoding HLA-C receptors, is highly diverse whereas the telomeric region encoding Bw4-specific KIR3DL1, lacks diversity in Africans. Present in the Ga-Adangbe are high frequencies of Bw4-bearing HLA-B*53:01 and Bw4-lacking HLA-B*35:01, which otherwise are identical. Balancing selection at key residues maintains numerous HLA-B allotypes having and lacking Bw4, and also those of stronger and weaker interaction with LILRB1, a KIR-related receptor. Correspondingly, there is a balance at key residues of KIR3DL1 that modulate its level of cell-surface expression. Thus, capacity to interact with NK cells synergizes with peptide binding diversity to drive HLA-B allele frequency distribution. These features of KIR and HLA are consistent with ongoing co-evolution and selection imposed by a pathogen endemic to West Africa. Because of the prevalence of malaria in the Ga-Adangbe and previous

  16. Reduction in renal blood flow following administration of norepinephrine and phenylephrine in septic rats treated with Kir6.1 ATP-sensitive and KCa1.1 calcium-activated K+ channel blockers.

    PubMed

    da Rosa Maggi Sant'Helena, Bruna; Guarido, Karla L; de Souza, Priscila; Crestani, Sandra; da Silva-Santos, J Eduardo

    2015-10-15

    We evaluated the effects of K+ channel blockers in the vascular reactivity of in vitro perfused kidneys, as well as on the influence of vasoactive agents in the renal blood flow of rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Both norepinephrine and phenylephrine had the ability to increase the vascular perfusion pressure reduced in kidneys of rats subjected to CLP at 18 h and 36 h before the experiments. The non-selective K+ channel blocker tetraethylammonium, but not the Kir6.1 blocker glibenclamide, normalized the effects of phenylephrine in kidneys from the CLP 18 h group. Systemic administration of tetraethylammonium, glibenclamide, or the KCa1.1 blocker iberiotoxin, did not change the renal blood flow in control or septic rats. Norepinephrine or phenylephrine also had no influence on the renal blood flow of septic animals, but its injection in rats from the CLP 18 h group previously treated with either glibenclamide or iberiotoxin resulted in an exacerbated reduction in the renal blood flow. These results suggest an abnormal functionality of K+ channels in the renal vascular bed in sepsis, and that the blockage of different subtypes of K+ channels may be deleterious for blood perfusion in kidneys, mainly when associated with vasoactive drugs.

  17. Reduction in renal blood flow following administration of norepinephrine and phenylephrine in septic rats treated with Kir6.1 ATP-sensitive and KCa1.1 calcium-activated K+ channel blockers.

    PubMed

    da Rosa Maggi Sant'Helena, Bruna; Guarido, Karla L; de Souza, Priscila; Crestani, Sandra; da Silva-Santos, J Eduardo

    2015-10-15

    We evaluated the effects of K+ channel blockers in the vascular reactivity of in vitro perfused kidneys, as well as on the influence of vasoactive agents in the renal blood flow of rats subjected to the cecal ligation and puncture (CLP) model of sepsis. Both norepinephrine and phenylephrine had the ability to increase the vascular perfusion pressure reduced in kidneys of rats subjected to CLP at 18 h and 36 h before the experiments. The non-selective K+ channel blocker tetraethylammonium, but not the Kir6.1 blocker glibenclamide, normalized the effects of phenylephrine in kidneys from the CLP 18 h group. Systemic administration of tetraethylammonium, glibenclamide, or the KCa1.1 blocker iberiotoxin, did not change the renal blood flow in control or septic rats. Norepinephrine or phenylephrine also had no influence on the renal blood flow of septic animals, but its injection in rats from the CLP 18 h group previously treated with either glibenclamide or iberiotoxin resulted in an exacerbated reduction in the renal blood flow. These results suggest an abnormal functionality of K+ channels in the renal vascular bed in sepsis, and that the blockage of different subtypes of K+ channels may be deleterious for blood perfusion in kidneys, mainly when associated with vasoactive drugs. PMID:26277325

  18. Elucidation of the Inhibitory Effect of Phytochemicals with Kir6.2 Wild-Type and Mutant Models Associated in Type-1 Diabetes through Molecular Docking Approach.

    PubMed

    Jagadeb, Manaswini; Konkimalla, V Badireenath; Rath, Surya Narayan; Das, Rohit Pritam

    2014-12-01

    Among all serious diseases globally, diabetes (type 1 and type 2) still poses a major challenge to the world population. Several target proteins have been identified, and the etiology causing diabetes has been reasonably well studied. But, there is still a gap in deciding on the choice of a drug, especially when the target is mutated. Mutations in the KCNJ11 gene, encoding the kir6.2 channel, are reported to be associated with congenital hyperinsulinism, having a major impact in causing type 1 diabetes, and due to the lack of its 3D structure, an attempt has been made to predict the structure of kir6.2, applying fold recognition methods. The current work is intended to investigate the affinity of four phytochemicals namely, curcumin (Curcuma longa), genistein (Genista tinctoria), piperine (Piper nigrum), and pterostilbene (Vitis vinifera) in a normal as well as in a mutant kir6.2 model by adopting a molecular docking methodology. The phytochemicals were docked in both wild and mutated kir6.2 models in two rounds: blind docking followed by ATP-binding pocket-specific docking. From the binding pockets, the common interacting amino acid residues participating strongly within the binding pocket were identified and compared. From the study, we conclude that these phytochemicals have strong affinity in both the normal and mutant kir6.2 model. This work would be helpful for further study of the phytochemicals above for the treatment of type 1 diabetes by targeting the kir6.2 channel. PMID:25705171

  19. Elucidation of the Inhibitory Effect of Phytochemicals with Kir6.2 Wild-Type and Mutant Models Associated in Type-1 Diabetes through Molecular Docking Approach

    PubMed Central

    Jagadeb, Manaswini; Konkimalla, V Badireenath; Das, Rohit Pritam

    2014-01-01

    Among all serious diseases globally, diabetes (type 1 and type 2) still poses a major challenge to the world population. Several target proteins have been identified, and the etiology causing diabetes has been reasonably well studied. But, there is still a gap in deciding on the choice of a drug, especially when the target is mutated. Mutations in the KCNJ11 gene, encoding the kir6.2 channel, are reported to be associated with congenital hyperinsulinism, having a major impact in causing type 1 diabetes, and due to the lack of its 3D structure, an attempt has been made to predict the structure of kir6.2, applying fold recognition methods. The current work is intended to investigate the affinity of four phytochemicals namely, curcumin (Curcuma longa), genistein (Genista tinctoria), piperine (Piper nigrum), and pterostilbene (Vitis vinifera) in a normal as well as in a mutant kir6.2 model by adopting a molecular docking methodology. The phytochemicals were docked in both wild and mutated kir6.2 models in two rounds: blind docking followed by ATP-binding pocket-specific docking. From the binding pockets, the common interacting amino acid residues participating strongly within the binding pocket were identified and compared. From the study, we conclude that these phytochemicals have strong affinity in both the normal and mutant kir6.2 model. This work would be helpful for further study of the phytochemicals above for the treatment of type 1 diabetes by targeting the kir6.2 channel. PMID:25705171

  20. Natural killer cell receptor genes in the family Equidae: not only Ly49.

    PubMed

    Futas, Jan; Horin, Petr

    2013-01-01

    Natural killer (NK) cells have important functions in immunity. NK recognition in mammals can be mediated through killer cell immunoglobulin-like receptors (KIR) and/or killer cell lectin-like Ly49 receptors. Genes encoding highly variable NK cell receptors (NKR) represent rapidly evolving genomic regions. No single conservative model of NKR genes was observed in mammals. Single-copy low polymorphic NKR genes present in one mammalian species may expand into highly polymorphic multigene families in other species. In contrast to other non-rodent mammals, multiple Ly49-like genes appear to exist in the horse, while no functional KIR genes were observed in this species. In this study, Ly49 and KIR were sought and their evolution was characterized in the entire family Equidae. Genomic sequences retrieved showed the presence of at least five highly conserved polymorphic Ly49 genes in horses, asses and zebras. These findings confirmed that the expansion of Ly49 occurred in the entire family. Several KIR-like sequences were also identified in the genome of Equids. Besides a previously identified non-functional KIR-Immunoglobulin-like transcript fusion gene (KIR-ILTA) and two putative pseudogenes, a KIR3DL-like sequence was analyzed. In contrast to previous observations made in the horse, the KIR3DL sequence, genomic organization and mRNA expression suggest that all Equids might produce a functional KIR receptor protein molecule with a single non-mutated immune tyrosine-based inhibition motif (ITIM) domain. No evidence for positive selection in the KIR3DL gene was found. Phylogenetic analysis including rhinoceros and tapir genomic DNA and deduced amino acid KIR-related sequences showed differences between families and even between species within the order Perissodactyla. The results suggest that the order Perissodactyla and its family Equidae with expanded Ly49 genes and with a potentially functional KIR gene may represent an interesting model for evolutionary biology of

  1. Natural Killer Cell Receptor Genes in the Family Equidae: Not only Ly49

    PubMed Central

    Futas, Jan; Horin, Petr

    2013-01-01

    Natural killer (NK) cells have important functions in immunity. NK recognition in mammals can be mediated through killer cell immunoglobulin-like receptors (KIR) and/or killer cell lectin-like Ly49 receptors. Genes encoding highly variable NK cell receptors (NKR) represent rapidly evolving genomic regions. No single conservative model of NKR genes was observed in mammals. Single-copy low polymorphic NKR genes present in one mammalian species may expand into highly polymorphic multigene families in other species. In contrast to other non-rodent mammals, multiple Ly49-like genes appear to exist in the horse, while no functional KIR genes were observed in this species. In this study, Ly49 and KIR were sought and their evolution was characterized in the entire family Equidae. Genomic sequences retrieved showed the presence of at least five highly conserved polymorphic Ly49 genes in horses, asses and zebras. These findings confirmed that the expansion of Ly49 occurred in the entire family. Several KIR-like sequences were also identified in the genome of Equids. Besides a previously identified non-functional KIR-Immunoglobulin-like transcript fusion gene (KIR-ILTA) and two putative pseudogenes, a KIR3DL-like sequence was analyzed. In contrast to previous observations made in the horse, the KIR3DL sequence, genomic organization and mRNA expression suggest that all Equids might produce a functional KIR receptor protein molecule with a single non-mutated immune tyrosine-based inhibition motif (ITIM) domain. No evidence for positive selection in the KIR3DL gene was found. Phylogenetic analysis including rhinoceros and tapir genomic DNA and deduced amino acid KIR-related sequences showed differences between families and even between species within the order Perissodactyla. The results suggest that the order Perissodactyla and its family Equidae with expanded Ly49 genes and with a potentially functional KIR gene may represent an interesting model for evolutionary biology of

  2. Natural killer cell receptor genes in the family Equidae: not only Ly49.

    PubMed

    Futas, Jan; Horin, Petr

    2013-01-01

    Natural killer (NK) cells have important functions in immunity. NK recognition in mammals can be mediated through killer cell immunoglobulin-like receptors (KIR) and/or killer cell lectin-like Ly49 receptors. Genes encoding highly variable NK cell receptors (NKR) represent rapidly evolving genomic regions. No single conservative model of NKR genes was observed in mammals. Single-copy low polymorphic NKR genes present in one mammalian species may expand into highly polymorphic multigene families in other species. In contrast to other non-rodent mammals, multiple Ly49-like genes appear to exist in the horse, while no functional KIR genes were observed in this species. In this study, Ly49 and KIR were sought and their evolution was characterized in the entire family Equidae. Genomic sequences retrieved showed the presence of at least five highly conserved polymorphic Ly49 genes in horses, asses and zebras. These findings confirmed that the expansion of Ly49 occurred in the entire family. Several KIR-like sequences were also identified in the genome of Equids. Besides a previously identified non-functional KIR-Immunoglobulin-like transcript fusion gene (KIR-ILTA) and two putative pseudogenes, a KIR3DL-like sequence was analyzed. In contrast to previous observations made in the horse, the KIR3DL sequence, genomic organization and mRNA expression suggest that all Equids might produce a functional KIR receptor protein molecule with a single non-mutated immune tyrosine-based inhibition motif (ITIM) domain. No evidence for positive selection in the KIR3DL gene was found. Phylogenetic analysis including rhinoceros and tapir genomic DNA and deduced amino acid KIR-related sequences showed differences between families and even between species within the order Perissodactyla. The results suggest that the order Perissodactyla and its family Equidae with expanded Ly49 genes and with a potentially functional KIR gene may represent an interesting model for evolutionary biology of

  3. The K+ channel KIR2.1 functions in tandem with proton influx to mediate sour taste transduction.

    PubMed

    Ye, Wenlei; Chang, Rui B; Bushman, Jeremy D; Tu, Yu-Hsiang; Mulhall, Eric M; Wilson, Courtney E; Cooper, Alexander J; Chick, Wallace S; Hill-Eubanks, David C; Nelson, Mark T; Kinnamon, Sue C; Liman, Emily R

    2016-01-12

    Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn(2+)-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K(+) current. We identify KIR2.1 as the acid-sensitive K(+) channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of Kir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K(+) current in amplifying the sensory response, entry of protons through the Zn(2+)-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K(+) channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.

  4. The K+ channel KIR2.1 functions in tandem with proton influx to mediate sour taste transduction

    PubMed Central

    Ye, Wenlei; Chang, Rui B.; Bushman, Jeremy D.; Tu, Yu-Hsiang; Mulhall, Eric M.; Wilson, Courtney E.; Cooper, Alexander J.; Chick, Wallace S.; Hill-Eubanks, David C.; Nelson, Mark T.; Kinnamon, Sue C.; Liman, Emily R.

    2016-01-01

    Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn2+-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K+ current. We identify KIR2.1 as the acid-sensitive K+ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of Kir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K+ current in amplifying the sensory response, entry of protons through the Zn2+-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K+ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids. PMID:26627720

  5. Functional Expression of Kir4.1 Channels in Spinal Cord Astrocytes

    PubMed Central

    OLSEN, M.L.; HIGASHIMORI, H.; CAMPBELL, S.L.; HABLITZ, J.J.; SONTHEIMER, H.

    2008-01-01

    Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild-type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ. PMID:16369934

  6. Humans differ from other hominids in lacking an activating NK cell receptor that recognizes the C1 epitope of MHC class I.

    PubMed

    Moesta, Achim K; Graef, Thorsten; Abi-Rached, Laurent; Older Aguilar, Anastazia M; Guethlein, Lisbeth A; Parham, Peter

    2010-10-01

    Modulation of human NK cell function by killer cell Ig-like receptors (KIR) and MHC class I is dominated by the bipartite interactions of inhibitory lineage III KIR with the C1 and C2 epitopes of HLA-C. In comparison, the ligand specificities and functional contributions of the activating lineage III KIR remain poorly understood. Using a robust, sensitive assay of KIR binding and a representative panel of 95 HLA class I targets, we show that KIR2DS1 binds C2 with ~50% the avidity of KIR2DL1, whereas KIR2DS2, KIR2DS3, and KIR2DS5 have no detectable avidity for C1, C2, or any other HLA class I epitope. In contrast, the chimpanzee has activating C1- and C2-specific lineage III KIR with strong avidity, comparable to those of their paired inhibitory receptors. One variant of chimpanzee Pt-KIR3DS2, the activating C2-specific receptor, has the same avidity for C2 as does inhibitory Pt-KIR3DL4, and a second variant has ~73% the avidity. Chimpanzee Pt-KIR3DS6, the activating C1-specific receptor, has avidity for C1 that is ~70% that of inhibitory Pt-KIR2DL6. In both humans and chimpanzees we observe an evolutionary trend toward reducing the avidity of the activating C1- and C2-specific receptors through selective acquisition of attenuating substitutions. However, the extent of attenuation has been extreme in humans, as exemplified by KIR2DS2, an activating C1-specific receptor that has lost all detectable avidity for HLA class I. Supporting such elimination of activating C1-specific receptors as a uniquely human phenomenon is the presence of a high-avidity activating C1-specific receptor (Gg-KIR2DSa) in gorilla.

  7. The Impact of KIR Polymorphism on the Risk of Developing Cancer: Not as Strong as Imagined?

    PubMed Central

    Augusto, Danillo G.

    2016-01-01

    The polymorphism of killer cell immunoglobulin-like receptors (KIR) has been associated with several diseases, including infection, autoimmunity and cancer. KIR molecules are a family of receptors expressed on the surface of natural killer cells (NK), frontline defense of innate immunity against microorganisms and neoplastic cells. Some studies have shown conflicting results concerning the role that KIR polymorphism plays in tumor susceptibility, particularly in leukemia and lymphoma. Interestingly, the presence of HLA ligands is sometimes strongly associated with several types of cancer and apparently is not related with their interaction with KIR. This manuscript briefly reviews the uncommon polymorphism of KIR and critically summarizes the recent findings with regards of the importance of KIR variation for cancer susceptibility. PMID:27446203

  8. Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

    PubMed Central

    Sicca, Federico; Ambrosini, Elena; Marchese, Maria; Sforna, Luigi; Servettini, Ilenio; Valvo, Giulia; Brignone, Maria Stefania; Lanciotti, Angela; Moro, Francesca; Grottesi, Alessandro; Catacuzzeno, Luigi; Baldini, Sara; Hasan, Sonia; D’Adamo, Maria Cristina; Franciolini, Fabio; Molinari, Paola; Santorelli, Filippo M.; Pessia, Mauro

    2016-01-01

    Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy. PMID:27677466

  9. Lack of KIR4.1 autoantibodies in Japanese patients with MS and NMO

    PubMed Central

    Higuchi, Osamu; Sakai, Waka; Maeda, Yasuhiro; Niino, Masaaki; Takahashi, Toshiyuki; Fukazawa, Toshiyuki; Kikuchi, Seiji; Fujihara, Kazuo; Matsuo, Hidenori

    2016-01-01

    Objectives: To examine anti-KIR4.1 antibodies by 2 different assays in Japanese patients with multiple sclerosis (MS) or neuromyelitis optica (NMO). Methods: One hundred sixty serum samples from 57 patients with MS, 40 patients with NMO/NMO spectrum disorder (NMOSD), and 50 healthy controls (all were Japanese) were tested with ELISA using a synthetic peptide of the first extracellular portion of human KIR4.1. In addition, we attempted to detect anti-KIR4.1 immunoglobulin G in the serum by the luciferase immunoprecipitation systems (LIPS) with the full length of human KIR4.1 produced in a human cell line, which is highly sensitive to single or multiple epitopes. Results: We failed to detect antibodies to the peptide fragment KIR4.183–120 in any case of MS and NMO/NMOSD using ELISA. Antibodies to the recombinant full length of KIR4.1 protein were detected in only 2 patients with MS and none in the patients with NMO/NMOSD by the LIPS assay. Conclusions: We developed 2 different methods (ELISA and LIPS) to measure autoantibodies to KIR4.1 in serum. We detected anti-KIR4.1 immunoglobulin G at a very low frequency in Japanese patients with MS or NMO/NMOSD. Serologic testing for human KIR4.1-specific antibodies is unlikely to improve the diagnosis of MS or NMO/NMOSD in Japanese patients. PMID:27489866

  10. Regulation of Kir2.1 channels by the Rho-GTPase, Rac1

    PubMed Central

    Boyer, Stephanie B.; Slesinger, Paul A.; Jones, S.V. Penelope

    2010-01-01

    Mutations in Kir2.1 inwardly rectifying potassium channels are associated with Andersen Syndrome, a disease characterized by potentially fatal cardiac arrhythmias. While several Andersen-associated mutations affect membrane expression, the cytoplasmic signals that regulate Kir2.1 trafficking are poorly understood. Here, we investigated whether the Rho-family of small GTPases regulates trafficking of Kir2.1 channels expressed in HEK-293 cells. Treatment with C. difficile toxin B, an inhibitor of Rho-family GTPases, or co-expression of the dominant-negative mutant of Rac1 (Rac1DN) increased Kir2.1 current density ~2-fold. However, the dominant-negative forms of other Rho-family GTPases, RhoA or Cdc42, did not alter Kir2.1 currents, suggesting a selective effect of Rac1 on Kir2.1 current density. Single-channel properties (γ, τo, τc) and total protein levels of Kir2.1 were unchanged with co-expression of Rac1DN; however, studies using TIRF microscopy and CFP-tagged Kir2.1 revealed increased channel surface expression. Immunohistochemical detection of extracellularly-tagged HA-Kir2.1 channels showed that Rac1DN reduced channel internalization when co-expressed. Finally, the dominant-negative mutant of dynamin, which interferes with endocytosis, occluded the Rac1DN–induced potentiation of Kir2.1 currents. These data suggest that inhibition of Rac1 increases Kir2.1 surface expression by interfering with endocytosis, likely via a dynamin-dependent pathway. Surprisingly, Rac1DN did not alter Kir2.2 current density or internalization, suggesting subunit specific modulation of Kir2.1 channels. Consistent with this, construction of Kir2.1/2.2 chimeras implicated the C-terminal domain of Kir2.1 in mediating the potentiating effect of Rac1DN. This novel pathway for regulating surface expression of cardiac Kir2.1 channels could have implications for normal and diseased cardiac states. PMID:18932198

  11. Kir7.1 immunoreactivity in canine choroid plexus tumors.

    PubMed

    Choi, Eun Ju; Sloma, Erica A; Miller, Andrew D

    2016-07-01

    Choroid plexus neoplasms are uncommon brain tumors in dogs. Choroid plexus carcinomas often spread diffusely throughout the ventricular system and subarachnoid space and, in aggressive forms, can mimic histologic patterns of other carcinomas, including being embedded in a desmoplastic reaction. Although choroid plexus tumors (CPTs) heterogeneously express pan-cytokeratin, little is known about other markers to identify choroid plexus and their associated tumors. Kir7.1, an inward-rectifier potassium channel, is reported to have high diagnostic utility in human neuropathology to distinguish CPTs from other primary brain tumors and cerebral metastases. To determine Kir7.1 expression in the dog brain, we analyzed the immunoreactivity of Kir7.1 in normal brain, gliomas, ependymomas, CPTs, meningiomas, and carcinomas. In normal brain tissue, the immunostaining was restricted to the choroid plexus where there was robust membrane immunoreactivity along the apical border of the cells with less intense cytoplasmic staining. Similar strong immunoreactivity was detected in 12 of 12 CPTs, whereas 5 of 5 gliomas, 4 of 5 ependymomas, 5 of 5 meningiomas, and 5 of 6 carcinomas had no immunoreactivity. One ependymoma and 1 nasal carcinoma with squamous metaplasia were up to 75% immunopositive, with moderate cytoplasmic and membranous immunoreactivity, but lacking the robust apical immunoreactivity pattern. Analysis for immunoreactivity in a tissue microarray failed to yield any other locations in which immunoreactivity was detected. These results, including the distinctive pattern of immunostaining in CPTs, suggest that Kir7.1 is an excellent marker for CPTs in the dog. PMID:27216721

  12. Permanent neonatal diabetes due to mutations in KCNJ11 encoding Kir6.2: patient characteristics and initial response to sulfonylurea therapy.

    PubMed

    Sagen, Jørn V; Raeder, Helge; Hathout, Eba; Shehadeh, Naim; Gudmundsson, Kolbeinn; Baevre, Halvor; Abuelo, Dianne; Phornphutkul, Chanika; Molnes, Janne; Bell, Graeme I; Gloyn, Anna L; Hattersley, Andrew T; Molven, Anders; Søvik, Oddmund; Njølstad, Pål R

    2004-10-01

    Permanent neonatal diabetes (PND) can be caused by mutations in the transcription factors insulin promoter factor (IPF)-1, eukaryotic translation initiation factor-2alpha kinase 3 (EIF2AK3), and forkhead box-P3 and in key components of insulin secretion: glucokinase (GCK) and the ATP-sensitive K(+) channel subunit Kir6.2. We sequenced the gene encoding Kir6.2 (KCNJ11) in 11 probands with GCK-negative PND. Heterozygous mutations were identified in seven probands, causing three novel (F35V, Y330C, and F333I) and two known (V59M and R201H) Kir6.2 amino acid substitutions. Only two probands had a family history of diabetes. Subjects with the V59M mutation had neurological features including motor delay. Three mutation carriers tested had an insulin secretory response to tolbutamide, but not to glucose or glucagon. Glibenclamide was introduced in increasing doses to investigate whether sulfonylurea could replace insulin. At a glibenclamide dose of 0.3-0.4 mg. kg(-1). day(-1), insulin was discontinued. Blood glucose did not deteriorate, and HbA(1c) was stable or fell during 2-6 months of follow-up. An oral glucose tolerance test performed in one subject revealed that glucose-stimulated insulin release was restored. Mutations in Kir6.2 were the most frequent cause of PND in our cohort. Apparently insulin-dependent patients with mutations in Kir6.2 may be managed on an oral sulfonylurea with sustained metabolic control rather than insulin injections, illustrating the principle of pharmacogenetics applied in diabetes treatment.

  13. Chromosomal location of a pollen fertility-restoring gene, Rf, for CMS in Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of A. galanthum Kar. et Kir. revealed by genomic in situ hybridization.

    PubMed

    Yamashita, Ken-Ichiro; Takatori, Yuka; Tashiro, Yosuke

    2005-06-01

    In a previous study, we developed cytoplasmic male sterile lines of Allium fistulosum possessing the cytoplasm of A. galanthum, a wild species, by continuous backcrossing. Furthermore, we reported the presence of a pollen fertility-restoring gene (Rf) for cytoplasmic male sterility (CMS) in A. fistulosum from segregation of pollen fertility of backcross progenies. In the present study, genomic in situ hybridization (GISH), using genomic DNA of A. galanthum as the probe DNA and that of A. fistulosum as the blocking DNA, was applied to F(1) hybrids between both species and backcross progenies to determine the chromosomal location of the Rf locus. By means of GISH, eight chromosomes from A. galanthum were clearly discriminated from those of A. fistulosum in the F(1) hybrids, and chromosome substitution process through continuous backcrossing was visualized. Interestingly, the chromosome region from A. galanthum, specific to male fertile plants, was detected in one chromosome of BC(4) to BC(7) generations. Based on the karyotype analysis of the male fertile plants, the chromosome was identified as the 5F chromosome. Our results confirm that the Rf locus is located on the 5F chromosome of the male fertile plants. This is the first report that identified the chromosomal location of the pollen fertility-restoring gene in A. fistulosum. PMID:15883793

  14. Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations

    PubMed Central

    González-Galarza, Faviel F.; Takeshita, Louise Y.C.; Santos, Eduardo J.M.; Kempson, Felicity; Maia, Maria Helena Thomaz; Silva, Andrea Luciana Soares da; Silva, André Luiz Teles e; Ghattaoraya, Gurpreet S.; Alfirevic, Ana; Jones, Andrew R.; Middleton, Derek

    2015-01-01

    It has been 12 years since the Allele Frequency Net Database (AFND; http://www.allelefrequencies.net) was first launched, providing the scientific community with an online repository for the storage of immune gene frequencies in different populations across the world. There have been a significant number of improvements from the first version, making AFND a primary resource for many clinical and scientific areas including histocompatibility, immunogenetics, pharmacogenetics and anthropology studies, among many others. The most widely used part of AFND stores population frequency data (alleles, genes or haplotypes) related to human leukocyte antigens (HLA), killer-cell immunoglobulin-like receptors (KIR), major histocompatibility complex class I chain-related genes (MIC) and a number of cytokine gene polymorphisms. AFND now contains >1400 populations from more than 10 million healthy individuals. Here, we report how the main features of AFND have been updated to include a new section on ‘HLA epitope’ frequencies in populations, a new section capturing the results of studies identifying HLA associations with adverse drug reactions (ADRs) and one for the examination of infectious and autoimmune diseases associated with KIR polymorphisms—thus extending AFND to serve a new user base in these growing areas of research. New criteria on data quality have also been included. PMID:25414323

  15. Carvedilol inhibits Kir2.3 channels by interference with PIP₂-channel interaction.

    PubMed

    Ferrer, Tania; Ponce-Balbuena, Daniela; López-Izquierdo, Angélica; Aréchiga-Figueroa, Ivan A; de Boer, Teun P; van der Heyden, Marcel A G; Sánchez-Chapula, José A

    2011-10-01

    Carvedilol, a β- and α-adrenoceptor blocker, is used to treat congestive heart failure, mild to moderate hypertension, and myocardial infarction. It has been proposed to block K(ATP) channels by binding to the bundle crossing region at a domain including cysteine at position 166, and thereby plugging the pore region. However, carvedilol was reported not to affect Kir2.1 channels, which lack 166 Cys. Here, we demonstrate that carvedilol inhibits Kir2.3 carried current by an alternative mechanism. Carvedilol inhibited Kir2.3 channels with at least 100 fold higher potency (IC(50)=0.49 μM) compared to that for Kir2.1 (IC(50)>50 μM). Kir2.3 channel inhibition was concentration-dependent and voltage-independent. Increasing Kir2.3 channel affinity for PIP(2), by a I213L point mutation, decreased the inhibitory effect of carvedilol more than twentyfold (IC(50)=11.1 μM). In the presence of exogenous PIP(2), Kir2.3 channel inhibition by carvedilol was strongly reduced (80 vs. 2% current inhibition). These results suggest that carvedilol, as other cationic amphiphilic drugs, inhibits Kir2.3 channels by interfering with the PIP(2)-channel interaction.

  16. The effect of pregnancy on the uterine NK cell KIR repertoire

    PubMed Central

    Male, Victoria; Sharkey, Andrew; Masters, Leanne; Kennedy, Philippa R; Farrell, Lydia E; Moffett, Ashley

    2011-01-01

    The major leukocyte population in the decidua during the first trimester of pregnancy consists of NK cells that express receptors capable of recognizing MHC class I molecules expressed by placental trophoblast. These include members of the killer immunoglobulin-like receptor (KIR) family, the two-domain KIR (KIR2D), which recognize HLA-C. Interactions between decidual NK (dNK) cell KIR2D and placental HLA-C contribute to the success of pregnancy and dNK cells express KIR2D at higher frequency than peripheral NK (pNK) cells. Thus, they are biased toward recognizing HLA-C. In order to investigate when this unusual KIR repertoire appears, we compared the phenotype of NK cells isolated from non-pregnant (endometrium) and pregnant (decidua) human uterine mucosa. Endometrial NK (eNK) cells did not express KIR2D at a higher level than matched pNK cells, so the bias toward HLA-C recognition occurs as a response to pregnancy. Furthermore, HLA-C expression was upregulated on uterine stromal cells as the mucosa transformed from endometrium to decidua at the onset of pregnancy. As uterine NK (uNK) cells can mature from NK precursors and acquire KIR expression in utero, the pregnancy-specific bias of uNK cells toward HLA-C recognition could arise as developing uNK cells interact with uterine stromal cells, which express higher levels of HLA-C during pregnancy. PMID:21739430

  17. Killer cell immunoglobulin-like receptor gene association with cryptorchidism.

    PubMed

    Niepiekło-Miniewska, Wanda; Kuśnierczyk, Piotr; Havrylyuk, Anna; Kamieniczna, Marzena; Nakonechnyy, Andrij; Chopyak, Valentyna; Kurpisz, Maciej

    2015-12-01

    Cryptorchidism is a condition where a testis persists in the abdominal cavity. Thus, due to elevated temperature we may expect induction of aberrant immune reactions depending on genetic constitution of individual. This may be reflected by development of anti-sperm antibodies (ASA) in cryptorchid males. Also, natural killer (NK) cells which belong to innate immunity may control adaptive immunity. Therefore, the gene system encoding polymorphic NK cell immunoglobulin receptors (KIRs) has been studied. 109 prepubertal boys with cryptorchidism and 136 ethnically matched young male donors were selected to study NK cell KIRs. DNA was isolated using automatic Maxwell(®) system from the peripheral venous blood drawn onto anticoagulant. Olerup SSP KIR Genotyping kit including Taq polymerase was used for detection of KIR genes. Human leukocyte antigen-C (HLA-C) groups, C1 and C2 were established using a Olerup SSP KIR HLA Ligand kit. KIR2DL2 (killer immunoglobulin-like receptor two-domain long 2) and KIR2DS2 (killer immunoglobulin-like receptor two-domain short 2) genes were less frequent in patients than in control individuals (corrected p values: 0.0110 and 0.0383, respectively). However, no significant differences were observed between ASA-positive and ASA-negative patients, or between bilateral or unilateral cryptorchidism. No association between KIR ligands C1 and C2, alone or together with KIR2DL2, was found. However, the results suggest that KIR2DL2+/KIR2DS2+ genotype may be, to some extent, protective against cryptorchidism.

  18. Gain-of-Function Mutation, S422L, in the KCNJ8-Encoded Cardiac KATP Channel Kir6.1 as a Pathogenic Substrate for J Wave Syndromes

    PubMed Central

    Medeiros-Domingo, Argelia; Tan, Bi-Hua; Crotti, Lia; Tester, David J.; Eckhardt, Lee; Cuoretti, Alessandra; Kroboth, Stacie L.; Song, Chunhua; Zhou, Qing; Kopp, Doug; Schwartz, Peter J.; Makielski, Jonathan C.; Ackerman, Michael J.

    2011-01-01

    Background J Wave Syndromes have emerged conceptually to encompass the pleiotropic expression of J point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). Recently, KCNJ8, which encodes the cardiac KATP Kir6.1 channel, has been implicated in ERS following the identification of a functionally uncharacterized missense mutation, S422L. Here, we sought to further explore KCNJ8 as a novel susceptibility gene for J wave syndromes. Methods Using PCR, DHPLC, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of KCNJ8 was performed in 101 unrelated patients with J wave syndromes including 87 with BrS and 14 with ERS. 600 healthy individuals were examined to assess allelic frequency for all variants detected. KCNJ8 mutation(s) were engineered by site directed mutagenesis and co-expressed heterologously with SUR2A in COS-1 cells. Ion currents were recorded using whole cell configuration of the patch-clamp technique. Results One BrS case and one ERS case hosted the identical missense mutation, S422L that was reported previously. KCNJ8-S422L involves a highly conserved residue and was absent in 1200 reference alleles. Both cases were negative for mutations in all known BrS- and ERS-susceptibility genes. The KATP current of Kir6.1-S422L mutation was increased significantly over the voltage range of 0 mV to 40 mV compared to Kir6.1-WT channels (p < 0.05, n=16-21). Conclusions These findings further implicate KCNJ8 as a novel J wave syndrome-susceptibility gene and a marked gain-of-function in the cardiac KATP Kir6.1 channel secondary to KCNJ8-S422L as a novel pathogenic mechanism for the phenotypic expression of both BrS and ERS. PMID:20558321

  19. Charges in the cytoplasmic pore control intrinsic inward rectification and single-channel properties in Kir1.1 and Kir2.1 channels.

    PubMed

    Chang, Hsueh-Kai; Yeh, Shih-Hao; Shieh, Ru-Chi

    2007-02-01

    An E224G mutation of the Kir2.1 channel generates intrinsic inward rectification and single-channel fluctuations in the absence of intracellular blockers. In this study, we showed that positively charged residues H226, R228 and R260, near site 224, regulated the intrinsic inward rectification and single-channel properties of the E224G mutant. By carrying out systematic mutations, we found that the charge effect on the intrinsic inward rectification and single-channel conductance is consistent with a long-range electrostatic mechanism. A Kir1.1 channel where the site equivalent to E224 in the Kir2.1 channel is a glycine residue does not show inward rectification or single-channel fluctuations. The G223K and N259R mutations of the Kir1.1 channel induced intrinsic inward rectification and reduced the single-channel conductance but did not generate large open-channel fluctuations. Substituting the cytoplasmic pore of the E224G mutant into the Kir1.1 channel induced open-channel fluctuations and intrinsic inward rectification. The single-channel conductance of the E224G mutant showed inward rectification. Also, a voltage-dependent gating mechanism decreased open probability during depolarization and contributed to the intrinsic inward rectification in the E224G mutant. In addition to an electrostatic effect, a close interaction of K(+) with channel pore may be required for generating open-channel fluctuations in the E224G mutant.

  20. A phase I trial of the anti-KIR antibody IPH2101 and lenalidomide in patients with relapsed/refractory multiple myeloma

    PubMed Central

    Benson, Don M.; Cohen, Adam D.; Jagannath, Sundar; Munshi, Nikhil C.; Spitzer, Gary; Hofmeister, Craig C.; Efebera, Yvonne A.; Andre, Pascale; Zerbib, Robert; Caligiuri, Michael A.

    2015-01-01

    PURPOSE Natural killer (NK) cells may play an important role in the immune response to multiple myeloma (MM); however, MM cells express killer immunoglobulin-like receptor (KIR) ligands to prevent NK cell cytotoxicity. Lenalidomide can expand and activate NK cells in parallel with its direct effects against MM; however, dexamethasone may impair these favorable immunomodulatory properties. IPH2101, a first-in-class anti-inhibitory KIR antibody, has acceptable safety and tolerability in MM as a single agent. The present work sought to characterize lenalidomide and IPH2101 as a novel, steroid-sparing, dual immune therapy for MM. EXPERIMENTAL DESIGN A phase I trial enrolled 15 patients in three cohorts. Lenalidomide was administered per os at 10mg on cohort 1 and 25mg on cohorts 2 and 3 days 1–21 on a 28-day cycle with IPH2101 given intravenously on day 1 of each cycle at 0.2 mg/kg on cohort 1, 1mg/kg on cohort 2, and 2mg/kg on cohort 3. No corticosteroids were utilized. The primary endpoint was safety, secondary endpoints included clinical activity, pharmacokinetics (PK) and pharmacodynamics (PD). RESULTS The biologic endpoint of full KIR occupancy was achieved across the IPH2101 dosing interval. PD and PK of IPH2101 with lenalidomide were similar to data from a prior single agent IPH2101 trial. Five serious adverse events (SAEs) were reported. Five objective responses occurred. No autoimmunity was seen. CONCLUSIONS These findings suggest that lenalidomide in combination with anti-inhibitory KIR therapy warrants further investigation in MM as a steroid-sparing, dual immune therapy. This trial was registered at www.clinicaltrials.gov (reference: NCT01217203). PMID:25999435

  1. Kir3 channel ontogeny – the role of Gβγ subunits in channel assembly and trafficking

    PubMed Central

    Zylbergold, Peter; Sleno, Rory; Khan, Shahriar M.; Jacobi, Ashley M.; Belhke, Mark A.; Hébert, Terence E.

    2014-01-01

    The role of Gβγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gβγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gβγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gβ1γ2, apart from its role in channel opening that it shares with other Gβγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gβ1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gβγ effects on Kir3 maturation and trafficking. PMID:24782712

  2. Neuronal and glial expression of inward rectifier potassium channel subunits Kir2.x in rat dorsal root ganglion and spinal cord.

    PubMed

    Murata, Yuzo; Yasaka, Toshiharu; Takano, Makoto; Ishihara, Keiko

    2016-03-23

    Inward rectifier K(+) channels of the Kir2.x subfamily play important roles in controlling the neuronal excitability. Although their cellular localization in the brain has been extensively studied, only a few studies have examined their expression in the spinal cord and peripheral nervous system. In this study, immunohistochemical analyses of Kir2.1, Kir2.2, and Kir2.3 expression were performed in rat dorsal root ganglion (DRG) and spinal cord using bright-field and confocal microscopy. In DRG, most ganglionic neurons expressed Kir2.1, Kir2.2 and Kir2.3, whereas satellite glial cells chiefly expressed Kir2.3. In the spinal cord, Kir2.1, Kir2.2 and Kir2.3 were all expressed highly in the gray matter of dorsal and ventral horns and moderately in the white matter also. Within the gray matter, the expression was especially high in the substantia gelatinosa (lamina II). Confocal images obtained using markers for neuronal cells, NeuN, and astrocytes, Sox9, showed expression of all three Kir2 subunits in both neuronal somata and astrocytes in lamina I-III of the dorsal horn and the lateral spinal nucleus of the dorsolateral funiculus. Immunoreactive signals other than those in neuronal and glial somata were abundant in lamina I and II, which probably located mainly in nerve fibers or nerve terminals. Colocalization of Kir2.1 and 2.3 and that of Kir2.2 and 2.3 were present in neuronal and glial somata. In the ventral horn, motor neurons and interneurons were also immunoreactive with the three Kir2 subunits. Our study suggests that Kir2 channels composed of Kir2.1-2.3 subunits are expressed in neuronal and glial cells in the DRG and spinal cord, contributing to sensory transduction and motor control. PMID:26854211

  3. Kir4.1 K+ channels are regulated by external cations.

    PubMed

    Edvinsson, Johan M; Shah, Anish J; Palmer, Lawrence G

    2011-01-01

    The inwardly rectifying potassium channel (Kir), Kir4.1 mediates spatial K(+)-buffering in the CNS. In this process the channel is potentially exposed to a large range of extracellular K(+) concentrations ([K(+)]o). We found that Kir4.1 is regulated by K(+)o. Increased [K(+)]o leads to a slow (mins) increase in the whole-cell currents of Xenopus oocytes expressing Kir4.1. Conversely, removing K(+) from the bath solution results in a slow decrease of the currents. This regulation is not coupled to the pHi-sensitive gate of the channel, nor does it require the presence of K67, a residue necessary for K(+)o-dependent regulation of Kir1.1. The voltage-dependent blockers Cs(+) and Ba(2+) substitute for K(+) and prevent deactivation of the channel in the absence of K(+)o. Cs(+) blocks and regulates the channel with similar affinity, consistent with the regulatory sites being in the selectivity-filter of the channel. Although both Rb(+) and NH4(+) permeate Kir4.1, only Rb(+) is able to regulate the channel. We conclude that Kir4.1 is regulated by ions interacting with specific sites in the selectivity filter. Using a kinetic model of the permeation process we show the plausibility of the channel's sensing the extracellular ionic environment through changes in the selectivity occupancy pattern, and that it is feasible for an ion with the selectivity properties of NH4(+) to permeate the channel without inducing these changes. PMID:21532341

  4. Hyperglycemia reduces functional expression of astrocytic Kir4.1 channels and glial glutamate uptake.

    PubMed

    Rivera-Aponte, D E; Méndez-González, M P; Rivera-Pagán, A F; Kucheryavykh, Y V; Kucheryavykh, L Y; Skatchkov, S N; Eaton, M J

    2015-12-01

    Diabetics are at risk for a number of serious health complications including an increased incidence of epilepsy and poorer recovery after ischemic stroke. Astrocytes play a critical role in protecting neurons by maintaining extracellular homeostasis and preventing neurotoxicity through glutamate uptake and potassium buffering. These functions are aided by the presence of potassium channels, such as Kir4.1 inwardly rectifying potassium channels, in the membranes of astrocytic glial cells. The purpose of the present study was to determine if hyperglycemia alters Kir4.1 potassium channel expression and homeostatic functions of astrocytes. We used q-PCR, Western blot, patch-clamp electrophysiology studying voltage and potassium step responses and a colorimetric glutamate clearance assay to assess Kir4.1 channel levels and homeostatic functions of rat astrocytes grown in normal and high glucose conditions. We found that astrocytes grown in high glucose (25 mM) had an approximately 50% reduction in Kir4.1 mRNA and protein expression as compared with those grown in normal glucose (5mM). These reductions occurred within 4-7 days of exposure to hyperglycemia, whereas reversal occurred between 7 and 14 days after return to normal glucose. The decrease in functional Kir channels in the astrocytic membrane was confirmed using barium to block Kir channels. In the presence of 100-μM barium, the currents recorded from astrocytes in response to voltage steps were reduced by 45%. Furthermore, inward currents induced by stepping extracellular [K(+)]o from 3 to 10mM (reflecting potassium uptake) were 50% reduced in astrocytes grown in high glucose. In addition, glutamate clearance by astrocytes grown in high glucose was significantly impaired. Taken together, our results suggest that down-regulation of astrocytic Kir4.1 channels by elevated glucose may contribute to the underlying pathophysiology of diabetes-induced CNS disorders and contribute to the poor prognosis after stroke

  5. Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A

    PubMed Central

    Surah-Narwal, S; Xu, S Z; McHugh, D; McDonald, R L; Hough, E; Cheong, A; Partridge, C; Sivaprasadarao, A; Beech, D J

    1999-01-01

    A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced.Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co-injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone.K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 μM glibenclamide. K+-current carried by hKir6.1+SUR1 was inhibited by the putative vascular-selective KATP channel inhibitor U37883A (IC50 32 μM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected.The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore-forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels. PMID:10516647

  6. Associations between genes for killer immunoglobulin-like receptors and their ligands in patients with solid tumors.

    PubMed

    Al Omar, Suliman; Middleton, Derek; Marshall, Ernie; Porter, Dawn; Xinarianos, George; Raji, Olaide; Field, John K; Christmas, Stephen E

    2010-10-01

    Killer immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) genotypes were analyzed from panels of lung (non-small-cell lung cancer [NSCLC] and small-cell lung cancer [SCLC]), colon, and kidney cancer patients and compared with normal control subjects. No significant differences were noted between KIR gene frequencies in patients compared with normal subjects. When combinations of KIR genes and their HLA ligands were considered, there were significant decreases in frequencies of both KIR2DL2 and KIR2DL3 in homozygotes for their ligand HLA-C1, and an increase in the frequency of KIR3DL1 and its ligand HLA-Bw4 in kidney cancer patients compared with controls. Both associations were partly attributable to changes in ligand frequencies alone. NSCLC patients showed a significant increase in the frequency of KIR2DL1 and its ligand HLA-C2 and a corresponding decrease in frequency of KIR2DL3 and its ligand HLA-C1 in homozygotes. In NSCLC, the Ile80 form of HLA-Bw4 was decreased in KIR3DL1+ HLA-Bw4+ patients, whereas in SCLC the Ile80 form was increased and the Thr80 form decreased in KIR3DS1+ HLA-Bw4+ patients. These findings are consistent with increased co-expression of high-affinity inhibitory KIRs and their ligands, potentially resulting in decreased natural killer cell function, and hence with natural killer cells having a protective role in lung and kidney cancer but not colon cancer.

  7. A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity.

    PubMed

    Sackin, Henry; Nanazashvili, Mikheil; Li, Hui; Palmer, Lawrence G; Walters, D Eric

    2010-01-01

    ROMK (Kir1.1) channels are important for K secretion and recycling in the collecting duct, connecting tubule and thick ascending limb of the mammalian nephron. We have identified a highly conserved Arg in the P loop of the channel near the selectivity filter that controls Rb/K selectivity. Mutation of this Arg to a Tyr (R128Y-Kir1.1b, R147Y-Kir1.1a) increased the macroscopic conductance ratio, G(Rb)/G(K) by 17 ± 3 fold and altered the selectivity sequence from NH(4) > K > Tl > Rb > Cs in wt-Kir1.1 to: Rb > Cs > Tl > NH(4) > K in R128Y, without significant change in the high K/Na permeability ratio of Kir1.1. R128M produced similar, but smaller, increases in Rb, Tl, NH(4) and Cs conductance relative to K. R128Y remained susceptible to block by both external Ba and the honeybee toxin, TPNQ, although R128Y had a reduced affinity for TPNQ, relative to wild-type. The effect of R128Y-Kir1.1b on the G(Rb)/G(K) ratio can be partly explained by a larger single-channel Rb conductance (12.4 ± 0.5 pS) than K conductance (<1.5 pS) in this mutant. The kinetics of R128Y gating at -120 mV with Rb as the permeant ion were similar to those of wt-Kir1.1 conducting Rb, but with a longer open time (129 ms vs. 6 ms for wt) and two closed states (13 ms, 905 ms), resulting in an open probability (Po) of 0.5, compared to a Po of 0.9 for wt-Kir1.1, which had a single closed state of 1 ms at -120 mV. Single-channel R128Y rectification was eliminated in excised, insideout patches with symmetrical Rb solutions. The large increase in the Rb/K conductance ratio, with no change in K/Na permeability or rectification, is consistent with R128Y-Kir1.1b causing a subtle change in the selectivity filter, perhaps by disruption of an intra-subunit salt bridge (R128-E118) near the filter. PMID:20458182

  8. A potential molecular target for morphological defects of fetal alcohol syndrome: Kir2.1.

    PubMed

    Bates, Emily A

    2013-06-01

    Fetal alcohol spectrum disorder (FASD) is a developmental disorder that affects up to 0.2% of births. FASD comprises severe cognitive and structural birth defects including cleft lip/palate, small jaw, wide-set eyes, dental abnormalities, digit abnormalities, small head, and short stature. Strict counseling guidelines stress abstaining from alcohol during pregnancy, but the prevalence of FASD persists. The lack of a convincing molecular target has hindered FASD research and treatment. Interestingly, mutations in an inwardly rectifying potassium channel, Kir2.1, cause a similar constellation of birth defects as in FASD. In other words, FASD phenocopies the traits conveyed by Kir2.1 mutations. Furthermore, alcohol directly binds to and modulates Kir2.1. Substantial evidence now suggests that alcohol targets Kir2.1 to cause the birth defects associated with FASD. This review compiles clinical, genetic, biochemical, electrophysiological, and molecular evidence that identifies Kir2.1 as a molecular target for FASD development and possibly therapeutic treatment.

  9. KIR4.1: K+ Channel Illusion or Reality in the Autoimmune Pathogenesis of Multiple Sclerosis

    PubMed Central

    Gu, Chen

    2016-01-01

    Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Many believe autoimmune pathogenesis plays a key role in MS, but its target(s) remains elusive. A recent study detected autoantibodies against KIR4.1, an ATP-sensitive, inward rectifier potassium channel, in nearly half of the MS patients examined. KIR4.1 channels are expressed in astrocytes. Together with aquaporin 4 (AQP4) water channels, they regulate astrocytic functions vital for myelination. Autoantibodies against AQP4 have been established as a key biomarker for neuromyelitis optica (NMO) and contributed to diagnostic and treatment strategy adjustments. Similarly, identification of KIR4.1 autoantibodies could have high therapeutic values in treating MS. Consistent with its potential role in MS, KIR4.1 dysfunction is implicated in several neurological disorders. However, the enrichment of KIR4.1 autoantibodies in MS patients is questioned by follow-up studies. Further, investigations are needed to clarify this controversy and unravel the underlying mechanisms of MS pathogenesis. PMID:27729847

  10. Active genes at the nuclear pore complex.

    PubMed

    Taddei, Angela

    2007-06-01

    The nucleus is spatially and functionally organized and its architecture is now seen as a key contributor to genome functions. A central component of this architecture is the nuclear envelope, which is studded with nuclear pore complexes that serve as gateways for communication between the nucleoplasm and cytoplasm. Although the nuclear periphery has traditionally been described as a repressive compartment and repository for gene-poor chromosome regions, several recent studies in yeast have demonstrated that repressive and activating domains can both be positioned at the periphery of the nucleus. Moreover, association with the nuclear envelope favors the expression of particular genes, demonstrating that nuclear organization can play an active role in gene regulation. PMID:17467257

  11. The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling.

    PubMed

    Oszmiana, Anna; Williamson, David J; Cordoba, Shaun-Paul; Morgan, David J; Kennedy, Philippa R; Stacey, Kevin; Davis, Daniel M

    2016-05-31

    Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.

  12. The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling.

    PubMed

    Oszmiana, Anna; Williamson, David J; Cordoba, Shaun-Paul; Morgan, David J; Kennedy, Philippa R; Stacey, Kevin; Davis, Daniel M

    2016-05-31

    Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling. PMID:27210755

  13. Adenosine Triphosphate-Sensitive Potassium Channel Kir Subunits Implicated in Cardioprotection by Diazoxide

    PubMed Central

    Henn, Matthew C; Janjua, M Burhan; Kanter, Evelyn M; Makepeace, Carol M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2015-01-01

    Background ATP-sensitive potassium (KATP) channel openers provide cardioprotection in multiple models. Ion flux at an unidentified mitochondrial KATP channel has been proposed as the mechanism. The renal outer medullary kidney potassium channel subunit, potassium inward rectifying (Kir)1.1, has been implicated as a mitochondrial channel pore-forming subunit. We hypothesized that subunit Kir1.1 is involved in cardioprotection (maintenance of volume homeostasis and contractility) of the KATP channel opener diazoxide (DZX) during stress (exposure to hyperkalemic cardioplegia [CPG]) at the myocyte and mitochondrial levels. Methods and Results Kir subunit inhibitor Tertiapin Q (TPN-Q) was utilized to evaluate response to stress. Mouse ventricular mitochondrial volume was measured in the following groups: isolation buffer; 200 μmol/L of ATP; 100 μmol/L of DZX+200 μmol/L of ATP; or 100 μmol/L of DZX+200 μmol/L of ATP+TPN-Q (500 or 100 nmol/L). Myocytes were exposed to Tyrode’s solution (5 minutes), test solution (Tyrode’s, cardioplegia [CPG], CPG+DZX, CPG+DZX+TPN-Q, Tyrode’s+TPN-Q, or CPG+TPN-Q), N=12 for all (10 minutes); followed by Tyrode’s (5 minutes). Volumes were compared. TPN-Q, with or without DZX, did not alter mitochondrial or myocyte volume. Stress (CPG) resulted in myocyte swelling and reduced contractility that was prevented by DZX. TPN-Q prevented the cardioprotection afforded by DZX (volume homeostasis and maintenance of contractility). Conclusions TPN-Q inhibited myocyte cardioprotection provided by DZX during stress; however, it did not alter mitochondrial volume. Because TPN-Q inhibits Kir1.1, Kir3.1, and Kir3.4, these data support that any of these Kir subunits could be involved in the cardioprotection afforded by diazoxide. However, these data suggest that mitochondrial swelling by diazoxide does not involve Kir1.1, 3.1, or 3.4. PMID:26304939

  14. Changes in aquaporin-4 and Kir4.1 expression in rats with inherited retinal dystrophy.

    PubMed

    Lassiale, S; Valamanesh, F; Klein, C; Hicks, D; Abitbol, M; Versaux-Botteri, C

    2016-07-01

    Muller glial cells (MGC) are essential for normal functioning of retina. They are especially involved in potassium (K+) and water homeostasis, via inwardly rectifying K+ (Kir 4.1) and aquaporin-4 (AQP4) channels respectively. Because MGC appear morphologically and functionally altered in most retinal pathologies, we studied the expression of AQP 4 and Kir 4.1 during the time course of progressive retinal degeneration in Royal College of Surgeons (RCS) rats, an animal model for the hereditary human retinal degenerative disease Retinitis pigmentosa. Simultaneous detection of AQP4 and Kir 4.1 was performed by quantitative real-time polymerase chain reaction (QRT-PCR), Western blot and immunohistochemistry at birth and during progression of the pathology. Although small quantities of AQP4 and Kir 4.1 mRNA were detected at birth (postnatal day (PNd) 0) in both control and dystrophic rat retinas, proteins could not be detected at this age. Detectable proteins appeared in the second week of postnatal life. From PNd15 onwards, the time course in the expression of both AQP4 and Kir 4.1 mRNAs and protein was similar in dystrophic and control rats, with a progressive increase peaking at PNd60 and a subsequent decrease by one year. AQP4 protein and mRNA content were significantly lowered in dystrophic compared to control rats. Kir 4.1 protein levels were also lower in dystrophic retinas, while mRNA concentrations were unchanged and/or slightly higher in dystrophic rats. The discrepancies between Kir4.1 mRNA and protein suggest perturbation in protein translation due to the pathology. AQP4 and Kir 4.1/vimentin co-immunolabeling showed that: 1) apical radial processes of some MGC invaded the subretinal zone, and 2) MGC morphology was distorted in advanced pathology. MGC became hypertrophic both during the pathology and also with age in control rats. In conclusion, our results confirm that this inherited photoreceptor degeneration also leads to progressive alterations in

  15. Acute exposure of methylglyoxal leads to activation of KATP channels expressed in HEK293 cells

    PubMed Central

    Yang, Yang; Konduru, Anuhya S; Cui, Ningren; Yu, Lei; Trower, Timothy C; Shi, Weiwei; Shi, Yun; Jiang, Chun

    2014-01-01

    Aim: Highly reactive carbonyl methylglyoxal (MGO) is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium (KATP) channel. In the present study we investigated the effects of MGO on the activity of KATP channels. Methods: Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2Δ36 (a truncated Kir6.2 isoform) alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software. Results: The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil (10 μmol/L) could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide (10 μmol/L). MGO (0.1-10 mmol/L) dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Kir6.2Δ36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO (3 mmol/L). Single channel recordings showed that MGO (3 mmol/L) markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered. Conclusion: Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits. PMID:24122011

  16. Functional effects of mutations at F35 in the NH2-terminus of Kir6.2 (KCNJ11), causing neonatal diabetes, and response to sulfonylurea therapy.

    PubMed

    Proks, Peter; Girard, Christophe; Baevre, Halvor; Njølstad, Pål R; Ashcroft, Frances M

    2006-06-01

    Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive K(+) channel (K(ATP) channel), cause neonatal diabetes. To date, all mutations increase whole-cell K(ATP) channel currents by reducing channel inhibition by MgATP. Here, we provide functional characterization of two mutations (F35L and F35V) at residue F35 of Kir6.2, which lies within the NH(2)-terminus. We further show that the F35V patient can be successfully transferred from insulin to sulfonylurea therapy. The patient has been off insulin for 24 months and shows improved metabolic control (mean HbA(1c) 7.58 before and 6.18% after sulfonylurea treatment; P < 0.007). Wild-type and mutant Kir6.2 were heterologously coexpressed with SUR1 in Xenopus oocytes. Whole-cell K(ATP) channel currents through homomeric and heterozygous F35V and F35L channels were increased due to a reduced sensitivity to inhibition by MgATP. The mutation also increased the open probability (P(O)) of homomeric F35 mutant channels in the absence of ATP. These effects on P(O) and ATP sensitivity were abolished in the absence of SUR1. Our results suggest that mutations at F35 cause permanent neonatal diabetes by affecting K(ATP) channel gating and thereby, indirectly, ATP inhibition. Heterozygous F35V channels were markedly inhibited by the sulfonylurea tolbutamide, accounting for the efficacy of sulfonylurea therapy in the patient.

  17. Mapping gene activity of Arabidopsis root hairs

    PubMed Central

    2013-01-01

    Background Quantitative information on gene activity at single cell-type resolution is essential for the understanding of how cells work and interact. Root hairs, or trichoblasts, tubular-shaped outgrowths of specialized cells in the epidermis, represent an ideal model for cell fate acquisition and differentiation in plants. Results Here, we provide an atlas of gene and protein expression in Arabidopsis root hair cells, generated by paired-end RNA sequencing and LC/MS-MS analysis of protoplasts from plants containing a pEXP7-GFP reporter construct. In total, transcripts of 23,034 genes were detected in root hairs. High-resolution proteome analysis led to the reliable identification of 2,447 proteins, 129 of which were differentially expressed between root hairs and non-root hair tissue. Dissection of pre-mRNA splicing patterns showed that all types of alternative splicing were cell type-dependent, and less complex in EXP7-expressing cells when compared to non-root hair cells. Intron retention was repressed in several transcripts functionally related to root hair morphogenesis, indicative of a cell type-specific control of gene expression by alternative splicing of pre-mRNA. Concordance between mRNA and protein expression was generally high, but in many cases mRNA expression was not predictive for protein abundance. Conclusions The integrated analysis shows that gene activity in root hairs is dictated by orchestrated, multilayered regulatory mechanisms that allow for a cell type-specific composition of functional components. PMID:23800126

  18. Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string.

    PubMed

    Milius, R P; Mack, S J; Hollenbach, J A; Pollack, J; Heuer, M L; Gragert, L; Spellman, S; Guethlein, L A; Trachtenberg, E A; Cooley, S; Bochtler, W; Mueller, C R; Robinson, J; Marsh, S G E; Maiers, M

    2013-08-01

    Knowledge of an individual's human leukocyte antigen (HLA) genotype is essential for modern medical genetics, and is crucial for hematopoietic stem cell and solid-organ transplantation. However, the high levels of polymorphism known for the HLA genes make it difficult to generate an HLA genotype that unambiguously identifies the alleles that are present at a given HLA locus in an individual. For the last 20 years, the histocompatibility and immunogenetics community has recorded this HLA genotyping ambiguity using allele codes developed by the National Marrow Donor Program (NMDP). While these allele codes may have been effective for recording an HLA genotyping result when initially developed, their use today results in increased ambiguity in an HLA genotype, and they are no longer suitable in the era of rapid allele discovery and ultra-high allele polymorphism. Here, we present a text string format capable of fully representing HLA genotyping results. This Genotype List (GL) String format is an extension of a proposed standard for reporting killer-cell immunoglobulin-like receptor (KIR) genotype data that can be applied to any genetic data that use a standard nomenclature for identifying variants. The GL String format uses a hierarchical set of operators to describe the relationships between alleles, lists of possible alleles, phased alleles, genotypes, lists of possible genotypes, and multilocus unphased genotypes, without losing typing information or increasing typing ambiguity. When used in concert with appropriate tools to create, exchange, and parse these strings, we anticipate that GL Strings will replace NMDP allele codes for reporting HLA genotypes.

  19. Direct Activation of β-Cell KATP Channels with a Novel Xanthine Derivative

    PubMed Central

    Raphemot, Rene; Swale, Daniel R.; Dadi, Prasanna K.; Jacobson, David A.; Cooper, Paige; Wojtovich, Andrew P.; Banerjee, Sreedatta; Nichols, Colin G.

    2014-01-01

    ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry. PMID:24646456

  20. Is Kir6.1 a subunit of mitoK{sub ATP}?

    SciTech Connect

    Brian Foster, D.; Rucker, Jasma J.; Marban, Eduardo

    2008-02-15

    The subunit composition of the mitochondrial ATP-sensitive K{sup +}-channel (mitoK{sub ATP}) is unknown, though some suspect a role for the inward rectifier, Kir6.1, based largely on antibody studies of heart mitochondria. To ascertain the molecular identity of mitoK{sub ATP} we therefore sought to purify this putative mitochondrial Kir6.1, and conclusively identify the subunits by mass spectrometry. Immunoblots, conducted with two commercially available antibodies, revealed two distinct signals in isolated heart mitochondria, of 51 and 48 kDa, respectively. Localization was confirmed by either immuno-gold electron microscopy or by immunofluorescence. Each putative Kir6.1 species was extracted, purified, and identified by LC-MS/MS. The 51 kDa band was identified as NADH-dehydrogenase flavoprotein 1, while the preponderant protein in the 48-kDa band was mitochondrial isocitrate dehydrogenase (NADP form). 1D-, 2D-, and native gel analyses were consistent with these assignments. The data suggest it is premature to assign Kir6.1 a role in mitoK{sub ATP} on the basis of immunoreactivity alone.

  1. Selection of an HLA-C*03:04-Restricted HIV-1 p24 Gag Sequence Variant Is Associated with Viral Escape from KIR2DL3+ Natural Killer Cells: Data from an Observational Cohort in South Africa

    PubMed Central

    Jimenez Cruz, Camilo A.; Garcia-Beltran, Wilfredo F.; Carlson, Jonathan M.; van Teijlingen, Nienke H.; Mann, Jaclyn K.; Jaggernath, Manjeetha; Kang, Seung-gu; Körner, Christian; Chung, Amy W.; Schafer, Jamie L.; Evans, David T.; Alter, Galit; Walker, Bruce D.; Goulder, Philip J.; Carrington, Mary; Hartmann, Pia; Pertel, Thomas; Zhou, Ruhong; Ndung’u, Thumbi; Altfeld, Marcus

    2015-01-01

    Background Viruses can evade immune surveillance, but the underlying mechanisms are insufficiently understood. Here, we sought to understand the mechanisms by which natural killer (NK) cells recognize HIV-1-infected cells and how this virus can evade NK-cell-mediated immune pressure. Methods and Findings Two sequence mutations in p24 Gag associated with the presence of specific KIR/HLA combined genotypes were identified in HIV-1 clade C viruses from a large cohort of infected, untreated individuals in South Africa (n = 392), suggesting viral escape from KIR+ NK cells through sequence variations within HLA class I—presented epitopes. One sequence polymorphism at position 303 of p24 Gag (TGag303V), selected for in infected individuals with both KIR2DL3 and HLA-C*03:04, enabled significantly better binding of the inhibitory KIR2DL3 receptor to HLA-C*03:04-expressing cells presenting this variant epitope compared to the wild-type epitope (wild-type mean 18.01 ± 10.45 standard deviation [SD] and variant mean 44.67 ± 14.42 SD, p = 0.002). Furthermore, activation of primary KIR2DL3+ NK cells from healthy donors in response to HLA-C*03:04+ target cells presenting the variant epitope was significantly reduced in comparison to cells presenting the wild-type sequence (wild-type mean 0.78 ± 0.07 standard error of the mean [SEM] and variant mean 0.63 ± 0.07 SEM, p = 0.012). Structural modeling and surface plasmon resonance of KIR/peptide/HLA interactions in the context of the different viral sequence variants studied supported these results. Future studies will be needed to assess processing and antigen presentation of the investigated HIV-1 epitope in natural infection, and the consequences for viral control. Conclusions These data provide novel insights into how viruses can evade NK cell immunity through the selection of mutations in HLA-presented epitopes that enhance binding to inhibitory NK cell receptors. Better understanding of the mechanisms by which HIV-1 evades

  2. Kir1.1 (ROMK) and Kv7.1 (KCNQ1/KvLQT1) are essential for normal gastric acid secretion: importance of functional Kir1.1.

    PubMed

    Vucic, Esad; Alfadda, Tariq; MacGregor, Gordon G; Dong, Ke; Wang, Tong; Geibel, John P

    2015-07-01

    Potassium channels comprise the apical leak pathway supplying extracellular K(+) for exchange with protons by the gastric H(+), K(+)-ATPase and provide potential therapeutic targets for inhibiting gastric acid secretion. The Kir1.1 (ROMK) potassium channel mediates the high capacity K(+) recycling necessary for NaCl reabsorption in the thick ascending limb of the kidney, and this channel exhibits functional and regulatory characteristic well suited for K(+) recycling by gastric parietal cells. We report here that Kir1.1 channels are required for gastric acid secretion and that this channel participates with Kv7.1 (KCNQ1/KvLQT1) in the potassium recycling process. We show that Kir1.1 colocalizes with the β-subunit of H(+), K(+)-ATPase in gastric parietal cells of Kir1.1 wild-type mice. In Kir1.1-deficient mice, gastric mucosal morphology, as well as parietal cell number, proliferation index, and ultrastructure were normal but secretagogue-stimulated gastric acid secretion in whole stomach and perfused gastric glands was absent. Luminal application of potassium-restored acid secretion in perfused gastric glands from Kir1.1-deficient as well as barium-blocked wild-type mice. In wild-type mice, both luminal Tertiapin-Q, an inhibitor of Kir1.1, as well as XE991, an inhibitor of Kv7.1, reduced proton secretion. We propose that Kir1.1 and Kv7.1 channels collaborate in potassium and current recycling across the apical pole of parietal cells. PMID:25127675

  3. Activities of Human Gene Nomenclature Committee

    SciTech Connect

    2002-07-16

    The objective of this project, shared between NIH and DOE, has been and remains to enable the medical genetics communities to use common names for genes that are discovered by different gene hunting groups, in different species. This effort provides consistent gene nomenclature and approved gene symbols to the community at large. This contributes to a uniform and consistent understanding of genomes, particularly the human as well as functional genomics based on comparisons between homologous genes in related species (human and mice).

  4. Microchip-Based Single-Cell Imaging Reveals That CD56dimCD57-KIR-NKG2A+ NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56dimCD57-KIR-NKG2A- NK Cells.

    PubMed

    Forslund, Elin; Sohlberg, Ebba; Enqvist, Monika; Olofsson, Per E; Malmberg, Karl-Johan; Önfelt, Björn

    2015-10-01

    NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(-) (lacking inhibitory receptors; IR(-)) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR(-) NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR(-) NK cells were generally more short-lived and IR(-) NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR(-) NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered. PMID:26320254

  5. Microchip-Based Single-Cell Imaging Reveals That CD56dimCD57-KIR-NKG2A+ NK Cells Have More Dynamic Migration Associated with Increased Target Cell Conjugation and Probability of Killing Compared to CD56dimCD57-KIR-NKG2A- NK Cells.

    PubMed

    Forslund, Elin; Sohlberg, Ebba; Enqvist, Monika; Olofsson, Per E; Malmberg, Karl-Johan; Önfelt, Björn

    2015-10-01

    NK cells are functionally educated by self-MHC specific receptors, including the inhibitory killer cell Ig-like receptors (KIRs) and the lectin-like CD94/NKG2A heterodimer. Little is known about how NK cell education influences qualitative aspects of cytotoxicity such as migration behavior and efficacy of activation and killing at the single-cell level. In this study, we have compared the behavior of FACS-sorted CD56(dim)CD57(-)KIR(-)NKG2A(+) (NKG2A(+)) and CD56(dim)CD57(-)KIR(-)NKG2A(-) (lacking inhibitory receptors; IR(-)) human NK cells by quantifying migration, cytotoxicity, and contact dynamics using microchip-based live cell imaging. NKG2A(+) NK cells displayed a more dynamic migration behavior and made more contacts with target cells than IR(-) NK cells. NKG2A(+) NK cells also more frequently killed the target cells once a conjugate had been formed. NK cells with serial killing capacity were primarily found among NKG2A(+) NK cells. Conjugates involving IR(-) NK cells were generally more short-lived and IR(-) NK cells did not become activated to the same extent as NKG2A(+) NK cells when in contact with target cells, as evident by their reduced spreading response. In contrast, NKG2A(+) and IR(-) NK cells showed similar dynamics in terms of duration of conjugation periods and NK cell spreading response in conjugates that led to killing. Taken together, these observations suggest that the high killing capacity of NKG2A(+) NK cells is linked to processes regulating events in the recognition phase of NK-target cell contact rather than events after cytotoxicity has been triggered.

  6. Modulation of T-bet and Eomes during Maturation of Peripheral Blood NK Cells Does Not Depend on Licensing/Educating KIR.

    PubMed

    Pradier, Amandine; Simonetta, Federico; Waldvogel, Sophie; Bosshard, Carine; Tiercy, Jean-Marie; Roosnek, Eddy

    2016-01-01

    Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94(neg)CD56(dim)NK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive. PMID:27605928

  7. Modulation of T-bet and Eomes during Maturation of Peripheral Blood NK Cells Does Not Depend on Licensing/Educating KIR

    PubMed Central

    Pradier, Amandine; Simonetta, Federico; Waldvogel, Sophie; Bosshard, Carine; Tiercy, Jean-Marie; Roosnek, Eddy

    2016-01-01

    Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94negCD56dimNK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive. PMID:27605928

  8. Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

    PubMed Central

    Antcliff, Jennifer F; Haider, Shozeb; Proks, Peter; Sansom, Mark S P; Ashcroft, Frances M

    2005-01-01

    ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the ‘slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state. PMID:15650751

  9. CIPP, a novel multivalent PDZ domain protein, selectively interacts with Kir4.0 family members, NMDA receptor subunits, neurexins, and neuroligins.

    PubMed

    Kurschner, C; Mermelstein, P G; Holden, W T; Surmeier, D J

    1998-06-01

    We report a novel multivalent PDZ domain protein, CIPP (for channel-interacting PDZ domain protein), which is expressed exclusively in brain and kidney. Within the brain, the highest CIPP mRNA levels were found in neurons of the cerebellum, inferior colliculus, vestibular nucleus, facial nucleus, and thalamus. Furthermore, we identified the inward rectifier K+ (Kir) channel, Kir4.1 (also called "Kir1.2"), as a cellular CIPP ligand. Among several other Kir channels tested, only the closely related Kir4.2 (or "Kir1.3") also interacted with CIPP. In addition, specific PDZ domains within CIPP associated selectively with the C-termini of N-methyl-D-aspartate subtypes of glutamate receptors, as well as neurexins and neuroligins, cell surface molecules enriched in synaptic membranes. Thus, CIPP may serve as a scaffold that brings structurally diverse but functionally connected proteins into close proximity at the synapse. The functional consequences of CIPP expression on Kir4.1 channels were studied using whole-cell voltage clamp techniques in Kir4.1 transfected COS-7 cells. On average, Kir4.1 current densities were doubled by cotransfection with CIPP.

  10. Oligoclonal band phenotypes in MS differ in their HLA class II association, while specific KIR ligands at HLA class I show association to MS in general.

    PubMed

    Gustavsen, Marte W; Viken, Marte K; Celius, Elisabeth G; Berge, Tone; Mero, Inger-Lise; Berg-Hansen, Pål; Aarseth, Jan H; Myhr, Kjell-Morten; Søndergaard, Helle B; Sellebjerg, Finn; Oturai, Annette B; Hillert, Jan; Alfredsson, Lars; Olsson, Tomas; Kockum, Ingrid; Lie, Benedicte A; Harbo, Hanne F

    2014-09-15

    Multiple sclerosis (MS) patients have been reported to have different HLA class II allele profiles depending on oligoclonal bands (OCBs) in the cerebrospinal fluid, but HLA class I alleles and killer cell immunoglobulin-like receptor (KIR) ligands have not been studied. We investigated the association of HLA alleles and KIR ligands according to OCB status in MS patients (n=3876). Specific KIR ligands were associated with patients when compared to controls (n=3148), supporting a role for NK cells in MS pathogenesis. HLA class I alleles and KIR ligands did not differ between OCB phenotypes, but HLA class II associations were convincingly replicated.

  11. Measurement of orexin (hypocretin) and substance P effects on constitutively active inward rectifier K(+) channels in brain neurons.

    PubMed

    Nakajima, Yasuko; Nakajima, Shigehiro

    2010-01-01

    Electrophysiological experiments in our laboratory have led to the discovery that the cholinergic neurons in the nucleus basalis in the rat forebrain possess constitutively active inward rectifier K(+) channels. Unlike cloned inward rectifier K(+) channels, these constitutively active inward rectifier K(+) channels were found to have unique properties, and thus were named "KirNB" (inward rectifier K(+) channels in the nucleus basalis). We found that slow excitatory transmitters, such as orexin (hypocretin) and substance P, suppress the KirNB channel, resulting in neuronal excitation. Furthermore, it was discovered that suppression of KirNB channels by these transmitters is through protein kinase C (PKC). This chapter describes detailed electrophysiological techniques for investigating the effects of orexin and substance P on constitutively active KirNB channels. For this purpose, we also present a method for culturing nucleus basalis cholinergic neurons in which KirNB channels exist. Then, we describe the procedures through which PKC has been determined to mediate inhibition of KirNB channels by orexin and substance P. There are probably many other transmitters which may produce effects on KirNB channels. This chapter will enable researchers to investigate the effects of such transmitters on KirNB channels and their roles in neuronal functions.

  12. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    EPA Science Inventory

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  13. Gene activation by induced DNA rearrangements

    SciTech Connect

    Schnipper, L.E.; Chan, V.; Sedivy, J.; Jat, P.; Sharp, P.A. )

    1989-12-01

    A murine cell line (EN/NIH) containing the retroviral vector ZIPNeoSV(x)1 that was modified by deletion of the enhancer elements in the viral long terminal repeats has been used as an assay system to detect induced DNA rearrangements that result in activation of a transcriptionally silent reporter gene encoded by the viral genome. The spontaneous frequency of G418 resistance is less than 10(-7), whereas exposure to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or the combination of UV irradiation plus TPA resulted in the emergence of drug resistant cell lines at a frequency of 5 per 10(6) and 67 per 10(6) cells, respectively. In several of the cell lines that were analyzed a low level of amplification of one of the two parental retroviral integrants was observed, whereas in others no alteration in the region of the viral genome was detected. To determine the effect of the SV40 large T antigen on induced DNA rearrangements, EN/NIH cells were transfected with a temperature sensitive (ts) mutant of SV40 T. Transfectants were maintained at the permissive temperature (33 degrees C) for varying periods of time (1-5 days) in order to vary SV40 T antigen exposure, after which they were shifted to 39.5 degrees C for selection in G418. The frequency of emergence of drug resistant cell clones increased with duration of exposure to large T antigen (9-52 per 10(6) cells over 1-5 days, respectively), and all cell lines analyzed demonstrated DNA rearrangements in the region of the neo gene. A novel 18-kilobase pair XbaI fragment was cloned from one cell line which revealed the presence of a 2.0-kilobase pair EcoRI segment containing an inverted duplication which hybridized to neo sequences. It is likely that the observed rearrangement was initiated by the specific binding of large T antigen to the SV40 origin of replication encoded within the viral genome.

  14. Spatial and temporal dissociation of AQP4 and Kir4.1 expression during induction of refractive errors

    PubMed Central

    Goodyear, Melinda J.; Crewther, Sheila G.; Murphy, Melanie J.; Giummarra, Loretta; Hazi, Agnes; Junghans, Barbara M.; Crewther, David P.

    2010-01-01

    Purpose Spatial co-localization of aquaporin water channels (AQP4) and inwardly rectifying potassium ion channels (Kir4.1) on the endfeet regions of glial cells has been suggested as the basis of functionally interrelated mechanisms of osmoregulation in brain edema. The aim of this study was to investigate the spatial and temporal changes in the expression of AQP4 and Kir4.1 channels in an avascular retina during the first week of the optical induction of refractive errors. Methods Three-day-old hatchling chicks were randomly assigned to three groups and either did not wear lenses or were monocularly goggled with ±10D lenses for varying times up to 7 days before biometric assessment. Retinal tissue was prepared either for western blot analysis to show the presence of the AQP4 and Kir4.1 protein in the chick retina or for immunolocalization using AQP4 and Kir4.1 antibodies to determine the regional distribution and intensity of labeling during the induction of refractive errors. Results As expected, ultrasonography demonstrated that all eyes showed rapid elongation post hatching. Negative lens-wearing eyes elongated faster than fellow eyes or normal non goggled eyes and became progressively more myopic with time post lensing. Positive lens-wearing eyes showed reduced ocular growth compared to normal controls and developed a hyperopic refraction. Quantitative immunohistochemistry revealed the upregulation of AQP4 channel expression on Müller cells in the retinal nerve fiber layer during the first 2 days of negative lens wear. Kir4.1 channel upregulation in the inner plexiform layer was only found on day 4 of positive lens wear during the development of refractive hyperopia. Conclusions These results indicate that the expression of AQP4 and Kir4.1 channels on Müller cells is associated with the changes in ocular volume seen during the induction of refractive errors. However, the sites of greatest expression and the temporal pattern of the upregulation of AQP4 and

  15. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia

    PubMed Central

    Cooley, Sarah; Weisdorf, Daniel J.; Guethlein, Lisbeth A.; Klein, John P.; Wang, Tao; Le, Chap T.; Marsh, Steven G. E.; Geraghty, Daniel; Spellman, Stephen; Haagenson, Michael D.; Ladner, Martha; Trachtenberg, Elizabeth; Parham, Peter

    2010-01-01

    Killer-cell immunoglobulin-like receptor (KIR) genes form a diverse, immunogenetic system. Group A and B KIR haplotypes have distinctive centromeric (Cen) and telomeric (Tel) gene-content motifs. Aiming to develop a donor selection strategy to improve transplant outcome, we compared the contribution of these motifs to the clinical benefit conferred by B haplotype donors. We KIR genotyped donors from 1409 unrelated transplants for acute myelogenous leukemia (AML; n = 1086) and acute lymphoblastic leukemia (ALL; n = 323). Donor KIR genotype influenced transplantation outcome for AML but not ALL. Compared with A haplotype motifs, centromeric and telomeric B motifs both contributed to relapse protection and improved survival, but Cen-B homozygosity had the strongest independent effect. With Cen-B/B homozygous donors the cumulative incidence of relapse was 15.4% compared with 36.5% for Cen-A/A donors (relative risk of relapse 0.34; 95% confidence interval 0.2-0.57; P < .001). Overall, significantly reduced relapse was achieved with donors having 2 or more B gene-content motifs (relative risk 0.64; 95% confidence interval 0.48-0.86; P = .003) for both HLA-matched and mismatched transplants. KIR genotyping of several best HLA-matched potential unrelated donors should substantially increase the frequency of transplants by using grafts with favorable KIR gene content. Adopting this practice could result in superior disease-free survival for patients with AML. PMID:20581313

  16. Increased frequency and function of KIR2DL1-3+ NK cells in primary HIV-1 infection are determined by HLA-C group haplotypes

    PubMed Central

    Körner, Christian; Granoff, Mitchell E.; Amero, Molly A.; Sirignano, Michael N.; Vaidya, Sagar A.; Jost, Stephanie; Allen, Todd M.; Rosenberg, Eric S.; Altfeld, Marcus

    2014-01-01

    Acquisition and maintenance of NK cell function is mediated by inhibitory killer-cell immunoglobulin-like receptors (KIR) through the interaction with HLA class I molecules. Recently, HLA-C expression levels were shown to be correlated with protection against multiple outcomes of HIV-1 infection; however the underlying mechanisms are poorly understood. As HLA-C is the natural ligand for the inhibitory receptors KIR2DL1 and KIR2DL2/3, we sought to determine whether HLA-C group haplotypes affect NK cell responses during primary HIV-1 infection. The phenotypes and functional capacity of NK cells derived from HIV-1(+) and HIV-1(-) individuals were assessed (N=42 and N=40, respectively). HIV-1 infection was associated with an increased frequency of KIR2DL1-3+ NK cells. Further analysis showed that KIR2DL1+ NK cells were selectively increased in individuals homozygous for HLA-C2, while HLA-C1-homozygous individuals displayed increased proportions of KIR2DL2/3+ NK cells. KIR2DL1-3+ NK cells were furthermore more polyfunctional during primary HIV-1 infection in individuals also encoding for their cognate HLA-C group haplotypes as measured by degranulation and cytokine production. These results identify a novel relationship between HLA-C and KIR2DL+ NK cell subsets and demonstrate that HLA-C-mediated licensing modulates NK cell responses to primary HIV-1 infection. PMID:25043727

  17. Association between KIR genotypes and HLA-B alleles on viral load in Southern Brazilian individuals infected by HIV-1 subtypes B and C.

    PubMed

    Fernandes-Cardoso, Juliana; Süffert, Theodoro Armando; Correa, Maria da Gloria; Jobim, Luiz Fernando Job; Jobim, Mariana; Salim, Patricia Hartstein; Arruda, Monica Barcelos; Boullosa, Lidia Theodoro; Tanuri, Amilcar; Porto, Luis Cristóvão; Ferreira, Orlando C

    2016-10-01

    There is a great variety of HIV-1 subtypes circulating in Brazil, including subtype C, whose prevalence is on the rise, particularly in the southern region. Many host and viral genetic factors may be involved in this trend. We evaluated the influence of human leukocyte antigen (HLA) class I alleles and killer-cell immunoglobulin-like receptor (KIR) genotypes on viral set point and T-CD4(+) parameters in 84 treatment-naïve HIV-1-positive individuals. Frequency data in the infected group were compared to data of 548 healthy control subjects. Individuals with the KIR AA genotype had a higher viral load (VL) than individuals with the KIR Bx genotype. The HIV-1 group was subdivided into three subgroups according to HLA-B allele presence: those with protection to disease alleles (HLA-B(+)), accelerated disease progression alleles (HLA-B(-)), or neither (HLA-B(o)) were grouped. We observed a significant effect of the HLA-B allele presence on VL. The HLA-B(+) group had significantly lower VL than the HLA-B(-) group and trended toward a lower VL than the HLA-B(o) group. There were significant differences between groups expressing extreme VL values: KIR-AA+HLA-B(-) vs. KIR Bx+HLA-B(+) and KIR-AA+HLA-B(o)vs. KIR Bx+HLA-B(+). The relationship of KIR/HLA host genetics with slow HIV disease progression in southern Brazil may be useful for vaccine developers, epidemiologists, and clinicians.

  18. Enhanced Excitability of Primary Sensory Neurons and Altered Gene Expression of Neuronal Ion Channels in Dorsal Root Ganglion in Paclitaxel-Induced Peripheral Neuropathy

    PubMed Central

    Zhang, Haijun; Dougherty, Patrick M.

    2014-01-01

    Background The mechanism of chemotherapy-induced peripheral neuropathy after paclitaxel treatment is not well understood. Given the poor penetration of paclitaxel into central nervous system, peripheral nervous system is most at risk. Methods Intrinsic membrane properties of dorsal root ganglion (DRG) neurons were studied by intracellular recordings. Multiple-gene real-time Polymerase Chain Reaction array was used to investigate gene expression of DRG neuronal ion channels. Results Paclitaxel increased the incidence of spontaneous activity from 4.8% to 27.1% in large and from 0% to 33.3% in medium-sized neurons. Paclitaxel decreased the rheobase (nA) from 1.6 ± 0.1 to 0.8 ± 0.1 in large, from 1.5 ± 0.2 to 0.6 ± 0.1 in medium-sized, and from 1.6 ± 0.2 to 1.0 ± 0.1 in small neurons. After paclitaxel, other characteristics of membrane properties in each group remained the same except that Aδ neurons showed shorter action potential fall time (ms) (1.0 ± 0.2, n = 10 vs. 1.8 ± 0.3, n = 9, paclitaxel vs. vehicle). Meanwhile, real-time polymerase chain reaction array revealed an alteration in expression of some neuronal ion channel genes including upregulation of HCN1 (fold change 1.76 ± 0.06) and Nav1.7 (1.26 ± 0.02) and downregulation of Kir channels (Kir1.1, 0.73 ± 0.05, Kir3.4, 0.66 ± 0.06) in paclitaxel-treated animals. Conclusions The increased neuronal excitability and the changes in gene expression of some neuronal ion channels in DRG may provide insight into the molecular and cellular basis of paclitaxel neuropathy, which may lead to novel therapeutic strategies. PMID:24534904

  19. Chromatin Remodeling Inactivates Activity Genes and Regulates Neural Coding

    PubMed Central

    Hill, Kelly K.; Hemberg, Martin; Reddy, Naveen C.; Cho, Ha Y.; Guthrie, Arden N.; Oldenborg, Anna; Heiney, Shane A.; Ohmae, Shogo; Medina, Javier F.; Holy, Timothy E.; Bonni, Azad

    2016-01-01

    Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating mRNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain. PMID:27418512

  20. HLA class I variation in Iranian Lur and Kurd populations: high haplotype and allotype diversity with an abundance of KIR ligands.

    PubMed

    Ashouri, E; Norman, P J; Guethlein, L A; Han, A S; Nemat-Gorgani, N; Norberg, S J; Ghaderi, A; Parham, P

    2016-09-01

    HLA-A, -B and -C alleles of 285 individuals, representing three Iranian Lur populations and one Iranian Kurd population were sequenced completely, yielding human leukocyte antigen (HLA) class I genotypes at high resolution and filling four fields of the official HLA nomenclature. Each population has 87-99 alleles, evenly distributed between the three HLA class I genes, 145 alleles being identified in total. These alleles were already known, named and deposited in the HLA database. The alleles form 316 different HLA A-B-C haplotypes, with each population having between 80 and 112 haplotypes. The four Iranian populations form a related group that is distinguished from other populations, including other Iranians. All four KIR ligands - the A3/11, Bw4, C1 and C2 epitopes - are well represented, particularly Bw4, which is carried by three high-frequency allotypes: HLA-A*24:02, HLA-A*32:01 and HLA-B*51:01. In the Lur and Kurd populations, between 82% and 94% of individuals have the Bw4 epitope, the ligand for KIR3DL1. HLA-B*51:01 is likely of Neandertal origin and associated with Behcet's disease, also known as the Silk Road disease. The Lordegan Lur have the highest frequency of HLA-B*51:01 in the world. This allele is present on 46 Lur and Kurd haplotypes. Present at lower frequency is HLA-B*51:08, which is also associated with Behcet's disease. In the four Iranian populations, 31 haplotypes encode both Bw4(+) HLA-A and Bw4(+) HLA-B, a dual combination of Bw4 epitopes that is relatively rare in other populations, worldwide. This study both demonstrates and emphasizes the value of studying HLA class I polymorphism at highest resolution in anthropologically well-defined populations. PMID:27558013

  1. Maternal activation of gap genes in the hover fly Episyrphus.

    PubMed

    Lemke, Steffen; Busch, Stephanie E; Antonopoulos, Dionysios A; Meyer, Folker; Domanus, Marc H; Schmidt-Ott, Urs

    2010-05-01

    The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, we used loss- and gain-of-function experiments in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. We find that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, we find that torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. We conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha).

  2. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels*

    PubMed Central

    Zhang, Roger S.; Wright, Jordan D.; Pless, Stephan A.; Nunez, John-Jose; Kim, Robin Y.; Li, Jenny B. W.; Yang, Runying; Ahern, Christopher A.; Kurata, Harley T.

    2015-01-01

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels. PMID:25934393

  3. A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

    PubMed

    Zhang, Roger S; Wright, Jordan D; Pless, Stephan A; Nunez, John-Jose; Kim, Robin Y; Li, Jenny B W; Yang, Runying; Ahern, Christopher A; Kurata, Harley T

    2015-06-19

    ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

  4. Genotypes of NK cell KIR receptors, their ligands, and Fcγ receptors in the response of neuroblastoma patients to Hu14.18-IL2 immunotherapy.

    PubMed

    Delgado, David C; Hank, Jacquelyn A; Kolesar, Jill; Lorentzen, David; Gan, Jacek; Seo, Songwon; Kim, Kyungmann; Shusterman, Suzanne; Gillies, Stephen D; Reisfeld, Ralph A; Yang, Richard; Gadbaw, Brian; DeSantes, Kenneth B; London, Wendy B; Seeger, Robert C; Maris, John M; Sondel, Paul M

    2010-12-01

    Response to immunocytokine (IC) therapy is dependent on natural killer cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR)/KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical antitumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Children's Oncology Group phase II trial. In this report, these patients were genotyped for KIR, HLA, and FcR alleles to determine whether KIR receptor-ligand mismatch or specific FcγR alleles were associated with antitumor response. DNA samples were available for 38 of 39 patients enrolled: 24 were found to have autologous KIR/KIR-ligand mismatch; 14 were matched. Of the 24 mismatched patients, 7 experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (P = 0.03). There was a trend toward patients with the FcγR2A 131-H/H genotype showing a higher response rate than other FcγR2A genotypes (P = 0.06). These analyses indicate that response or improvement of relapsed/refractory NBL patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for natural killer cells in this clinical response.

  5. Modeling the Activity of Single Genes

    NASA Technical Reports Server (NTRS)

    Mjolsness, Eric; Gibson, Michael

    1999-01-01

    The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large

  6. Imprinted control of gene activity in Drosophila.

    PubMed

    Golic, K G; Golic, M M; Pimpinelli, S

    1998-11-19

    Genetic imprinting is defined as a reversible, differential marking of genes or chromosomes that is determined by the sex of the parent from whom the genetic material is inherited [1]. Imprinting was first observed in insects where, in some species, most notably among the coccoids (scale insects and allies), the differential marking of paternally and maternally transmitted chromosome sets leads to inactivation or elimination of paternal chromosomes [2]. Imprinting is also widespread in plants and mammals [3,4], in which paternally and maternally inherited alleles may be differentially expressed. Despite imprinting having been discovered in insects, clear examples of parental imprinting are scarce in the model insect species Drosophila melanogaster. We describe a case of imprint-mediated control of gene expression in Drosophila. The imprinted gene - the white+ eye-color gene - is expressed at a low level when transmitted by males, and at a high level when transmitted by females. Thus, in common with coccoids, Drosophila is capable of generating an imprint, and can respond to that imprint by silencing the paternal allele. PMID:9822579

  7. Carcinogen-induced trans activation of gene expression

    SciTech Connect

    Kleinberger, T.; Flint, Y.B.; Blank, M.; Etkin, S.; Lavi, S.

    1988-03-01

    The authors report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later.

  8. Carcinogen-induced trans activation of gene expression.

    PubMed Central

    Kleinberger, T; Flint, Y B; Blank, M; Etkin, S; Lavi, S

    1988-01-01

    We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later. Images PMID:2835673

  9. A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

    PubMed

    Xie, Wensheng; Silvers, Robert; Ouellette, Michael; Wu, Zining; Lu, Quinn; Li, Hu; Gallagher, Kathleen; Johnson, Kathy; Montoute, Monica

    2016-01-01

    Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase. PMID:27316998

  10. Dietary Methanol Regulates Human Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Sheshukova, Ekaterina V.; Kosorukov, Vyacheslav S.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  11. Robust, synergistic regulation of human gene expression using TALE activators.

    PubMed

    Maeder, Morgan L; Linder, Samantha J; Reyon, Deepak; Angstman, James F; Fu, Yanfang; Sander, Jeffry D; Joung, J Keith

    2013-03-01

    Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

  12. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

    PubMed

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E

    2016-01-01

    Inwardly rectifying K(+) (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K(+) ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG).

  13. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

    PubMed

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E

    2016-01-01

    Inwardly rectifying K(+) (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K(+) ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG). PMID:27439597

  14. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel

    PubMed Central

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E.

    2016-01-01

    Inwardly rectifying K+ (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K+ ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG). PMID:27439597

  15. Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK-293 cells

    PubMed Central

    Gopalakrishnan, Murali; Molinari, Eduardo J; Shieh, Char-Chang; Monteggia, Lisa M; Roch, Jean-Marc; Whiteaker, Kristi L; Scott, Victoria E S; Sullivan, James P; Brioni, Jorge D

    2000-01-01

    The pharmacological properties of KATP channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K+ channel Kir6.2 were characterized in HEK-293 cells.[3H]-Glyburide (glibenclamide) bound to transfected cells with a Bmax value of 18.5 pmol mg−1 protein and with a KD value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain.Functional activity of KATP channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 μM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC50=0.92 nM).Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered 86Rb+ efflux from transfected cells that was sensitive to inhibition by glyburide (IC50=3.6 nM).Diazoxide, but not levcromakalim, evoked concentration-dependent decreases in DiBAC4(3) fluorescence responses with an EC50 value of 14.1 μM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities.In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native KATP channels described in pancreatic and neuronal tissues. PMID:10742287

  16. A synergistic blocking effect of Mg2+ and spermine on the inward rectifier K+ (Kir2.1) channel pore

    PubMed Central

    Huang, Chiung-Wei; Kuo, Chung-Chin

    2016-01-01

    Inward rectifier K+ channels (Kir2.1) exhibit an extraordinary rectifying feature in the current–voltage relationship. We have previously showed that the bundle–crossing region of the transmembrane domain constitutes the crucial segment responsible for the polyamine block. In this study, we demonstrated that the major blocking effect of intracellular Mg2+ on Kir2.1 channels is also closely correlated with K+ current flow, and the coupled movements of Mg2+ and K+ seem to happen in the same flux–coupling segment of the pore as polyamines. With a preponderant outward K+ flow, intracellular Mg2+ would also be pushed to and thus stay at the outermost site of a flux–coupling segment in the bundle–crossing region of Kir2.1 channels to block the pore, although with a much lower apparent affinity than spermine (SPM). However, in contrast to the evident possibilities of outward exit of SPM through the channel pore especially during strong membrane depolarization, intracellular Mg2+ does not seem to traverse the Kir2.1 channel pore in any case. Intracellular Mg2+ and SPM therefore may have a synergistic action on the pore–blocking effect, presumably via prohibition of the outward exit of the higher–affinity blocking SPM by the lower–affinity Mg2+. PMID:26869275

  17. Massive Activation of Archaeal Defense Genes during Viral Infection

    PubMed Central

    Voet, Marleen; Sismeiro, Odile; Dillies, Marie-Agnes; Jagla, Bernd; Coppée, Jean-Yves; Sezonov, Guennadi; Forterre, Patrick; van der Oost, John; Lavigne, Rob

    2013-01-01

    Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo. PMID:23698312

  18. Transcriptional activation of heat-shock genes in eukaryotes.

    PubMed

    Tanguay, R M

    1988-06-01

    Prokaryotes and eukaryotes respond to thermal or various chemical stresses by the rapid induction of a group of genes collectively referred to as the heat shock genes. In eucaryotes, the expression of these genes is primarily regulated at the transcriptional level. The early observations that transfected heat shock genes were inducible in heterologous systems suggested the existence of common regulatory elements in these ubiquitous genes. Sequence analysis of cloned Drosophila heat shock genes revealed a conserved 14 base pair (bp) inverted repeat, which is essential for heat induction. This regulatory sequence, referred to as the heat shock element (HSE), is found in multiple imperfect copies upstream of the TATA box of all heat shock genes. While studies in heterologous systems indicated that a single copy of HSE was sufficient for inducibility, further analysis in homologous assays suggests that multiple HSE can act in a cooperative way and that the efficiency of transcriptional activation is related, within limits, to the number of HSE. Comparative analysis of heat shock genes reveals that HSE can be positioned at different distances from the TATA box in either orientation, a behavior reminiscent of enhancer elements. However, the presence of HSE does not necessarily confer heat inducibility, as shown by their presence in the constitutively expressed but non-heat-inducible homologous cognate genes. Footprinting and nuclease mapping have been used to show that a protein factor (HSTF: heat shock transcription factor) binds to the HSE element, activating heat shock gene transcription in a dose-dependent manner. The recent progress in the isolation and characterization of HSTF in Drosophila, yeast, and human cells is reviewed. Finally, different models suggested to account for the positive regulation of heat shock genes by the HSTF are presented.

  19. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  20. Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis.

    PubMed Central

    Lam, E; Green, P J; Wong, M; Chua, N H

    1989-01-01

    We have investigated the effects of protein synthesis inhibitors on light-induced expression of two plant nuclear genes, Cab and rbcS, in wheat, pea and transgenic tobacco. Light activation of these two genes is very sensitive to cycloheximide, an inhibitor of cytoplasmic protein synthesis but not to chloramphenicol, an inhibitor of organellar protein synthesis. Studies with chimeric gene constructs in transgenic tobacco seedlings show that cycloheximide exerts its effect at the transcriptional level. As a control, we show that the expression of the cauliflower mosaic virus (CaMV) 35S promoter is enhanced by cycloheximide treatment, irrespective of the coding sequence used. Escape-time analyses with green wheat seedlings show that the cycloheximide block for Cab gene expression is after the primary signal transduction step linked to phytochrome photoconversion. Our results suggest that phytochrome activation of Cab and rbcS is mediated by a labile protein factor(s) synthesized on cytoplasmic ribosomes. Images PMID:2583082

  1. A model for the topology of active ribosomal RNA genes.

    PubMed

    Denissov, Serguei; Lessard, Frédéric; Mayer, Christine; Stefanovsky, Victor; van Driel, Marc; Grummt, Ingrid; Moss, Tom; Stunnenberg, Hendrik G

    2011-03-01

    The Christmas tree view of active ribosomal RNA (rRNA) genes suggests a gene topology in which a large number of nascent rRNA transcripts are prevented from intertwining. The way in which this is achieved has remained unclear. By using a combination of chromatin immunoprecipitation and chromosome conformation capture techniques, we show that the promoter, upstream region and terminator R3 of active rRNA genes are held together spatially throughout the cell cycle, forming a stable core around which the transcribed region is organized. We suggest a new core-helix model for the topology of rRNA genes, that provides a structural basis for the productive synthesis or rRNA.

  2. Targeted Gene Activation Using RNA-Guided Nucleases.

    PubMed

    Brown, Alexander; Woods, Wendy S; Perez-Pinera, Pablo

    2017-01-01

    The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors. PMID:27662880

  3. Killer cell immunoglobulin-like receptor genes in Latvian patients with type 1 diabetes mellitus and healthy controls.

    PubMed

    Nikitina-Zake, Liene; Rajalingham, Raja; Rumba, Ingrida; Sanjeevi, Carani B

    2004-12-01

    T1DM is very common in Sweden and is positively associated with HLA class II genes. Approximately 89% of the newly diagnosed patients carry the high-risk HLA DR4-DQ8 and DR3-DQ2. The remaining 11% develop T1DM without them. This can be due to involvement of other genes and environmental factors. Natural killer (NK) cells of the innate immune system are important in antiviral and antitumor immunity. They are implicated in the etiology of autoimmune T1DM. Human NK cells express killer cell immunoglobulin-like receptors (KIR) that belong to the polymorphic multigene family in chromosome 19q3.4. They modulate NK cell response by interacting with HLA class I. In addition, polymorphic MICA in HLA class I interacts with non-polymorphic NKG2D receptor on NK cells. We have studied, in addition to HLA-DR and -DQ, genes of the innate immune system MICA and KIR in Latvian patients (n = 98) with T1DM and controls (n = 100). They were genotyped using standard PCR-based typing methods. MICA allele 5 is positively associated with T1DM. KIR2DL2 and KIR2DS2 were both positively associated. Combined association of MICA4 and KIR2DL2 gave an odds ration (OR) of 26.7. However, the combined risk of KIR2DL2 and HLA class II genes, HLADR3 (OR = 73.4), DR4 (OR = 66.8), and DR3 and DR4 (OR = 88.3), was higher. The maximum risk was when KIR2DL2, MICA5, and DR3/DR4 were in combination. In conclusion, our results suggest that a balance between innate and acquired immunity is important, and an imbalance coud lead to T1DM.

  4. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation

    PubMed Central

    Alam, S.; Rangaswamy, D.; Prakash, S.; Sharma, R. K.; Khan, M. I.; Sonawane, A.; Agrawal, S.

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival. PMID:25684869

  5. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation.

    PubMed

    Alam, S; Rangaswamy, D; Prakash, S; Sharma, R K; Khan, M I; Sonawane, A; Agrawal, S

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

  6. T-cell activation and early gene response in dogs.

    PubMed

    Mortlock, Sally-Anne; Wei, Jerry; Williamson, Peter

    2015-01-01

    T-cells play a crucial role in canine immunoregulation and defence against invading pathogens. Proliferation is fundamental to T-cell differentiation, homeostasis and immune response. Initiation of proliferation following receptor mediated stimuli requires a temporally programmed gene response that can be identified as immediate-early, mid- and late phases. The immediate-early response genes in T-cell activation engage the cell cycle machinery and promote subsequent gene activation events. Genes involved in this immediate-early response in dogs are yet to be identified. The present study was undertaken to characterise the early T-cell gene response in dogs to improve understanding of the genetic mechanisms regulating immune function. Gene expression profiles were characterised using canine gene expression microarrays and quantitative reverse transcription PCR (qRT-PCR), and paired samples from eleven dogs. Significant functional annotation clusters were identified following stimulation with phytohemagluttinin (PHA) (5μg/ml), including the Toll-like receptor signaling pathway and phosphorylation pathways. Using strict statistical criteria, 13 individual genes were found to be differentially expressed, nine of which have ontologies that relate to proliferation and cell cycle control. These included, prostaglandin-endoperoxide synthase 2 (PTGS2/COX2), early growth response 1 (EGR1), growth arrest and DNA damage-inducible gene (GADD45B), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), V-FOS FBJ murine osteosarcoma viral oncogene homolog (FOS), early growth response 2 (EGR2), hemogen (HEMGN), polo-like kinase 2 (PLK2) and polo-like kinase 3 (PLK3). Differential gene expression was re-examined using qRT-PCR, which confirmed that EGR1, EGR2, PMAIP1, PTGS2, FOS and GADD45B were significantly upregulated in stimulated cells and ALAS2 downregulated. PTGS2 and EGR1 showed the highest levels of response in these dogs. Both of these genes are involved in cell cycle

  7. High Intensity Focused Ultrasound induced Gene Activation in Solid Tumors

    NASA Astrophysics Data System (ADS)

    Liu, Yunbo; Kon, Takashi; Li, Chuanyuan; Zhong, Pei

    2006-05-01

    In this work, the feasibility of using high intensity focused ultrasound (HIFU) to activate trans-gene expression in a mouse tumor model was investigated. 4T1 cancer cells were implanted subcutaneously in the hind limbs of Balb/C mice and adenovirus luciferase gene vectors under the control of heat shock protein 70B promoter (Adeno-hsp70B-Luc) were injected intratumoraly for gene transfection. One day following the virus injection, the transfected tumors were heated to a peak temperature of 55, 65, 75, and 85°C, respectively, in 10s at multiple sites around the center of the tumor using a HIFU transducer operated at either 1.1-MHz (fundamental) or 3.3-MHz (3rd harmonic) frequency. Inducible luciferase gene expression was found to vary from 15-fold to 120-fold of the control group following 1.1-MHz HIFU exposure. The maximum gene activation was produced at a peak temperature of 65˜75°C one day following HIFU exposure and decayed gradually to baseline level within 7 days. The inducible gene activation produced by 3.3-MHz HIFU exposure (75°C-10s) was found to be comparable to that produced by hyperthermia (42°C-30min). Altogether, these results demonstrate the feasibility of using HIFU as a simple and versatile physical means to regulate trans-gene expression in vivo. This unique feature may be explored in the future for a synergistic combination of HIFU-induced thermal ablation with heat-induced gene therapy for improved cancer therapy.

  8. Insights into the structural nature of the transition state in the Kir channel gating pathway.

    PubMed

    Fowler, Philip W; Bollepalli, Murali K; Rapedius, Markus; Nematian-Ardestani, Ehsan; Shang, Lijun; Sansom, Mark Sp; Tucker, Stephen J; Baukrowitz, Thomas

    2014-01-01

    In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a 'catalytic' effect upon the channel gating kinetics. PMID:25483285

  9. Insights into the structural nature of the transition state in the Kir channel gating pathway

    PubMed Central

    Fowler, Philip W; Bollepalli, Murali K; Rapedius, Markus; Nematian-Ardestani, Ehsan; Shang, Lijun; Sansom, Mark SP; Tucker, Stephen J; Baukrowitz, Thomas

    2014-01-01

    In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the center of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics. PMID:25483285

  10. Identification of Interferon-Stimulated Genes with Antiretroviral Activity.

    PubMed

    Kane, Melissa; Zang, Trinity M; Rihn, Suzannah J; Zhang, Fengwen; Kueck, Tonya; Alim, Mudathir; Schoggins, John; Rice, Charles M; Wilson, Sam J; Bieniasz, Paul D

    2016-09-14

    Interferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs. PMID:27631702

  11. Identification of Interferon-Stimulated Genes with Antiretroviral Activity.

    PubMed

    Kane, Melissa; Zang, Trinity M; Rihn, Suzannah J; Zhang, Fengwen; Kueck, Tonya; Alim, Mudathir; Schoggins, John; Rice, Charles M; Wilson, Sam J; Bieniasz, Paul D

    2016-09-14

    Interferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs.

  12. Estrogen-dependent transcriptional activation and vitellogenin gene memory.

    PubMed

    Edinger, R S; Mambo, E; Evans, M I

    1997-12-01

    The concept of hepatic memory suggests that a gene responds more rapidly to a second exposure of an inducer than it does during the initial activation. To determine how soon estrogen-dependent DNA/protein interactions occur during the primary response, in vivo dimethylsulfate footprinting was carried out using genomic DNA amplified by ligation-mediated PCR. When estrogen was added to disrupted cells from a hormone-naive liver, changes within and around the estrogen response elements occurred within seconds, indicating a direct and rapid effect on this estrogen-responsive promoter that had never before been activated. Because this effect was so rapid relative to the delayed onset of mRNA accumulation during the primary response, run-on transcription assays were used to determine the transcription profiles for four of the yolk protein genes during the primary and secondary responses to estrogen. As with the accumulation of mRNA, the onset of transcription was delayed for all of these genes after a primary exposure to estrogen. Interestingly, after the secondary exposure to estrogen, the vitellogenin I, vitellogenin II, and very low density apolipoprotein II genes displayed a more rapid onset of transcription, whereas the primary and secondary profiles of apolipoprotein B transcription in response to estrogen were identical. Because the apoB gene is constitutively expressed in the absence of estrogen, and the vitellogenins are quiescent before the administration of the hormone, hepatic memory most likely represents a relatively stable event in the transition to an active state of a gene that is committed for tissue-specific expression.

  13. Metallothionein gene activation in the earthworm (Lumbricus rubellus).

    PubMed

    Höckner, M; Dallinger, R; Stürzenbaum, S R

    2015-05-01

    In order to cope with changing environmental conditions, organisms require highly responsive stress mechanisms. Heavy metal stress is handled by metallothioneins (MTs), the regulation of which is evolutionary conserved in insects and vertebrates and involves the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) positioned in the promoter of MT genes. However, in most invertebrate phyla, the transcriptional activation of MTs is different and the exact mechanism is still unknown. Interestingly, although MREs are typically present also in invertebrate MT gene promoters, MTF-1 is notably absent. Here we use Lumbricus rubellus, the red earthworm, to study the elusive mechanism of wMT-2 activation in control and Cd-exposed conditions. EMSA and DNase I footprinting approaches were used to pinpoint functional binding sites within the wMT-2 promoter region, which revealed that the cAMP responsive element (CRE) is a promising candidate which may act as a transcriptional activator of invertebrate MTs.

  14. Prenatal protein malnutrition decreases KCNJ3 and 2DG activity in rat prefrontal cortex.

    PubMed

    Amaral, A C; Jakovcevski, M; McGaughy, J A; Calderwood, S K; Mokler, D J; Rushmore, R J; Galler, J R; Akbarian, S A; Rosene, D L

    2015-02-12

    Prenatal protein malnutrition (PPM) in rats causes enduring changes in brain and behavior including increased cognitive rigidity and decreased inhibitory control. A preliminary gene microarray screen of PPM rat prefrontal cortex (PFC) identified alterations in KCNJ3 (GIRK1/Kir3.1), a gene important for regulating neuronal excitability. Follow-up with polymerase chain reaction and Western blot showed decreased KCNJ3 expression in the PFC, but not hippocampus or brainstem. To verify localization of the effect to the PFC, baseline regional brain activity was assessed with (14)C-2-deoxyglucose. Results showed decreased activation in the PFC but not hippocampus. Together these findings point to the unique vulnerability of the PFC to the nutritional insult during early brain development, with enduring effects in adulthood on KCNJ3 expression and baseline metabolic activity. PMID:25446346

  15. Prenatal Protein Malnutrition Decreases KCNJ3 and 2DG Activity in Rat Prefrontal Cortex

    PubMed Central

    Amaral, A.C.; Jakovcevski, M.; McGaughy, J.A.; Calderwood, S.K.; Mokler, D.J.; Rushmore, R.J.; Galler, J.R.; Akbarian, S.A.; Rosene, D.L.

    2014-01-01

    Prenatal protein malnutrition (PPM) in rats causes enduring changes in brain and behavior including increased cognitive rigidity and decreased inhibitory control. A preliminary gene microarray screen of PPM rat prefrontal cortex (PFC) identified alterations in KCNJ3 (GIRK1/Kir3.1), a gene important for regulating neuronal excitability. Follow-up with polymerase chain reaction and Western blot showed decreased KCNJ3 expression in PFC, but not hippocampus or brainstem. To verify localization of the effect to the PFC, baseline regional brain activity was assessed with 14C-2-deoxyglucose. Results showed decreased activation in PFC but not hippocampus. Together these findings point to the unique vulnerability of the PFC to the nutritional insult during early brain development, with enduring effects in adulthood on KCNJ3 expression and baseline metabolic activity. PMID:25446346

  16. Prenatal protein malnutrition decreases KCNJ3 and 2DG activity in rat prefrontal cortex.

    PubMed

    Amaral, A C; Jakovcevski, M; McGaughy, J A; Calderwood, S K; Mokler, D J; Rushmore, R J; Galler, J R; Akbarian, S A; Rosene, D L

    2015-02-12

    Prenatal protein malnutrition (PPM) in rats causes enduring changes in brain and behavior including increased cognitive rigidity and decreased inhibitory control. A preliminary gene microarray screen of PPM rat prefrontal cortex (PFC) identified alterations in KCNJ3 (GIRK1/Kir3.1), a gene important for regulating neuronal excitability. Follow-up with polymerase chain reaction and Western blot showed decreased KCNJ3 expression in the PFC, but not hippocampus or brainstem. To verify localization of the effect to the PFC, baseline regional brain activity was assessed with (14)C-2-deoxyglucose. Results showed decreased activation in the PFC but not hippocampus. Together these findings point to the unique vulnerability of the PFC to the nutritional insult during early brain development, with enduring effects in adulthood on KCNJ3 expression and baseline metabolic activity.

  17. The mechanism of inward rectification in Kir channels: A novel kinetic model with non-equilibrium thermodynamics approach.

    PubMed

    Hsieh, Chi-Pan; Chiang, Cheng-Chin; Huang, Chiung-Wei

    2016-05-01

    The mechanisms of the strong inward rectification in inward rectifier K(+) (Kir) channels are controversial because the drop in electrical potential due to the movement of the blocker and coupling ions is insufficient to explain the steep voltage-dependent block near the equilibrium potential. Here, we study the "driving force"-dependent block in Kir channels with a novel approach incorporating concepts from the non-equilibrium thermodynamics of small systems, and computer kinetic simulations based on the experimental data of internal Ba(2+) block on Kir2.1 channels. The steep exponential increase in the apparent binding rate near the equilibrium potential is explained, when the encounter frequency is construed as the likelihood of transfer events down or against the electrochemical potential gradient. The exponent of flux ratio, nf=2.62, implies that the blockage of the internal blocker may be coupled with the outward transport of 2 to 3K(+) ions. The flux-coupled block in the single-file multi-ion pore can be demonstrated by the concentration gradient alone, as well as when the driving force is the electrochemical potential difference across the membrane.

  18. Mechanism for attenuated outward conductance induced by mutations in the cytoplasmic pore of Kir2.1 channels

    NASA Astrophysics Data System (ADS)

    Chang, Hsueh-Kai; Iwamoto, Masayuki; Oiki, Shigetoshi; Shieh, Ru-Chi

    2015-12-01

    Outward currents through Kir2.1 channels regulate the electrical properties of excitable cells. These currents are subject to voltage-dependent attenuation by the binding of polyamines to high- and low-affinity sites, which leads to inward rectification, thereby controlling cell excitability. To examine the effects of positive charges at the low-affinity site in the cytoplasmic pore on inward rectification, we studied a mutant Kir channel (E224K/H226E) and measured single-channel currents and streaming potentials (Vstream), the latter provide the ratio of water to ions queued in a single-file permeation process in the selectivity filter. The water-ion coupling ratio was near one at a high K+ concentration ([K+]) for the wild-type channel and increased substantially as [K+] decreased. On the other hand, fewer ions occupied the selectivity filter in the mutant at all [K+]. A model for the Kir channel involving a K+ binding site in the wide pore was introduced. Model analyses revealed that the rate constants associated with the binding and release to and from the wide-pore K+ binding site was modified in the mutant. These effects lead to the reduced contribution of a conventional two-ion permeation mode to total conductance, especially at positive potentials, thereby inward rectification.

  19. Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

    PubMed

    Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

    2015-08-01

    Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

  20. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    PubMed

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

  1. Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

    PubMed

    Adelman, Karen; Wei, Wenxiang; Ardehali, M Behfar; Werner, Janis; Zhu, Bing; Reinberg, Danny; Lis, John T

    2006-01-01

    The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure. PMID:16354696

  2. Cis and trans activation of adenovirus IVa2 gene transcription.

    PubMed Central

    Natarajan, V; Salzman, N P

    1985-01-01

    The transcriptional control region of the adenovirus IVa2 promoter was analyzed by cloning this promoter in front of a gene coding for bacterial chloramphenicol acetyl transferase (CATase) and estimating levels of CATase and IVa2 promoter specific RNA synthesized after transfection. To produce detectable amounts of CATase with the IVa2 promoter, an enhancer has to be present in cis. In the absence of enhancer sequences, the adenovirus E1A gene can not stimulate CATase synthesis. When cells were transfected with plasmids containing enhancer sequences and various IVa2 mutant promoters upstream of the CAT gene, we observed that CATase activity was not reduced significantly even after deletion of all sequences upstream of the RNA initiation site. Synthesis of IVa2 specific RNA was dependent on plasmids containing an enhancer (SV40 72 bp repeat) that was present in cis. In the absence of enhancer sequences, co-transfection to provide the adenovirus E1A gene in trans also stimulated IVa2 RNA synthesis. When HeLa cells were transfected with various deletion mutants with an enhancer in cis it was seen that sequences -38 to -64 base pairs upstream of the RNA initiation site are necessary for efficient transcription. The E1A gene in trans and an enhancer in cis have an additive effect on RNA synthesis from both IVa2 and major late promoters. The basis for the conflicting results between transcription and CATase synthesis is discussed. Images PMID:2989786

  3. Adaptation of muscle gene expression to changes in contractile activity

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Babij, P.; Thomason, D. B.; Wong, T. S.; Morrison, P. R.

    1987-01-01

    A review of the existing literature regarding the effects of different types of physical activities on the gene expression of adult skeletal muscles leads us to conclude that each type of exercise training program has, as a result, a different phenotype, which means that there are multiple mechanisms, each producing a unique phenotype. A portion of the facts which support this position is presented and interpreted here. [Abstract translated from the original French by NASA].

  4. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  5. trans activation of gene expression by v-myb.

    PubMed Central

    Ibanez, C E; Lipsick, J S

    1990-01-01

    The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation. Images PMID:2325652

  6. Titanium nanotubes activate genes related to bone formation in vitro

    PubMed Central

    Pozio, Alfonso; Palmieri, Annalisa; Girardi, Ambra; Cura, Francesca; Carinci, Francesco

    2012-01-01

    Background: Titanium is used worldwide to make osseointegrable devices, thanks to its favorable characteristics as mechanical proprieties and biocompatibility, demonstrated by in vivo studies with animal models and clinical trials over a forty-year period. However, the exact genetic effect of the titanium layer on cells is still not well characterized. Materials and Methods: To investigate how titanium nanotubes stimulate osteoblasts differentiation and proliferation, some osteoblast genes (SP7, RUNX2, COL3A1, COL1A1, ALPL, SPP1 and FOSL1) were analyzed by quantitative Real Time RT- PCR. Results: After 15 days, osteoblasts cultivated on titanium naotube showed the up-regulation of bone related genes SP7, ENG, FOSL1 and SPP1 and the down-regulation of RUNX2, COL3A1, COL1A1, and ALPL. After 30 days of treatment, the bone related genes SP7, ENG, FOSL1 and RUNX2 were up-regulated while COL3A1, COL1A1, ALPL and SPP1 were down-regulated. Conclusions: Our results, demonstrates that titanium nanotubes can lead to osteoblast differentiation and extracellular matrix deposition and mineralization in dental pulp stem cells by the activation of osteoblast related genes SPP1, FOSL1 and RUNX2. PMID:23814577

  7. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

  8. Hormonal activity of polycyclic musks evaluated by reporter gene assay.

    PubMed

    Mori, Taiki; Iida, Mitsuru; Ishibashi, Hiroshi; Kohra, Shinya; Takao, Yuji; Takemasa, Takehiro; Arizono, Koji

    2007-01-01

    Synthetic musk fragrance compounds, such as polycyclic musks (PCMs), are a group of chemicals used extensively as personal care products, and can be found in the environment and the human body. PCMs, such as 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta-gamma-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyltetralin (AHTN), are known to have agonistic activities toward human estrogen receptor alpha (hERalpha) and hERbeta, and have antagonistic activity toward the human androgen receptor (hAR), as shown in several reporter gene assays. However, little is known about the interaction of PCMs with the human thyroid hormone receptor (hTR), and the hormonal effects of other PCMs except for HHCB and AHTN. In this study, we focus on the interactions of six PCMs, namely, HHCB, AHTN, 4-acetyl-1,1-dimethyl-6-tert-butyl-indan (ADBI), 6-acetyl-1,1,2,3,3,5-hexamethylindan (AHMI), 6,7-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone (DPMI), and 5-acetyl-1,1,2,6-tetramethyl-3-isopropy-lindan (ATII) with hERalpha, hAR, and hTRbeta by in vitro reporter gene assay using Chinese hamster ovary cells. All the samples were found to be agonists toward hERalpha, whereas no agonistic activities of these PCMs for hAR and hTRbeta were observed. No antagonistic activities for hERalpha and hTRbeta were observed at the concentrations tested. However, several PCMs, namely, HHCB, AHTN, ATII, ADBI, and AHMI, showed dose-dependent antagonistic activities for hAR, and the IC50 values of these compounds were estimated to be 1.0 x 10(-7), 1.5 x 10(-7), 1.4 x 10(-7), 9.8 x 10(-6), and 1.4 x 10(-7) M, respectively. The results suggest that these PCMs interact with hERalpha and hAR but have no hormonal effect on hTRbeta. This is the first report on the agonistic and antagonistic activities of ATII, ADBI, AHMI, and DPMI for hERalpha and hAR as determined by in vitro reporter gene assay using stably transfected Chinese hamster ovary cells.

  9. Gene-regulatory activity of alpha-tocopherol.

    PubMed

    Rimbach, Gerald; Moehring, Jennifer; Huebbe, Patricia; Lodge, John K

    2010-03-01

    Vitamin E is an essential vitamin and a lipid soluble antioxidant, at least, under in vitro conditions. The antioxidant properties of vitamin E are exerted through its phenolic hydroxyl group, which donates hydrogen to peroxyl radicals, resulting in the formation of stable lipid species. Beside an antioxidant role, important cell signalling properties of vitamin E have been described. By using gene chip technology we have identified alpha-tocopherol sensitive molecular targets in vivo including christmas factor (involved in the blood coagulation) and 5alpha-steroid reductase type 1 (catalyzes the conversion of testosterone to 5alpha-dihydrotestosterone) being upregulated and gamma-glutamyl-cysteinyl synthetase (the rate limiting enzyme in GSH synthesis) being downregulated due to alpha-tocopherol deficiency. Alpha-tocopherol regulates signal transduction cascades not only at the mRNA but also at the miRNA level since miRNA 122a (involved in lipid metabolism) and miRNA 125b (involved in inflammation) are downregulated by alpha-tocopherol. Genetic polymorphisms may determine the biological and gene-regulatory activity of alpha-tocopherol. In this context we have recently shown that genes encoding for proteins involved in peripheral alpha-tocopherol transport and degradation are significantly affected by the apoE genotype.

  10. ATP-sensitive K(+) channels (Kir6.1/SUR1) regulate gap junctional coupling in cochlear-supporting cells.

    PubMed

    Blödow, Alexander; Begandt, Daniela; Bader, Almke; Becker, Annegret; Burghard, Alice; Kühne, Daniela; Kral, Andrej; Ngezahayo, Anaclet

    2016-07-01

    Using the double whole-cell patch-clamp technique, we found that the absence of intracellular ATP led to gap junction uncoupling in cochlear-supporting Hensen cells. The uncoupling was observed as a progressive reduction of the gap junctional electrical conductance from a starting value of approximately 40 nS to less than 0.04 nS within 10-20 min. The conductance rundown was partly avoided by at least 3 mM ATP and completely suppressed by 5 mM ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP), the non-hydrolysable ATP analog, in the pipette filling solution, suggesting that ATP was needed as ligand and not as a hydrolysable energy supplier or substrate for enzymatic reactions. The effect of intracellular ATP was mimicked by the external application of barium, a nonselective blocker of inwardly rectifying K(+) (Kir) channels, and glibenclamide, an inhibitor of the ATP-sensitive Kir channels (KATP). Moreover a Ba(2+)-sensitive whole-cell inward current was observed in absence of internal ATP. We propose that the internal ATP kept the KATP channels in a closed state, thereby maintaining the gap junction coupling of Hensen cells. The immunostaining of guinea pig cochlear tissue revealed for the first time the expression of the KATP channel subunits Kir6.1 and SUR1 in Hensen cells and supported the proposed hypothesis. The results suggest that KATP channels, as regulator of the gap junction coupling in Hensen cells, could be the physiological link between the metabolic state of the supporting cells and K(+) recycling in the organ of Corti. PMID:27030354

  11. ATP-sensitive K(+) channels (Kir6.1/SUR1) regulate gap junctional coupling in cochlear-supporting cells.

    PubMed

    Blödow, Alexander; Begandt, Daniela; Bader, Almke; Becker, Annegret; Burghard, Alice; Kühne, Daniela; Kral, Andrej; Ngezahayo, Anaclet

    2016-07-01

    Using the double whole-cell patch-clamp technique, we found that the absence of intracellular ATP led to gap junction uncoupling in cochlear-supporting Hensen cells. The uncoupling was observed as a progressive reduction of the gap junctional electrical conductance from a starting value of approximately 40 nS to less than 0.04 nS within 10-20 min. The conductance rundown was partly avoided by at least 3 mM ATP and completely suppressed by 5 mM ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP), the non-hydrolysable ATP analog, in the pipette filling solution, suggesting that ATP was needed as ligand and not as a hydrolysable energy supplier or substrate for enzymatic reactions. The effect of intracellular ATP was mimicked by the external application of barium, a nonselective blocker of inwardly rectifying K(+) (Kir) channels, and glibenclamide, an inhibitor of the ATP-sensitive Kir channels (KATP). Moreover a Ba(2+)-sensitive whole-cell inward current was observed in absence of internal ATP. We propose that the internal ATP kept the KATP channels in a closed state, thereby maintaining the gap junction coupling of Hensen cells. The immunostaining of guinea pig cochlear tissue revealed for the first time the expression of the KATP channel subunits Kir6.1 and SUR1 in Hensen cells and supported the proposed hypothesis. The results suggest that KATP channels, as regulator of the gap junction coupling in Hensen cells, could be the physiological link between the metabolic state of the supporting cells and K(+) recycling in the organ of Corti.

  12. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells.

    PubMed

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-08-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. PMID:27235552

  13. A Bayesian Framework for the Classification of Microbial Gene Activity States.

    PubMed

    Disselkoen, Craig; Greco, Brian; Cook, Kaitlyn; Koch, Kristin; Lerebours, Reginald; Viss, Chase; Cape, Joshua; Held, Elizabeth; Ashenafi, Yonatan; Fischer, Karen; Acosta, Allyson; Cunningham, Mark; Best, Aaron A; DeJongh, Matthew; Tintle, Nathan

    2016-01-01

    Numerous methods for classifying gene activity states based on gene expression data have been proposed for use in downstream applications, such as incorporating transcriptomics data into metabolic models in order to improve resulting flux predictions. These methods often attempt to classify gene activity for each gene in each experimental condition as belonging to one of two states: active (the gene product is part of an active cellular mechanism) or inactive (the cellular mechanism is not active). These existing methods of classifying gene activity states suffer from multiple limitations, including enforcing unrealistic constraints on the overall proportions of active and inactive genes, failing to leverage a priori knowledge of gene co-regulation, failing to account for differences between genes, and failing to provide statistically meaningful confidence estimates. We propose a flexible Bayesian approach to classifying gene activity states based on a Gaussian mixture model. The model integrates genome-wide transcriptomics data from multiple conditions and information about gene co-regulation to provide activity state confidence estimates for each gene in each condition. We compare the performance of our novel method to existing methods on both simulated data and real data from 907 E. coli gene expression arrays, as well as a comparison with experimentally measured flux values in 29 conditions, demonstrating that our method provides more consistent and accurate results than existing methods across a variety of metrics. PMID:27555837

  14. A Bayesian Framework for the Classification of Microbial Gene Activity States

    PubMed Central

    Disselkoen, Craig; Greco, Brian; Cook, Kaitlyn; Koch, Kristin; Lerebours, Reginald; Viss, Chase; Cape, Joshua; Held, Elizabeth; Ashenafi, Yonatan; Fischer, Karen; Acosta, Allyson; Cunningham, Mark; Best, Aaron A.; DeJongh, Matthew; Tintle, Nathan

    2016-01-01

    Numerous methods for classifying gene activity states based on gene expression data have been proposed for use in downstream applications, such as incorporating transcriptomics data into metabolic models in order to improve resulting flux predictions. These methods often attempt to classify gene activity for each gene in each experimental condition as belonging to one of two states: active (the gene product is part of an active cellular mechanism) or inactive (the cellular mechanism is not active). These existing methods of classifying gene activity states suffer from multiple limitations, including enforcing unrealistic constraints on the overall proportions of active and inactive genes, failing to leverage a priori knowledge of gene co-regulation, failing to account for differences between genes, and failing to provide statistically meaningful confidence estimates. We propose a flexible Bayesian approach to classifying gene activity states based on a Gaussian mixture model. The model integrates genome-wide transcriptomics data from multiple conditions and information about gene co-regulation to provide activity state confidence estimates for each gene in each condition. We compare the performance of our novel method to existing methods on both simulated data and real data from 907 E. coli gene expression arrays, as well as a comparison with experimentally measured flux values in 29 conditions, demonstrating that our method provides more consistent and accurate results than existing methods across a variety of metrics. PMID:27555837

  15. Production of the Ramoplanin Activity Analogue by Double Gene Inactivation

    PubMed Central

    Han, Jungang; Chen, Junsheng; Shao, Lei; Zhang, Junliang; Dong, Xiaojing; Liu, Pengyu; Chen, Daijie

    2016-01-01

    Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin. PMID:27149627

  16. Identification of Three Interactions to Determine the Conformation Change and to Maintain the Function of Kir2.1 Channel Protein

    NASA Astrophysics Data System (ADS)

    Li, Jun-Wei; Xiao, Shao-Ying; Xie, Xiao-Xiao; Yu, Hui; Zhang, Hai-Lin; Zhan, Yong; An, Hai-Long

    2015-02-01

    We find that a conserved mutation residue Glu to residue Asp (E303D), which both have the same polar and charged properties, makes Kir2.1 protein lose its function. To understand the mechanism, we identify three interactions which control the conformation change and maintain the function of the Kir2.1 protein by combining homology modeling and molecular dynamics with targeted molecular dynamics. We find that the E303D mutation weakens these interactions and results in the loss of the related function. Our data indicate that not only the amino residues but also the interactions determine the function of proteins.

  17. Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method.

    PubMed

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.

  18. Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

    PubMed Central

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K.; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H.; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars. PMID:23658605

  19. Isolation and characterization of a novel B cell activation gene

    SciTech Connect

    Hong, J.X.; Wilson, G.L.; Fox, C.H.; Kehrl, J.H. )

    1993-05-01

    Using subtractive cDNA cloning, the authors have isolated a series of cDNA clones that are differentially expressed between B and T lymphocytes. Whereas some of the isolated cDNA are from known B cell-specific genes, many of them represent previously uncharacterized genes. One of these unknown genes was denoted as BL34. Northern blot analysis performed with the BL34 cDNA revealed a 1.6-kb mRNA transcript that was present at low levels in RNA extracted from resting B lymphocytes, but whose expression was markedly increased in RNA prepared from mitogen-activated B cells. Similarly, RNA prepared from several B cell lines treated with phorbol myristate acetate (PMA) contained high levels of BL34 mRNA. In contrast, RNA from purified T cells treated with phytohemagglutinin and PMA had undetectable amounts of BL34 mRNA. In addition, high levels of BL34 mRNA were detected in RNA purified from PBMC of a patient with B cell acute lymphocytic leukemia. Southern blot analysis of human DNA from various tissues and cells lines demonstrated that BL34 is a single-copy gene without evidence of rearrangement. Two full length BL34 cDNA were sequenced, and an open reading frame of 588 bp was identified that was predicted to encode for a 196 amino acid protein. Searches of several protein data bases failed to find any homologous proteins. To directly analyze the expression of BL34 mRNA in lymphoid tissues in situ, hybridization studies with human tonsil tissue sections were performed. BL34 mRNA was detected in a portion of the cells in the germinal center region and adjacent to the mantle region. Further characterization of the BL34 gene and its protein should lead to insights to its role in B cell function and the consequences of its over-expression in acute lymphocytic leukemia. 26 refs., 6 figs., 1 tab.

  20. KIR3DL2 binds to HLA-B27 dimers and free heavy chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis

    PubMed Central

    Wong-Baeza, Isabel; Ridley, Anna; Shaw, Jackie; Hatano, Hiroko; Rysnik, Oliwia; McHugh, Kirsty; Piper, Christopher; Brackenbridge, Simon; Fernandes, Ricardo; Chan, Anthoni; Bowness, Paul; Kollnberger, Simon

    2013-01-01

    1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC. PMID:23440420

  1. Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages

    PubMed Central

    Saavedra, Nicolás; Cuevas, Alejandro; Cavalcante, Marcela F.; Dörr, Felipe A.; Saavedra, Kathleen; Zambrano, Tomás; Abdalla, Dulcineia S. P.; Salazar, Luis A.

    2016-01-01

    Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques. PMID:27119082

  2. Activation of silenced cytokine gene promoters by the synergistic effect of TBP-TALE and VP64-TALE activators.

    PubMed

    Anthony, Kim; More, Abhijit; Zhang, Xiaoliu

    2014-01-01

    Recent work has shown that the combinatorial use of multiple TALE activators can selectively activate certain cellular genes in inaccessible chromatin regions. In this study, we aimed to interrogate the activation potential of TALEs upon transcriptionally silenced immune genes in the context of non-immune cells. We designed a unique strategy, in which a single TALE fused to the TATA-box binding protein (TBP-TALE) is coupled with multiple VP64-TALE activators. We found that our strategy is significantly more potent than multiple TALE activators alone in activating expression of IL-2 and GM-CSF in diverse cell origins in which both genes are otherwise completely silenced. Chromatin analysis revealed that the gene activation was due in part to displacement of a distinctly positioned nucleosome. These studies provide a novel epigenetic mechanism for artificial gene induction and have important implications for targeted cancer immunotherapy, DNA vaccine development, as well as rational design of TALE activators.

  3. Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

    PubMed Central

    De Vos, P; Lefebvre, A M; Miller, S G; Guerre-Millo, M; Wong, K; Saladin, R; Hamann, L G; Staels, B; Briggs, M R; Auwerx, J

    1996-01-01

    The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter. PMID:8770873

  4. Thiazolidinediones repress ob gene expression in rodents via activation of peroxisome proliferator-activated receptor gamma.

    PubMed

    De Vos, P; Lefebvre, A M; Miller, S G; Guerre-Millo, M; Wong, K; Saladin, R; Hamann, L G; Staels, B; Briggs, M R; Auwerx, J

    1996-08-15

    The ob gene product, leptin, is a signaling factor regulating body weight and energy balance. ob gene expression in rodents is increased in obesity and is regulated by feeding patterns and hormones, such as insulin and glucocorticoids. In humans with gross obesity, ob mRNA levels are higher, but other modulators of human ob expression are unknown. In view of the importance of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte differentiation, we analyzed whether ob gene expression is subject to regulation by factors activating PPARs. Treatment of rats with the PPARalpha activator fenofibrate did not change adipose tissue and body weight and had no significant effect on ob mRNA levels. However, administration of the thiazolidinedione BRL49653, a PPARgamma ligand, increased food intake and adipose tissue weight while reducing ob mRNA levels in rats in a dose-dependent manner. The inhibitory action of the thiazolidinedione BRL49653 on ob mRNA levels was also observed in vitro. Thiazolidinediones reduced the expression of the human ob promoter in primary adipocytes, however, in undifferentiated 3T3-L1 preadipocytes lacking endogenous PPARgamma, cotransfection of PPARgamma was required to observe the decrease. In conclusion, these data suggest that PPARgamma activators reduce ob mRNA levels through an effect of PPARgamma on the ob promoter.

  5. Identification of Novel Gene Targets and Functions of p21-Activated Kinase 1 during DNA Damage by Gene Expression Profiling

    PubMed Central

    Motwani, Mona; Li, Da-Qiang; Horvath, Anelia; Kumar, Rakesh

    2013-01-01

    P21-activated kinase 1 (PAK1), a serine/threonine protein kinase, modulates many cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, PAK1 also affects gene transcription due to its nuclear localization and association with chromatin. It is now recognized that PAK1 kinase activity and its nuclear translocation are rapidly stimulated by ionizing radiation (IR), and that PAK1 activation is a component of the DNA damage response. Owing to the role of PAK1 in the cell survival, its association with the chromatin, and now, stimulation by ionizing radiation, we hypothesize that PAK1 may be contributing to modulation of genes with roles in cellular processes that might be important in the DNA damage response. The purpose of this study was to identify new PAK1 targets in response to ionizing radiation with putative role in the DNA damage response. We examined the effect of IR on the gene expression patterns in the murine embryonic fibroblasts with or without Pak1 using microarray technology. Differentially expressed transcripts were identified using Gene Spring GX 10.0.2. Pathway, network, functional analyses and gene family classification were carried out using Kyoto Encyclopedia of Genes and Genomes (KEGG), Ingenuity Pathway, Gene Ontology and PANTHER respectively. Selective targets of PAK1 were validated by RT-qPCR. For the first time, we provide a genome-wide analysis of PAK1 and identify its targets with potential roles in the DNA damage response. Gene Ontology analysis identified genes in the IR-stimulated cells that were involved in cell cycle arrest and cell death. Pathway analysis revealed p53 pathway being most influenced by IR responsive, PAK1 targets. Gene family of transcription factors was over represented and gene networks involved in DNA replication, repair and cellular signaling were identified. In brief, this study identifies novel PAK1 dependent IR responsive genes which reveal new aspects of PAK1

  6. Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels

    PubMed Central

    Sepúlveda, Francisco V.; Pablo Cid, L.; Teulon, Jacques; Niemeyer, María Isabel

    2015-01-01

    K+ channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K+ channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K+ homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K+-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge. PMID:25540142

  7. Development of a Selective Small-Molecule Inhibitor of Kir1.1, the Renal Outer Medullary Potassium ChannelS⃞

    PubMed Central

    Bhave, Gautam; Chauder, Brian A.; Liu, Wen; Dawson, Eric S.; Kadakia, Rishin; Nguyen, Thuy T.; Lewis, L. Michelle; Meiler, Jens; Weaver, C. David; Satlin, Lisa M.; Lindsley, Craig W.

    2011-01-01

    The renal outer medullary potassium (K+) channel, ROMK (Kir1.1), is a putative drug target for a novel class of loop diuretic that would lower blood volume and pressure without causing hypokalemia. However, the lack of selective ROMK inhibitors has hindered efforts to assess its therapeutic potential. In a high-throughput screen for small-molecule modulators of ROMK, we previously identified a potent and moderately selective ROMK antagonist, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (VU590), that also inhibits Kir7.1. Because ROMK and Kir7.1 are coexpressed in the nephron, VU590 is not a good probe of ROMK function in the kidney. Here we describe the development of the structurally related inhibitor 2,2′-oxybis(methylene)bis(5-nitro-1H-benzo[d]imidazole) (VU591), which is as potent as VU590 but is selective for ROMK over Kir7.1 and more than 65 other potential off-targets. VU591 seems to block the intracellular pore of the channel. The development of VU591 may enable studies to explore the viability of ROMK as a diuretic target. PMID:20926757

  8. Flow- and voltage-dependent blocking effect of ethosuximide on the inward rectifier K⁺ (Kir2.1) channel.

    PubMed

    Huang, Chiung-Wei; Kuo, Chung-Chin

    2015-08-01

    Absence seizures are manifestations of abnormal thalamocortical oscillations characterized by spike-and-wave complexes in EEG. Ethosuximide (ETX) is one of the principal medications against absence seizures. We investigate the effect of ETX on the Kir2.1 channel, a prototypical inward rectifier K(+) channel possibly playing an important role in the setting of neuronal membrane potential. We demonstrate that the outward currents of Kir2.1 channels are significantly inhibited by intracellular ETX. We further show that the movement of neutral molecule ETX in the Kir2.1 channel is accompanied by ∼1.2 K(+), giving rise to the vivid voltage dependence of ETX unbinding rate. Moreover, the apparent affinity (K d ) of ETX in the channels are decreased by single-point mutations involving M183, E224, and S165, and especially by double mutations involving T141/S165, which always also disrupt the flux-coupling feature of ETX block. Molecular dynamics simulation demonstrates narrowing of the pore at ∼D172 by binding of ETX to S165 or T141. ETX block of the Kir2.1 channels may cause a modest but critical depolarization of the relevant neurons, decreasing available T-type Ca(2+) channels and consequently lessening pathological thalamocortical burst discharges.

  9. Molecular aspects of structure, gating, and physiology of pH-sensitive background K2P and Kir K+-transport channels.

    PubMed

    Sepúlveda, Francisco V; Pablo Cid, L; Teulon, Jacques; Niemeyer, María Isabel

    2015-01-01

    K(+) channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K(+) channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K(+) homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K(+)-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.

  10. Conserved structure and adjacent location of the thrombin receptor and protease-activated receptor 2 genes define a protease-activated receptor gene cluster.

    PubMed Central

    Kahn, M.; Ishii, K.; Kuo, W. L.; Piper, M.; Connolly, A.; Shi, Y. P.; Wu, R.; Lin, C. C.; Coughlin, S. R.

    1996-01-01

    BACKGROUND: Thrombin is a serine protease that elicits a variety of cellular responses. Molecular cloning of a thrombin receptor revealed a G protein-coupled receptor that is activated by a novel proteolytic mechanism. Recently, a second protease-activated receptor was discovered and dubbed PAR2. PAR2 is highly related to the thrombin receptor by sequence and, like the thrombin receptor, is activated by cleavage of its amino terminal exodomain. Also like the thrombin receptor, PAR2 can be activated by the hexapeptide corresponding to its tethered ligand sequence independent of receptor cleavage. Thus, functionally, the thrombin receptor and PAR2 constitute a fledgling receptor family that shares a novel proteolytic activation mechanism. To further explore the relatedness of the two known protease-activated receptors and to examine the possibility that a protease-activated gene cluster might exist, we have compared the structure and chromosomal locations of the thrombin receptor and PAR2 genes. MATERIALS AND METHODS: The genomic structures of the two protease-activated receptor genes were determined by analysis of lambda phage, P1 bacteriophage, and bacterial artificial chromosome (BAC) genomic clones. Chromosomal location was determined with fluorescent in situ hybridization (FISH) on metaphase chromosomes, and the relative distance separating the two genes was evaluated both by means of two-color FISH and analysis of YACs and BACs containing both genes. RESULTS: Analysis of genomic clones revealed that the two protease-activated receptor genes share a two-exon genomic structure in which the first exon encodes 5'-untranslated sequence and signal peptide, and the second exon encodes the mature receptor protein and 3'-untranslated sequence. The two receptor genes also share a common locus with the two human genes located at 5q13 and the two mouse genes at 13D2, a syntenic region of the mouse genome. These techniques also suggest that the physical distance separating

  11. Mining functional relationships in feature subspaces from gene expression profiles and drug activity profiles.

    PubMed

    Bao, Lei; Guo, Tao; Sun, Zhirong

    2002-04-10

    In an effort to determine putative functional relationships between gene expression patterns and drug activity patterns of 60 human cancer cell lines, a novel method was developed to discover local associations within cell line subsets. The association of drug-gene pairs is an explorative way of discovering gene markers that predict clinical tumor sensitivity to therapy. Nine drug-gene networks were discovered, as well as dozens of gene-gene and drug-drug networks. Three drug-gene networks with well studied members were discussed and the literature shows that hypothetical functional relationships exist. Therefore, this method enables the gathering of new information beyond global associations.

  12. Behavioral science and the study of gene-nutrition and gene-physical activity interactions in obesity research.

    PubMed

    Faith, Myles S

    2008-12-01

    This report summarizes emerging opportunities for behavioral science to help advance the field of gene-environment and gene-behavior interactions, based on presentations at The National Cancer Institute (NCI) Workshop, "Gene-Nutrition and Gene-Physical Activity Interactions in the Etiology of Obesity." Three opportunities are highlighted: (i) designing potent behavioral "challenges" in experiments, (ii) determining viable behavioral phenotypes for genetics studies, and (iii) identifying specific measures of the environment or environmental exposures. Additional points are underscored, including the need to incorporate novel findings from neuroimaging studies regarding motivation and drive for eating and physical activity. Advances in behavioral science theory and methods can play an important role in advancing understanding of gene-brain-behavior relationships in obesity onset.

  13. The association of the immune response genes to human papillomavirus-related cervical disease in a Brazilian population.

    PubMed

    Marangon, Amanda Vansan; Guelsin, Gláucia Andreia Soares; Visentainer, Jeane Eliete Laguila; Borelli, Sueli Donizete; Watanabe, Maria Angélica Ehara; Consolaro, Márcia Edilaine Lopes; Caleffi-Ferracioli, Katiany Rizzieri; Rudnick, Cristiane Conceição Chagas; Sell, Ana Maria

    2013-01-01

    The genetic variability of the host contributes to the risk of human papillomavirus (HPV)-related cervical disease. Immune response genes to HPV must be investigated to define patients with the highest risk of developing malignant disease. The aim of this study was to investigate the association of polymorphic immune response genes, namely KIR, HLA class I and II, and single-nucleotide polymorphisms (SNPs) of cytokines with HPV-related cervical disease. We selected 79 non-related, admixed Brazilian women from the state of Paraná, southern region of Brazil, who were infected with high carcinogenic risk HPV and present cervical intraepithelial neoplasia grade 3 (CIN3), and 150 HPV-negative women from the same region matched for ethnicity. KIR genes were genotyped using an in-house PCR-SSP. HLA alleles were typed using a reverse sequence-specific oligonucleotide technique. SNPs of TNF -308G>A, IL6 -174G>C, IFNG +874T>A, TGFB1 +869T>C +915G>C, and IL10 -592C>A -819C>T -1082G>A were evaluated using PCR-SSP. The KIR genes were not associated with HPV, although some pairs of i(inhibitory)KIR-ligands occurred more frequently in patients, supporting a role for NK in detrimental chronic inflammatory and carcinogenesis. Some HLA haplotypes were associated with HPV. The associations of INFG and IL10 SNPs potentially reflect impaired or invalid responses in advanced lesions. PMID:23936772

  14. Sensation seeking genes and physical activity in youth

    PubMed Central

    Wilkinson, Anna V.; Gabriel, Kelley Pettee; Wang, Jian; Bondy, Melissa L.; Dong, Qiong; Wu, Xifeng; Shete, Sanjay; Spitz, Margaret R.

    2012-01-01

    Many studies examining genetic influences on physical activity (PA) have evaluated the impact of single nucleotide polymorphisms (SNPs) related to the development of lifestyle-related chronic diseases, under the hypothesis that they would be associated with PA. However, PA is a multi-determined behavior and associated with a multitude of health consequences. Thus, examining a broader range of candidate genes associated with a boarder range of PA correlates may provide new insights into the genetic underpinnings of PA. In this study we focus on one such correlate – sensation seeking behavior. Participants (N=1,130 Mexican origin youth) provided a saliva sample and data on PA and sensation seeking tendencies in 2008–09. Participants were genotyped for 630 functional and tagging variants in the dopamine, serotonin, and cannabinoid pathways. Overall 30% of participants (males – 37.6%; females – 22.0%) reported ≥60 minutes of PA on five out of seven days. After adjusting for gender, age and population stratification, and applying the Bayesian False Discovery Probability approach for assessing noteworthiness, four gene variants were significantly associated with PA. In a multivariable model, being male, having higher sensation seeking tendencies and at least one copy of the minor allele for SNPs in ACE (rs8066276 OR=1.44; p=0.012) and TPH2 (rs11615016 OR=1.73; p=0.021) were associated with increased likelihood of meeting PA recommendations. Participants with at least one copy of the minor allele for SNPs in SNAP25 (rs363035 OR=0.53; p=0.005) and CNR1 (rs6454672 OR=0.62; p=0.022) have decreased likelihood of meeting PA recommendations. Our findings extend current knowledge of the complex relationship between PA and possible genetic underpinnings. PMID:23190435

  15. Epigenetic signature and enhancer activity of the human APOE gene.

    PubMed

    Yu, Chang-En; Cudaback, Eiron; Foraker, Jessica; Thomson, Zachary; Leong, Lesley; Lutz, Franziska; Gill, James Anthony; Saxton, Aleen; Kraemer, Brian; Navas, Patrick; Keene, C Dirk; Montine, Thomas; Bekris, Lynn M

    2013-12-15

    The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ε2/ε3/ε4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ε4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ε2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ε2/ε3/ε4 allele-carrying 3'-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ε2/ε3/ε4 allele-specific manner. These findings implicate a novel functional role for a 3'-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.

  16. Epigenetic signature and enhancer activity of the human APOE gene

    PubMed Central

    Yu, Chang-En; Cudaback, Eiron; Foraker, Jessica; Thomson, Zachary; Leong, Lesley; Lutz, Franziska; Gill, James Anthony; Saxton, Aleen; Kraemer, Brian; Navas, Patrick; Keene, C. Dirk; Montine, Thomas; Bekris, Lynn M.

    2013-01-01

    The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ɛ2/ɛ3/ɛ4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ɛ4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ɛ2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ɛ2/ɛ3/ɛ4 allele-carrying 3′-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ɛ2/ɛ3/ɛ4 allele-specific manner. These findings implicate a novel functional role for a 3′-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI. PMID:23892237

  17. The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

    PubMed Central

    Dyar, Kenneth A.; Ciciliot, Stefano; Tagliazucchi, Guidantonio Malagoli; Pallafacchina, Giorgia; Tothova, Jana; Argentini, Carla; Agatea, Lisa; Abraham, Reimar; Ahdesmäki, Miika; Forcato, Mattia; Bicciato, Silvio; Schiaffino, Stefano; Blaauw, Bert

    2015-01-01

    Objective Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. Methods We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. Results We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. Conclusions We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression. PMID:26629406

  18. Membrane channel gene expression in human costal and articular chondrocytes.

    PubMed

    Asmar, A; Barrett-Jolley, R; Werner, A; Kelly, R; Stacey, M

    2016-04-01

    Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca(2+) activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  19. Membrane channel gene expression in human costal and articular chondrocytes

    PubMed Central

    Asmar, A.; Barrett-Jolley, R.; Werner, A.; Kelly, R.; Stacey, M.

    2016-01-01

    ABSTRACT Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Schönberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human articular and costal (teenage and fetal origin) cartilages. Notably, we found 14 ion channel genes commonly expressed between articular and both types of costal cartilage chondrocytes. There were several other ion channel genes expressed only in articular (6 genes) or costal chondrocytes (5 genes). Significant differences in expression of BEST1 and KCNJ2 (Kir2.1) were observed between fetal and teenage costal cartilage. Interestingly, the large Ca2+ activated potassium channel (BKα, or KCNMA1) was very highly expressed in all chondrocytes examined. Expression of the gap junction genes for Panx1, GJA1 (Cx43) and GJC1 (Cx45) was also observed in chondrocytes from all cartilage samples. Together, this data highlights similarities between chondrocyte membrane channel gene expressions in cells derived from different anatomical sites, and may imply that common electrophysiological signaling pathways underlie cellular control. The high expression of a range of mechanically and metabolically sensitive membrane channels suggest that chondrocyte mechanotransduction may be more complex than previously thought. PMID:27116676

  20. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    PubMed Central

    Horlbeck, Max A; Gilbert, Luke A; Villalta, Jacqueline E; Adamson, Britt; Pak, Ryan A; Chen, Yuwen; Fields, Alexander P; Park, Chong Yon; Corn, Jacob E; Kampmann, Martin; Weissman, Jonathan S

    2016-01-01

    We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 PMID:27661255

  1. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    EPA Science Inventory

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  2. Network activity-independent coordinated gene expression program for synapse assembly

    PubMed Central

    Valor, Luis M.; Charlesworth, Paul; Humphreys, Lawrence; Anderson, Chris N. G.; Grant, Seth G. N.

    2007-01-01

    Global biological datasets generated by genomics, transcriptomics, and proteomics provide new approaches to understanding the relationship between the genome and the synapse. Combined transcriptome analysis and multielectrode recordings of neuronal network activity were used in mouse embryonic primary neuronal cultures to examine synapse formation and activity-dependent gene regulation. Evidence for a coordinated gene expression program for assembly of synapses was observed in the expression of 642 genes encoding postsynaptic and plasticity proteins. This synaptogenesis gene expression program preceded protein expression of synapse markers and onset of spiking activity. Continued expression was followed by maturation of morphology and electrical neuronal networks, which was then followed by the expression of activity-dependent genes. Thus, two distinct sequentially active gene expression programs underlie the genomic programs of synapse function. PMID:17360580

  3. Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava.

    PubMed

    Cohn, Megan; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J

    2016-08-01

    Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems. PMID:26575863

  4. Comparison of gene activation by two TAL effectors from Xanthomonas axonopodis pv. manihotis reveals candidate host susceptibility genes in cassava.

    PubMed

    Cohn, Megan; Morbitzer, Robert; Lahaye, Thomas; Staskawicz, Brian J

    2016-08-01

    Xanthomonas axonopodis pv. manihotis (Xam) employs transcription activator-like (TAL) effectors to promote bacterial growth and symptom formation during infection of cassava. TAL effectors are secreted via the bacterial type III secretion system into plant cells, where they are directed to the nucleus, bind DNA in plant promoters and activate the expression of downstream genes. The DNA-binding activity of TAL effectors is carried out by a central domain which contains a series of repeat variable diresidues (RVDs) that dictate the sequence of bound nucleotides. TAL14Xam668 promotes virulence in Xam strain Xam668 and has been shown to activate multiple cassava genes. In this study, we used RNA sequencing to identify the full target repertoire of TAL14Xam668 in cassava, which includes over 50 genes. A subset of highly up-regulated genes was tested for activation by TAL14CIO151 from Xam strain CIO151. Although TAL14CIO151 and TAL14Xam668 differ by only a single RVD, they display differential activation of gene targets. TAL14CIO151 complements the TAL14Xam668 mutant defect, implying that shared target genes are important for TAL14Xam668 -mediated disease susceptibility. Complementation with closely related TAL effectors is a novel approach to the narrowing down of biologically relevant susceptibility genes of TAL effectors with multiple targets. This study provides an example of how TAL effector target activation by two strains within a single species of Xanthomonas can be dramatically affected by a small change in RVD-nucleotide affinity at a single site, and reflects the parameters of RVD-nucleotide interaction determined using designer TAL effectors in transient systems.

  5. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, E.E.; Roessler, P.G.

    1999-07-27

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities. 8 figs.

  6. Isolated gene encoding an enzyme with UDP-glucose pyrophosphorylase and phosphoglucomutase activities from Cyclotella cryptica

    DOEpatents

    Jarvis, Eric E.; Roessler, Paul G.

    1999-01-01

    The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme. The gene, isolated from Cyclotella cryptica, encodes a multifunctional enzyme that has both UDP-glucose pyrophosphorylase and phosphoglucomutase activities.

  7. Luciferase as a reporter of gene activity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since their development and introduction in the early days of plant genetic engineering, reporter genes have established a proven track record as effective tools for exploring the molecular underpinnings of gene regulation. When driven by appropriate genetic control systems (e.g. transcriptional pr...

  8. SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil

    PubMed Central

    Shindo, Takashi; Yamada, Mitsuhiko; Isomoto, Shojiro; Horio, Yoshiyuki; Kurachi, Yoshihisa

    1998-01-01

    The classical ATP sensitive K+ (KATP) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K+ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K+ channel openers, such as pinacidil and nicorandil.In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the KATP channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method.In the cell-attached recordings (145 mM K+ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K+ channel in both the transfected cells.In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR2B/Kir6.2 and SUR2A/Kir6.2 channels (EC50 of ∼2 and ∼10 μM, respectively). On the other hand, nicorandil activated the SUR2B/Kir6.2 channel >100 times more potently than the SUR2A/Kir6.2 (EC50 of ∼10 μM and >500 μM, respectively).Thus, nicorandil, but not pinacidil, preferentially activates the KATP channels containing SUR2B. Because SUR2A and SUR2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR2B may play a critical role in the action of nicorandil on the vascular type classical KATP channel. PMID:9692785

  9. TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

    PubMed

    Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

    2014-01-24

    Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells.

  10. Activation of Developmentally Mutated Human Globin Genes by Cell Fusion

    NASA Astrophysics Data System (ADS)

    Papayannopoulou, Thalia; Enver, Tariq; Takegawa, Susumu; Anagnou, Nicholas P.; Stamatoyannopoulos, George

    1988-11-01

    Human fetal globin genes are not expressed in hybrid cells produced by the fusion of normal human lymphocytes with mouse erythroleukemia cells. In contrast, when lymphocytes from persons with globin gene developmental mutations (hereditary persistence of fetal hemoglobin) are used for these fusions, fetal globin is expressed in the hybrid cells. Thus, mutations of developmental origin can be reconstituted in vitro by fusing mutant lymphoid cells with differentiated cell lines of the proper lineage. This system can readily be used for analyses, such as globin gene methylation, that normally require large numbers of pure nucleated erythroid cells, which are difficult to obtain.

  11. Interplay between stochasticity and negative feedback leads to pulsed dynamics and distinct gene activity patterns

    NASA Astrophysics Data System (ADS)

    Zambrano, Samuel; Bianchi, Marco E.; Agresti, Alessandra; Molina, Nacho

    2015-08-01

    Gene expression is an inherently stochastic process that depends on the structure of the biochemical regulatory network in which the gene is embedded. Here we study the dynamical consequences of the interplay between stochastic gene switching and the widespread negative feedback regulatory loop in a simple model of a biochemical regulatory network. Using a simplified hybrid simulation approach, in which only the gene activation is modeled stochastically, we find that stochasticity in gene switching by itself can induce pulses in the system, providing also analytical insights into their origin. Furthermore, we find that this simple network is able to reproduce both exponential and peaked distributions of gene active and inactive times similar to those that have been observed experimentally. This simplified hybrid simulation approach also allows us to link these patterns to the dynamics of the system for each gene state.

  12. Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies.

    PubMed

    Savkovic, S D; Koutsouris, A; Wu, G; Hecht, G

    2000-09-01

    Pathogenic microbes influence gene regulation in eukaryotic hosts. Reporter gene studies can define the roles of promoter regulatory sequences. The effect of pathogenic bacteria on reporter genes has not been examined. The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems. Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter. Cells were infected with enteropathogenic E. coli or Salmonella typhimurium, and the reporter activity was assessed. Luc activity significantly decreased following infection, regardless of the promoter. The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system. Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation. Neither beta-gal nor CAT activity was altered by infection. Disruption of type III secretion prevented the loss of luc activity. We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation.

  13. Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes.

    PubMed

    German, Donovan P; Foti, Dolly M; Heras, Joseph; Amerkhanian, Hooree; Lockwood, Brent L

    2016-01-01

    Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result. PMID:27327179

  14. Bidirectional Transcription Directs Both Transcriptional Gene Activation and Suppression in Human Cells

    PubMed Central

    Morris, Kevin V.; Santoso, Sharon; Turner, Anne-Marie; Pastori, Chiara; Hawkins, Peter G.

    2008-01-01

    Small RNAs targeted to gene promoters in human cells have been shown to modulate both transcriptional gene suppression and activation. However, the mechanism involved in transcriptional activation has remained poorly defined, and an endogenous RNA trigger for transcriptional gene silencing has yet to be identified. Described here is an explanation for siRNA-directed transcriptional gene activation, as well as a role for non-coding antisense RNAs as effector molecules driving transcriptional gene silencing. Transcriptional activation of p21 gene expression was determined to be the result of Argonaute 2–dependent, post-transcriptional silencing of a p21-specific antisense transcript, which functions in Argonaute 1–mediated transcriptional control of p21 mRNA expression. The data presented here suggest that in human cells, bidirectional transcription is an endogenous gene regulatory mechanism whereby an antisense RNA directs epigenetic regulatory complexes to a sense promoter, resulting in RNA-directed epigenetic gene regulation. The observations presented here support the notion that epigenetic silencing of tumor suppressor genes, such as p21, may be the result of an imbalance in bidirectional transcription levels. This imbalance allows the unchecked antisense RNA to direct silent state epigenetic marks to the sense promoter, resulting in stable transcriptional gene silencing. PMID:19008947

  15. Elevated Gene Copy Number Does Not Always Explain Elevated Amylase Activities in Fishes.

    PubMed

    German, Donovan P; Foti, Dolly M; Heras, Joseph; Amerkhanian, Hooree; Lockwood, Brent L

    2016-01-01

    Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result.

  16. Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene.

    PubMed

    Zong, Chenghang; So, Lok-hang; Sepúlveda, Leonardo A; Skinner, Samuel O; Golding, Ido

    2010-11-30

    The ability of living cells to maintain an inheritable memory of their gene-expression state is key to cellular differentiation. Bacterial lysogeny serves as a simple paradigm for long-term cellular memory. In this study, we address the following question: in the absence of external perturbation, how long will a cell stay in the lysogenic state before spontaneously switching away from that state? We show by direct measurement that lysogen stability exhibits a simple exponential dependence on the frequency of activity bursts from the fate-determining gene, cI. We quantify these gene-activity bursts using single-molecule-resolution mRNA measurements in individual cells, analyzed using a stochastic mathematical model of the gene-network kinetics. The quantitative relation between stability and gene activity is independent of the fine details of gene regulation, suggesting that a quantitative prediction of cell-state stability may also be possible in more complex systems. PMID:21119634

  17. A Drosophila Adh gene can be activated in trans by an enhancer.

    PubMed Central

    Rothberg, I; Hotaling, E; Sofer, W

    1991-01-01

    The ability of a segment of the Drosophila Adh gene to produce ADH activity in larvae is dependent upon the presence of a 53 bp sequence (called NS1) located between 289 and 341 bp upstream of the larval transcription start site. This sequence behaves like an enhancer in that it can stimulate gene activity when it is placed at various distances from, or on either side of, an Adh gene. Like a typical enhancer, NS1 does not ordinarily function in trans. However, when an Adh gene lacking NS1 is placed on one plasmid, and a second gene carrying NS1 is placed on another, and the two plasmids are interlocked in a catenane, both genes are active. This finding supports the mechanism of loop-mediated enhancer action. Images PMID:1945848

  18. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    PubMed

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  19. Transposable Elements Contribute to Activation of Maize Genes in Response to Abiotic Stress

    PubMed Central

    Makarevitch, Irina; Waters, Amanda J.; West, Patrick T.; Stitzer, Michelle; Hirsch, Candice N.; Ross-Ibarra, Jeffrey; Springer, Nathan M.

    2015-01-01

    Transposable elements (TEs) account for a large portion of the genome in many eukaryotic species. Despite their reputation as “junk” DNA or genomic parasites deleterious for the host, TEs have complex interactions with host genes and the potential to contribute to regulatory variation in gene expression. It has been hypothesized that TEs and genes they insert near may be transcriptionally activated in response to stress conditions. The maize genome, with many different types of TEs interspersed with genes, provides an ideal system to study the genome-wide influence of TEs on gene regulation. To analyze the magnitude of the TE effect on gene expression response to environmental changes, we profiled gene and TE transcript levels in maize seedlings exposed to a number of abiotic stresses. Many genes exhibit up- or down-regulation in response to these stress conditions. The analysis of TE families inserted within upstream regions of up-regulated genes revealed that between four and nine different TE families are associated with up-regulated gene expression in each of these stress conditions, affecting up to 20% of the genes up-regulated in response to abiotic stress, and as many as 33% of genes that are only expressed in response to stress. Expression of many of these same TE families also responds to the same stress conditions. The analysis of the stress-induced transcripts and proximity of the transposon to the gene suggests that these TEs may provide local enhancer activities that stimulate stress-responsive gene expression. Our data on allelic variation for insertions of several of these TEs show strong correlation between the presence of TE insertions and stress-responsive up-regulation of gene expression. Our findings suggest that TEs provide an important source of allelic regulatory variation in gene response to abiotic stress in maize. PMID:25569788

  20. Acidity-Activated Shielding Strategies of Cationic Gene Delivery for Cancer Therapy.

    PubMed

    Xia, Jialiang; Feng, Zongcai; Yang, Hongyan; Lin, Sanqing; Han, Bing

    2016-01-01

    Cationic gene vectors increased attractive for gene therapy. However, unstable systemic circulation due to the interaction of gene delivery system with blood cells limited the further application. Therefore, pH sensitive shielding systems were exploited, by which, the positive surface charge density of polyplexes was reduced, circulation time was improved and pH-triggered targeting delivery was promised. This mini review mainly focuses on the development of solid tumors pH environment activated shielding systems for cationic gene vectors. This shielding strategy shows great potential for enhancing efficient gene transporting and achieving better therapeutic effects in acidic tumor treatment.

  1. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

    PubMed

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-03-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

  2. Ancient Genetic Signatures of Orang Asli Revealed by Killer Immunoglobulin-Like Receptor Gene Polymorphisms.

    PubMed

    NurWaliyuddin, Hanis Z A; Norazmi, Mohd N; Edinur, Hisham A; Chambers, Geoffrey K; Panneerchelvam, Sundararajulu; Zafarina, Zainuddin

    2015-01-01

    The aboriginal populations of Peninsular Malaysia, also known as Orang Asli (OA), comprise three major groups; Semang, Senoi and Proto-Malays. Here, we analyzed for the first time KIR gene polymorphisms for 167 OA individuals, including those from four smallest OA subgroups (Che Wong, Orang Kanaq, Lanoh and Kensiu) using polymerase chain reaction-sequence specific primer (PCR-SSP) analyses. The observed distribution of KIR profiles of OA is heterogenous; Haplotype B is the most frequent in the Semang subgroups (especially Batek) while Haplotype A is the most common type in the Senoi. The Semang subgroups were clustered together with the Africans, Indians, Papuans and Australian Aborigines in a principal component analysis (PCA) plot and shared many common genotypes (AB6, BB71, BB73 and BB159) observed in these other populations. Given that these populations also display high frequencies of Haplotype B, it is interesting to speculate that Haplotype B may be generally more frequent in ancient populations. In contrast, the two Senoi subgroups, Che Wong and Semai are displaced toward Southeast Asian and African populations in the PCA scatter plot, respectively. Orang Kanaq, the smallest and the most endangered of all OA subgroups, has lost some degree of genetic variation, as shown by their relatively high frequency of the AB2 genotype (0.73) and a total absence of KIR2DL2 and KIR2DS2 genes. Orang Kanaq tradition that strictly prohibits intermarriage with outsiders seems to have posed a serious threat to their survival. This present survey is a demonstration of the value of KIR polymorphisms in elucidating genetic relationships among human populations. PMID:26565719

  3. Ancient Genetic Signatures of Orang Asli Revealed by Killer Immunoglobulin-Like Receptor Gene Polymorphisms

    PubMed Central

    NurWaliyuddin, Hanis Z. A.; Norazmi, Mohd N.; Edinur, Hisham A.; Chambers, Geoffrey K.; Panneerchelvam, Sundararajulu; Zafarina, Zainuddin

    2015-01-01

    The aboriginal populations of Peninsular Malaysia, also known as Orang Asli (OA), comprise three major groups; Semang, Senoi and Proto-Malays. Here, we analyzed for the first time KIR gene polymorphisms for 167 OA individuals, including those from four smallest OA subgroups (Che Wong, Orang Kanaq, Lanoh and Kensiu) using polymerase chain reaction-sequence specific primer (PCR-SSP) analyses. The observed distribution of KIR profiles of OA is heterogenous; Haplotype B is the most frequent in the Semang subgroups (especially Batek) while Haplotype A is the most common type in the Senoi. The Semang subgroups were clustered together with the Africans, Indians, Papuans and Australian Aborigines in a principal component analysis (PCA) plot and shared many common genotypes (AB6, BB71, BB73 and BB159) observed in these other populations. Given that these populations also display high frequencies of Haplotype B, it is interesting to speculate that Haplotype B may be generally more frequent in ancient populations. In contrast, the two Senoi subgroups, Che Wong and Semai are displaced toward Southeast Asian and African populations in the PCA scatter plot, respectively. Orang Kanaq, the smallest and the most endangered of all OA subgroups, has lost some degree of genetic variation, as shown by their relatively high frequency of the AB2 genotype (0.73) and a total absence of KIR2DL2 and KIR2DS2 genes. Orang Kanaq tradition that strictly prohibits intermarriage with outsiders seems to have posed a serious threat to their survival. This present survey is a demonstration of the value of KIR polymorphisms in elucidating genetic relationships among human populations. PMID:26565719

  4. Short and long-term changes in gene expression mediated by the activation of TLR9

    PubMed Central

    Klaschik, Sven; Tross, Debra; Shirota, Hidekazu; Klinman, Dennis M.

    2009-01-01

    CpG DNA binds to Toll-like receptor 9 to stimulate a strong innate immune response. The magnitude, duration and scope of CpG-induced changes in gene expression is incompletely understood despite extensive studies of TLR9 mediated signal transduction pathways. In particular, the prolonged effects of CpG DNA on gene activation have not been investigated despite evidence that a single dose of CpG DNA alters immune reactivity for several weeks. This study used gene expression analysis to monitor changes in mRNA levels for 14 days, and identified the genes, pathways and functional groups triggered in vivo following CpG DNA administration. Two discrete peaks of gene activation (at 3 hr and 5 days) were observed after CpG injection. Both the behavior and function of genes activated during the second peak differed from those triggered shortly after CpG administration. Initial gene up-regulation corresponded to a period when TLR9 ligation stimulated genes functionally associated with the generation of innate and adaptive immune responses (e.g. the NF-kB and B-cell receptor pathways). The second peak reflected processes associated with cell division (e.g., cell cycle and DNA replication & repair). The complex bimodal pattern of gene expression elicited by CpG DNA administration provides novel insights into the long term effects of TLR9 engagement on genes associated with immunity and cell proliferation. PMID:20005572

  5. Laughter up-regulates the genes related to NK cell activity in diabetes.

    PubMed

    Hayashi, Takashi; Tsujii, Satoru; Iburi, Tadao; Tamanaha, Tamiko; Yamagami, Keiko; Ishibashi, Rieko; Hori, Miyo; Sakamoto, Shigeko; Ishii, Hitoshi; Murakami, Kazuo

    2007-12-01

    To elucidate the sustainable effects of laughter on gene expression, we recruited type 2 diabetic patients who were in-patient for receiving self-management education and examined time-dependent regulation for gene expression by laughter. Two-day experiment was performed. On one day, the patients watched comic video and laughed together with hospital staffs. On the other day, they participated in an inpatient diabetes educational program. Blood samples were collected before and 1.5, 4 h after watching comic video or spending lecture time, and changes in gene expression were comprehensively analyzed by microarray technique. Of the 41,000 genes analyzed, the laughter relatively up-regulated 39 genes, among which, 27 genes were relatively increased in the expression for all the observation period after watching comic video. By functional classification of these genes, 14 genes were found to be related to natural killer cell activity. No genes were included that are directly involved in blood glucose regulation, though successive suppression of postprandial blood glucose levels was observed. These results suggest that the laughter influences the expression of many genes classified into immune responses, and may contribute to amelioration of postprandial blood glucose elevation through a modulation of NK cell activity caused by up-regulation of relating genes.

  6. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  7. Fur-Mediated Activation of Gene Transcription in the Human Pathogen Neisseria gonorrhoeae

    PubMed Central

    Yu, Chunxiao

    2012-01-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  8. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity.

    PubMed

    Ahmed, Afsar U; Williams, Bryan R G; Hannigan, Gregory E

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  9. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity.

    PubMed

    Ahmed, Afsar U; Williams, Bryan R G; Hannigan, Gregory E

    2015-11-11

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding.

  10. The dependence of Ag+ block of a potassium channel, murine kir2.1, on a cysteine residue in the selectivity filter.

    PubMed

    Dart, C; Leyland, M L; Barrett-Jolley, R; Shelton, P A; Spencer, P J; Conley, E C; Sutcliffe, M J; Stanfield, P R

    1998-08-15

    Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.

  11. Crystal Structure of the Eukaryotic Strong Inward-Rectifier K[superscript +] Channel Kir2.2 at 3.1 Å Resolution

    SciTech Connect

    Tao, Xiao; Avalos, Jose L.; Chen, Jiayun; MacKinnon, Roderick

    2010-03-29

    Inward-rectifier potassium (K{sup +}) channels conduct K{sup +} ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells, and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb{sup +}), strontium (Sr{sup 2+}), and europium (Eu{sup 3+}) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel's extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K{sup +} channel family.

  12. Impact of physical activity and doping on epigenetic gene regulation.

    PubMed

    Schwarzenbach, Heidi

    2011-10-01

    To achieve success in sports, many athletes consume doping substances, such as anabolic androgenic steroids and growth hormones, and ignore the negative influence of these drugs on their health. Apart from the unethical aspect of doping in sports, it is essential to consider the tremendous risk it represents to their physical condition. The abuse of pharmaceuticals which improve athletic performance may alter the expression of specific genes involved in muscle and bone metabolism by epigenetic mechanisms, such as DNA methylation and histone modifications. Moreover, excessive and relentless training to increase the muscle mass, may also have an influence on the health of the athletes. This stress releases neurotransmitters and growth factors, and may affect the expression of endogenous genes by DNA methylation, too. This paper focuses on the relationship between epigenetic mechanisms and sports, highlights the potential consequences of abuse of doping drugs on gene expression, and describes methods to molecularly detect epigenetic changes of gene markers reflecting the physiological or metabolic effects of doping agents.

  13. Activation and Characterization of a Cryptic Polycyclic Tetramate Macrolactam Biosynthetic Gene Cluster

    PubMed Central

    Luo, Yunzi; Huang, Hua; Liang, Jing; Wang, Meng; Lu, Lu; Shao, Zengyi; Cobb, Ryan E.; Zhao, Huimin

    2014-01-01

    Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of them have not been characterized. Here we apply a plug and play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three potential PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) which resembles iterative PKSs known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products. PMID:24305602

  14. Transcriptomic Sequencing Reveals a Set of Unique Genes Activated by Butyrate-Induced Histone Modification.

    PubMed

    Li, Cong-Jun; Li, Robert W; Baldwin, Ransom L; Blomberg, Le Ann; Wu, Sitao; Li, Weizhong

    2016-01-01

    Butyrate is a nutritional element with strong epigenetic regulatory activity as a histone deacetylase inhibitor. Based on the analysis of differentially expressed genes in the bovine epithelial cells using RNA sequencing technology, a set of unique genes that are activated only after butyrate treatment were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network, using Ingenuity Pathways Analysis, indicated that these genes activated by butyrate treatment are related to major cellular functions, including cell morphological changes, cell cycle arrest, and apoptosis. Our results offered insight into the butyrate-induced transcriptomic changes and will accelerate our discerning of the molecular fundamentals of epigenomic regulation. PMID:26819550

  15. Transcriptomic Sequencing Reveals a Set of Unique Genes Activated by Butyrate-Induced Histone Modification

    PubMed Central

    Li, Cong-Jun; Li, Robert W.; Baldwin, Ransom L.; Blomberg, Le Ann; Wu, Sitao; Li, Weizhong

    2016-01-01

    Butyrate is a nutritional element with strong epigenetic regulatory activity as a histone deacetylase inhibitor. Based on the analysis of differentially expressed genes in the bovine epithelial cells using RNA sequencing technology, a set of unique genes that are activated only after butyrate treatment were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network, using Ingenuity Pathways Analysis, indicated that these genes activated by butyrate treatment are related to major cellular functions, including cell morphological changes, cell cycle arrest, and apoptosis. Our results offered insight into the butyrate-induced transcriptomic changes and will accelerate our discerning of the molecular fundamentals of epigenomic regulation. PMID:26819550

  16. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

    PubMed

    Harashima, S; Hinnebusch, A G

    1986-11-01

    GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

  17. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    DOEpatents

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  18. Lymphocyte activation gene 3 and coronary artery disease

    PubMed Central

    Golden, Diana; Kolmakova, Antonina; Sura, Sunitha; Vella, Anthony T.; Manichaikul, Ani; Wang, Xin-Qun; Bielinski, Suzette J.; Taylor, Kent D.; Chen, Yii-Der Ida; Rich, Stephen S.

    2016-01-01

    BACKGROUND: The lipoprotein scavenger receptor BI (SCARB1) rs10846744 noncoding variant is significantly associated with atherosclerotic disease independently of traditional cardiovascular risk factors. We identified a potentially novel connection between rs10846744, the immune checkpoint inhibitor lymphocyte activation gene 3 (LAG3), and atherosclerosis. METHODS: In vitro approaches included flow cytometry, lipid raft isolation, phosphosignaling, cytokine measurements, and overexpressing and silencing LAG3 protein. Fasting plasma LAG3 protein was measured in hyperalphalipoproteinemic (HALP) and Multi-Ethnic Study of Atherosclerosis (MESA) participants. RESULTS: In comparison with rs10846744 reference (GG homozygous) cells, LAG3 protein levels by flow cytometry (P < 0.001), in lipid rafts stimulated and unstimulated (P = 0.03), and phosphosignaling downstream of B cell receptor engagement of CD79A (P = 0.04), CD19 (P = 0.04), and LYN (P = 0.001) were lower in rs10846744 risk (CC homozygous) cells. Overexpressing LAG3 protein in risk cells and silencing LAG3 in reference cells confirmed its importance in phosphosignaling. Secretion of TNF-α was higher (P = 0.04) and IL-10 was lower (P = 0.04) in risk cells. Plasma LAG3 levels were lower in HALP carriers of the CC allele (P < 0.0001) and by race (P = 0.004). In MESA, race (P = 0.0005), age (P = 0.003), lipid medications (P = 0.03), smoking history (P < 0.0001), and rs10846744 genotype (P = 0.002) were independent predictors of plasma LAG3. In multivariable regression models, plasma LAG3 was significantly associated with HDL-cholesterol (HDL-C) (P = 0.007), plasma IL-10 (P < 0.0001), and provided additional predictive value above the Framingham risk score (P = 0.04). In MESA, when stratified by high HDL-C, plasma LAG3 was associated with coronary heart disease (CHD) (odds ratio 1.45, P = 0.004). CONCLUSION: Plasma LAG3 is a potentially novel independent predictor of HDL-C levels and CHD risk. FUNDING: This work was

  19. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    NASA Astrophysics Data System (ADS)

    Golubnitchaya-Labudová, O.; Portele, A.; Vaçata, V.; Lubec, G.; Rink, H.; Höfer, M.

    1997-10-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the insert of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9.

  20. BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks.

    PubMed

    Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M Mallar; Banaschewski, Tobias; Barker, Gareth J; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W; Smolka, Michael N; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D

    2015-06-12

    During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function.

  1. BRAIN NETWORKS. Correlated gene expression supports synchronous activity in brain networks.

    PubMed

    Richiardi, Jonas; Altmann, Andre; Milazzo, Anna-Clare; Chang, Catie; Chakravarty, M Mallar; Banaschewski, Tobias; Barker, Gareth J; Bokde, Arun L W; Bromberg, Uli; Büchel, Christian; Conrod, Patricia; Fauth-Bühler, Mira; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny; Heinz, Andreas; Lemaître, Hervé; Mann, Karl F; Martinot, Jean-Luc; Nees, Frauke; Paus, Tomáš; Pausova, Zdenka; Rietschel, Marcella; Robbins, Trevor W; Smolka, Michael N; Spanagel, Rainer; Ströhle, Andreas; Schumann, Gunter; Hawrylycz, Mike; Poline, Jean-Baptiste; Greicius, Michael D

    2015-06-12

    During rest, brain activity is synchronized between different regions widely distributed throughout the brain, forming functional networks. However, the molecular mechanisms supporting functional connectivity remain undefined. We show that functional brain networks defined with resting-state functional magnetic resonance imaging can be recapitulated by using measures of correlated gene expression in a post mortem brain tissue data set. The set of 136 genes we identify is significantly enriched for ion channels. Polymorphisms in this set of genes significantly affect resting-state functional connectivity in a large sample of healthy adolescents. Expression levels of these genes are also significantly associated with axonal connectivity in the mouse. The results provide convergent, multimodal evidence that resting-state functional networks correlate with the orchestrated activity of dozens of genes linked to ion channel activity and synaptic function. PMID:26068849

  2. Exploring the transcription factor activity in high-throughput gene expression data using RLQ analysis

    PubMed Central

    2013-01-01

    Background Interpretation of gene expression microarray data in the light of external information on both columns and rows (experimental variables and gene annotations) facilitates the extraction of pertinent information hidden in these complex data. Biologists classically interpret genes of interest after retrieving functional information from a subset of genes of interest. Transcription factors play an important role in orchestrating the regulation of gene expression. Their activity can be deduced by examining the presence of putative transcription factors binding sites in the gene promoter regions. Results In this paper we present the multivariate statistical method RLQ which aims to analyze microarray data where additional information is available on both genes and samples. As an illustrative example, we applied RLQ methodology to analyze transcription factor activity associated with the time-course effect of steroids on the growth of primary human lung fibroblasts. RLQ could successfully predict transcription factor activity, and could integrate various other sources of external information in the main frame of the analysis. The approach was validated by means of alternative statistical methods and biological validation. Conclusions RLQ provides an efficient way of extracting and visualizing structures present in a gene expression dataset by directly modeling the link between experimental variables and gene annotations. PMID:23742070

  3. Cluster Analysis of Tumor Suppressor Genes in Canine Leukocytes Identifies Activation State

    PubMed Central

    Daly, Julie-Anne; Mortlock, Sally-Anne; Taylor, Rosanne M.; Williamson, Peter

    2015-01-01

    Cells of the immune system undergo activation and subsequent proliferation in the normal course of an immune response. Infrequently, the molecular and cellular events that underlie the mechanisms of proliferation are dysregulated and may lead to oncogenesis, leading to tumor formation. The most common forms of immunological cancers are lymphomas, which in dogs account for 8%–20% of all cancers, affecting up to 1.2% of the dog population. Key genes involved in negatively regulating proliferation of lymphocytes include a group classified as tumor suppressor genes (TSGs). These genes are also known to be associated with progression of lymphoma in humans, mice, and dogs and are potential candidates for pathological grading and diagnosis. The aim of the present study was to analyze TSG profiles in stimulated leukocytes from dogs to identify genes that discriminate an activated phenotype. A total of 554 TSGs and three gene set collections were analyzed from microarray data. Cluster analysis of three subsets of genes discriminated between stimulated and unstimulated cells. These included 20 most upregulated and downregulated TSGs, TSG in hallmark gene sets significantly enriched in active cells, and a selection of candidate TSGs, p15 (CDKN2B), p18 (CDKN2C), p19 (CDKN1A), p21 (CDKN2A), p27 (CDKN1B), and p53 (TP53) in the third set. Analysis of two subsets suggested that these genes or a subset of these genes may be used as a specialized PCR set for additional analysis. PMID:27478369

  4. Neuritin: a gene induced by neural activity and neurotrophins that promotes neuritogenesis.

    PubMed

    Naeve, G S; Ramakrishnan, M; Kramer, R; Hevroni, D; Citri, Y; Theill, L E

    1997-03-18

    Neural activity and neurotrophins induce synaptic remodeling in part by altering gene expression. A cDNA encoding a glycosylphoshatidylinositol-anchored protein was identified by screening for hippocampal genes that are induced by neural activity. This molecule, named neuritin, is expressed in postmitotic-differentiating neurons of the developing nervous system and neuronal structures associated with plasticity in the adult. Neuritin message is induced by neuronal activity and by the activity-regulated neurotrophins BDNF and NT-3. Purified recombinant neuritin promotes neurite outgrowth and arborization in primary embryonic hippocampal and cortical cultures. These data implicate neuritin as a downstream effector of activity-induced neurite outgrowth. PMID:9122250

  5. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  6. lasA and lasB genes of Pseudomonas aeruginosa: analysis of transcription and gene product activity.

    PubMed Central

    Toder, D S; Ferrell, S J; Nezezon, J L; Rust, L; Iglewski, B H

    1994-01-01

    The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced. Its function and significance have been studied by genetic approaches (D. S. Toder, M. J. Gambello, and B. H. Iglewski, Mol. Microbiol. 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J. E. Peters and D. R. Galloway, J. Bacteriol. 172:2236-2240, 1990). To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1. Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity. Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present. Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity. We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64. Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB. Images PMID:8132339

  7. Contracting human skeletal muscle maintains the ability to blunt α1-adrenergic vasoconstriction during KIR channel and Na+/K+-ATPase inhibition

    PubMed Central

    Crecelius, Anne R; Kirby, Brett S; Hearon, Christopher M; Luckasen, Gary J; Larson, Dennis G; Dinenno, Frank A

    2015-01-01

    Sympathetic vasoconstriction in contracting skeletal muscle is blunted relative to that which occurs in resting tissue; however, the mechanisms underlying this ‘functional sympatholysis’ remain unclear in humans. We tested the hypothesis that α1-adrenergic vasoconstriction is augmented during exercise following inhibition of inwardly rectifying potassium (KIR) channels and Na+/K+-ATPase (BaCl2 + ouabain). In young healthy humans, we measured forearm blood flow (Doppler ultrasound) and calculated forearm vascular conductance (FVC) at rest, during steady-state stimulus conditions (pre-phenylephrine), and after 2 min of phenylephrine (PE; an α1-adrenoceptor agonist) infusion via brachial artery catheter in response to two different stimuli: moderate (15% maximal voluntary contraction) rhythmic handgrip exercise or adenosine infusion. In Protocol 1 (n = 11 subjects) a total of six trials were performed in three conditions: control (saline), combined enzymatic inhibition of nitric oxide (NO) and prostaglandin (PG) synthesis (l-NMMA + ketorolac) and combined inhibition of NO, PGs, KIR channels and Na+/K+-ATPase (l-NMMA + ketorolac + BaCl2 + ouabain). In Protocol 2 (n = 6) a total of four trials were performed in two conditions: control (saline), and combined KIR channel and Na+/K+-ATPase inhibition. All trials occurred after local β-adrenoceptor blockade (propranolol). PE-mediated vasoconstriction was calculated (%ΔFVC) in each condition. Contrary to our hypothesis, despite attenuated exercise hyperaemia of ∼30%, inhibition of KIR channels and Na+/K+-ATPase, combined with inhibition of NO and PGs (Protocol 1) or alone (Protocol 2) did not enhance α1-mediated vasoconstriction during exercise (Protocol 1: −27 ± 3%; P = 0.2 vs. control, P = 0.4 vs.l-NMMA + ketorolac; Protocol 2: −21 ± 7%; P = 0.9 vs. control). Thus, contracting human skeletal muscle maintains the ability to blunt α1-adrenergic vasoconstriction during

  8. Manganese peroxidase gene transcription in Phanerochaete chrysosporium: Activation by manganese

    SciTech Connect

    Brown, J.A.; Alic, M. Gold, M.H. )

    1991-07-01

    The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, the authors demonstrate unequivocally that Mn regulates mnp gene transcription. The amount of mnp mRNA in cells of 4-day-old nitrogen-limited cultures is a direct function of the concentration of Mn in the culture medium up to a maximum of 180 {mu}M. Addition of Mn to nitrogen-limited Mn-deficient secondary metabolic (4-, 5-, and 6-day-old) cultures results in the appearance of mnp mRNA within 40 min. The appearance of this message is completely inhibited by the RNA synthesis inhibitor dactinomycin but not by the protein synthesis inhibitor cycloheximide. Furthermore, the amount of mnp mRNA produced is a direct function of the concentration of added Mn. In contrast, addition of Mn to low-nitrogen Mn-deficient 2- or 3-day-old cultures does not result in the appearance of mnp mRNA. Manganese peroxidase protein is detected by specific immunoprecipitation of the in vitro translation products of poly(A) RNA isolated from Mn-supplemented (but nor from Mn-deficient) cells. All of these results demonstrate that Mn, the substrate for the enzyme, regulates mnp gene transcription via a growth-stage-specific and concentration-dependent mechanism.

  9. The Mediator Subunit MED16 Transduces NRF2-Activating Signals into Antioxidant Gene Expression

    PubMed Central

    Sekine, Hiroki; Okazaki, Keito; Ota, Nao; Shima, Hiroki; Katoh, Yasutake; Suzuki, Norio; Igarashi, Kazuhiko; Ito, Mitsuhiro

    2015-01-01

    The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying NRF2-dependent transcriptional activation, we purified the NRF2 nuclear protein complex and identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated with NRF2. Disruption of Med16 significantly attenuated the electrophile-induced expression of NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of NRF2-activated genes exhibited blunted inductions by electrophiles in Med16-deficient cells compared to wild-type cells, which strongly argues that MED16 is a major contributor supporting NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of the RNA polymerase II C-terminal domain was absent in Med16-deficient cells, suggesting that MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 indeed turned out to be essential for cytoprotection against oxidative insults. Thus, the KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant response through the robust activation of NRF2 target genes. PMID:26572828

  10. RNA silencing of genes involved in Alzheimer's disease enhances mitochondrial function and synaptic activity.

    PubMed

    Manczak, Maria; Reddy, P Hemachandra

    2013-12-01

    An age-dependent increase in mRNA levels of the amyloid precursor protein (APP), the microtubule-associated protein Tau, and voltage-dependent anion channel 1 (VDAC1) genes are reported to be toxic to neurons affected by Alzheimer's disease (AD). However, the underlying toxic nature of these genes is not completely understood. The purpose of our study was to determine the effects of RNA silencing of APP, Tau, and VDAC1 genes in AD pathogenesis. Using human neuroblastoma (SHSY5Y) cells, we first silenced RNA for APP, Tau, and VDAC1 genes, and then performed real-time RT-PCR analysis to measure mRNA levels of 34 genes that are involved in AD pathogenesis. Using biochemical assays, we also assessed mitochondrial function by measuring levels of H2O2 production, lipid peroxidation, cytochrome c oxidase activity, ATP production, and GTPase enzymatic activity. We found that increased mRNA expression of synaptic function and mitochondrial fission genes, and reduced levels of mitochondrial fusion genes in RNA silenced the SHSY5Y cells for APP, Tau and VDAC1 genes relative to the control SHSY5Y cells. In addition, RNA-silenced APP, Tau, and VDAC1 genes in SHSY5Y cells showed reduced levels of H2O2 production, lipid peroxidation, fission-linked GTPase activity, and increased cytochrome oxidase activity and ATP production. These findings suggest that a reduction of human APP, Tau, and VDAC1 may enhance synaptic activity, may improve mitochondrial maintenance and function, and may protect against toxicities of AD-related genes. Thus, these findings also suggest that the reduction of APP, Tau, and VDAC1 mRNA expressions may have therapeutic value for patients with AD.

  11. Xenobiotics shape the physiology and gene expression of the active human gut microbiome

    PubMed Central

    Maurice, Corinne Ferrier; Haiser, Henry Joseph; Turnbaugh, Peter James

    2012-01-01

    SUMMARY The human gut contains trillions of microorganisms that influence our health by metabolizing xenobiotics, including host-targeted drugs and antibiotics. Recent efforts have characterized the diversity of this host-associated community, but it remains unclear which microorganisms are active and what perturbations influence this activity. Here, we combine flow cytometry, 16S rRNA gene sequencing, and metatranscriptomics to demonstrate that the gut contains a distinctive set of active microorganisms, primarily Firmicutes. Short-term exposure to a panel of xenobiotics significantly affected the physiology, structure, and gene expression of this active gut microbiome. Xenobiotic-responsive genes were found across multiple bacterial phyla, encoding antibiotic resistance, drug metabolism, and stress response pathways. These results demonstrate the power of moving beyond surveys of microbial diversity to better understand metabolic activity, highlight the unintended consequences of xenobiotics, and suggest that attempts at personalized medicine should consider inter-individual variations in the active human gut microbiome. PMID:23332745

  12. Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

    PubMed

    Pandian, Ganesh N; Taniguchi, Junichi; Junetha, Syed; Sato, Shinsuke; Han, Le; Saha, Abhijit; AnandhaKumar, Chandran; Bando, Toshikazu; Nagase, Hiroki; Vaijayanthi, Thangavel; Taylor, Rhys D; Sugiyama, Hiroshi

    2014-01-24

    The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

  13. Transcriptomic Analysis of Musca domestica to Reveal Key Genes of the Prophenoloxidase-Activating System.

    PubMed

    Li, Dianxiang; Liang, Yongli; Wang, Xianwei; Wang, Lei; Qi, Mei; Yu, Yang; Luan, Yuanyuan

    2015-09-01

    The proPO system regulates melanization in arthropods. However, the genes that are involved in the proPO system in housefly Musca domestica remain unclear. Thus, this study analyzed the combined transcriptome obtained from M. domestica larvae, pupae, and adults that were either normal or bacteria-challenged by an Escherichia coli and Staphylococcus aureus mixture. A total of 54,821,138 clean reads (4.93 Gb) were yielded by Illumina sequencing, which were de novo assembled into 89,842 unigenes. Of the 89,842 unigenes, based on a similarity search with known genes in other insects, 24 putative genes related to the proPO system were identified. Eight of the identified genes encoded for peptidoglycan recognition receptors, two encoded for prophenoloxidases, three encoded for prophenoloxidase-activating enzymes, and 11 encoded for serine proteinase inhibitors. The expression levels of these identified genes were investigated by qRT-PCR assay, which were consistent with expected activation process of the proPO system, and their activation functions were confirmed by the measurement of phenoloxidase activity in bacteria-infected larvae after proPO antibody blockage, suggesting these candidate genes might have potentially different roles in the activation of proPO system. Collectively, this study has provided the comprehensive transcriptomic data of an insect and some fundamental basis toward achieving understanding of the activation mechanisms and immune functions of the proPO system in M. domestica.

  14. Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

    SciTech Connect

    Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J. )

    1988-08-01

    Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5{prime} flanking region of the gene revealed a perfect TATA box at position {minus}28 to position {minus}23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5{prime} flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk{sup {minus}} fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides {minus}305 and +75 of the plasminogen activator inhibitor type 1 gene.

  15. Transcriptomic Analysis of Musca domestica to Reveal Key Genes of the Prophenoloxidase-Activating System

    PubMed Central

    Li, Dianxiang; Liang, Yongli; Wang, Xianwei; Wang, Lei; Qi, Mei; Yu, Yang; Luan, Yuanyuan

    2015-01-01

    The proPO system regulates melanization in arthropods. However, the genes that are involved in the proPO system in housefly Musca domestica remain unclear. Thus, this study analyzed the combined transcriptome obtained from M. domestica larvae, pupae, and adults that were either normal or bacteria-challenged by an Escherichia coli and Staphylococcus aureus mixture. A total of 54,821,138 clean reads (4.93 Gb) were yielded by Illumina sequencing, which were de novo assembled into 89,842 unigenes. Of the 89,842 unigenes, based on a similarity search with known genes in other insects, 24 putative genes related to the proPO system were identified. Eight of the identified genes encoded for peptidoglycan recognition receptors, two encoded for prophenoloxidases, three encoded for prophenoloxidase-activating enzymes, and 11 encoded for serine proteinase inhibitors. The expression levels of these identified genes were investigated by qRT-PCR assay, which were consistent with expected activation process of the proPO system, and their activation functions were confirmed by the measurement of phenoloxidase activity in bacteria-infected larvae after proPO antibody blockage, suggesting these candidate genes might have potentially different roles in the activation of proPO system. Collectively, this study has provided the comprehensive transcriptomic data of an insect and some fundamental basis toward achieving understanding of the activation mechanisms and immune functions of the proPO system in M. domestica. PMID:26156588

  16. Insights into GATA-1 Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

    PubMed Central

    Yu, Ming; Riva, Laura; Xie, Huafeng; Schindler, Yocheved; Moran, Tyler B.; Cheng, Yong; Yu, Duonan; Hardison, Ross; Weiss, Mitchell J; Orkin, Stuart H.; Bernstein, Bradley E.; Fraenkel, Ernest; Cantor, Alan B.

    2009-01-01

    Summary The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo. PMID:19941827

  17. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression.

  18. Potato tuber cytokinin oxidase/dehydrogenase genes: biochemical properties, activity, and expression during tuber dormancy progression.

    PubMed

    Suttle, Jeffrey C; Huckle, Linda L; Lu, Shunwen; Knauber, Donna C

    2014-03-15

    The enzymatic and biochemical properties of the proteins encoded by five potato cytokinin oxidase/dehydrogenase (CKX)-like genes functionally expressed in yeast and the effects of tuber dormancy progression on StCKX expression and cytokinin metabolism were examined in lateral buds isolated from field-grown tubers. All five putative StCKX genes encoded proteins with in vitro CKX activity. All five enzymes were maximally active at neutral to slightly alkaline pH with 2,6-dichloro-indophenol as the electron acceptor. In silico analyses indicated that four proteins were likely secreted. Substrate dependence of two of the most active enzymes varied; one exhibiting greater activity with isopentenyl-type cytokinins while the other was maximally active with cis-zeatin as a substrate. [(3)H]-isopentenyl-adenosine was readily metabolized by excised tuber buds to adenine/adenosine demonstrating that CKX was active in planta. There was no change in apparent in planta CKX activity during either natural or chemically forced dormancy progression. Similarly although expression of individual StCKX genes varied modestly during tuber dormancy, there was no clear correlation between StCKX gene expression and tuber dormancy status. Thus although CKX gene expression and enzyme activity are present in potato tuber buds throughout dormancy, they do not appear to play a significant role in the regulation of cytokinin content during tuber dormancy progression. PMID:24594397

  19. NF-Y activates genes of metabolic pathways altered in cancer cells.

    PubMed

    Benatti, Paolo; Chiaramonte, Maria Luisa; Lorenzo, Mariangela; Hartley, John A; Hochhauser, Daniel; Gnesutta, Nerina; Mantovani, Roberto; Imbriano, Carol; Dolfini, Diletta

    2016-01-12

    The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

  20. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  1. Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

    PubMed Central

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-01-01

    We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. PMID:21689637

  2. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes

    PubMed Central

    Kato, Lucia; Begum, Nasim A.; Burroughs, A. Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O.; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-01-01

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites. PMID:22308462

  3. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

    PubMed

    Kato, Lucia; Begum, Nasim A; Burroughs, A Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-02-14

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

  4. Opposing LSD1 complexes function in developmental gene activation and repression programmes.

    PubMed

    Wang, Jianxun; Scully, Kathleen; Zhu, Xiaoyan; Cai, Ling; Zhang, Jie; Prefontaine, Gratien G; Krones, Anna; Ohgi, Kenneth A; Zhu, Ping; Garcia-Bassets, Ivan; Liu, Forrest; Taylor, Havilah; Lozach, Jean; Jayes, Friederike L; Korach, Kenneth S; Glass, Christopher K; Fu, Xiang-Dong; Rosenfeld, Michael G

    2007-04-19

    Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1--a component of the CoREST-CtBP co-repressor complex--is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

  5. Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

    PubMed Central

    Liu, H T; Baserga, R; Mercer, W E

    1985-01-01

    We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical. Images PMID:2427924

  6. Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

    SciTech Connect

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-07-08

    Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

  7. Differential regulation of plasminogen activator and inhibitor gene transcription by the tumor suppressor p53.

    PubMed Central

    Kunz, C; Pebler, S; Otte, J; von der Ahe, D

    1995-01-01

    The ability of p53 to activate or repress transcription suggests that its biological function as tumor suppressor is in part accomplished by regulating a number of genes including such required for inhibition of cell growth. We here give evidence that p53 also may regulate genes responsible for the proteolytic degradation of the extracellular matrix, which is considered a crucial feature for local invasion and metastasis of neoplastic cells. An important and highly regulated cascade of such proteolytic events involves the plasminogen activator system. We show that wild-type p53 represses transcription from the enhancer and promoter of the human urokinase-type (u-PA) and the tissue-type plasminogen activator (t-PA) gene through a non-DNA binding mechanism. Oncogenic mutants lost the repressing activity. In contrast, wild-type but not mutant p53 specifically binds to and activates the promoter of the plasminogen activator inhibitor type-1 (PAI-1) gene. Interestingly, one of the p53 mutants (273his) inhibited PAI-1 promoter activity. Our results suggest that altered function of oncogenic forms of p53 may lead to altered expression of the plasminogen activators and their inhibitor(s) and thus to altered activation of the plasminogen/plasmin system during tumor progression. Images PMID:7479001

  8. AIRE activated tissue specific genes have histone modifications associated with inactive chromatin.

    PubMed

    Org, Tõnis; Rebane, Ana; Kisand, Kai; Laan, Martti; Haljasorg, Uku; Andreson, Reidar; Peterson, Pärt

    2009-12-15

    The Autoimmune Regulator (AIRE) protein is expressed in thymic medullary epithelial cells, where it promotes the ectopic expression of tissue-restricted antigens needed for efficient negative selection of developing thymocytes. Mutations in AIRE cause APECED syndrome, which is characterized by a breakdown of self-tolerance. The molecular mechanism by which AIRE increases the expression of a variety of different genes remains unknown. Here, we studied AIRE-regulated genes using whole genome expression analysis and chromatin immunoprecipitation. We show that AIRE preferentially activates genes that are tissue-specific and characterized by low levels of initial expression in stably transfected HEK293 cell model and mouse thymic medullary epithelial cells. In addition, the AIRE-regulated genes lack active chromatin marks, such as histone H3 trimethylation (H3K4me3) and acetylation (AcH3), on their promoters. We also show that during activation by AIRE, the target genes acquire histone H3 modifications associated with transcription and RNA polymerase II. In conclusion, our data show that AIRE is able to promote ectopic gene expression from chromatin associated with histone modifications characteristic to inactive genes.

  9. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  10. Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.

    PubMed

    Warren, Curtis R; Kassir, Elias; Spurlin, James; Martinez, Jerahme; Putnam, Nicholas H; Farach-Carson, Mary C

    2015-01-01

    The heparan sulfate proteoglycan 2 (HSPG2)/perlecan gene is ancient and conserved in all triploblastic species. Its presence maintains critical cell boundaries in tissue and its large (up to ~900 kDa) modular structure has prompted speculation about the evolutionary origin of the gene. The gene's conservation amongst basal metazoans is unclear. After the recent sequencing of their genomes, the cnidarian Nematostella vectensis and the placozoan Trichoplax adhaerens have become favorite models for studying tissue regeneration and the evolution of multicellularity. More ancient basal metazoan phyla include the poriferan and ctenophore, whose evolutionary relationship has been clarified recently. Our in silico and PCR-based methods indicate that the HSPG2 gene is conserved in both the placozoan and cnidarian genomes, but not in those of the ctenophores and only partly in poriferan genomes. HSPG2 also is absent from published ctenophore and Capsaspora owczarzaki genomes. The gene in T. adhaerens is encoded as two separate but genetically juxtaposed genes that house all of the constituent pieces of the mammalian HSPG2 gene in tandem. These genetic constituents are found in isolated genes of various poriferan species, indicating a possible intronic recombinatory mechanism for assembly of the HSPG2 gene. Perlecan's expression during wound healing and boundary formation is conserved, as expression of the gene was activated during tissue regeneration and reformation of the basement membrane of N. vectensis. These data indicate that the complex HSPG2 gene evolved concurrently in a common ancestor of placozoans, cnidarians and bilaterians, likely along with the development of differentiated cell types separated by acellular matrices, and is activated to reestablish these tissue borders during wound healing. PMID:25876075

  11. Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.

    PubMed

    Warren, Curtis R; Kassir, Elias; Spurlin, James; Martinez, Jerahme; Putnam, Nicholas H; Farach-Carson, Mary C

    2015-01-01

    The heparan sulfate proteoglycan 2 (HSPG2)/perlecan gene is ancient and conserved in all triploblastic species. Its presence maintains critical cell boundaries in tissue and its large (up to ~900 kDa) modular structure has prompted speculation about the evolutionary origin of the gene. The gene's conservation amongst basal metazoans is unclear. After the recent sequencing of their genomes, the cnidarian Nematostella vectensis and the placozoan Trichoplax adhaerens have become favorite models for studying tissue regeneration and the evolution of multicellularity. More ancient basal metazoan phyla include the poriferan and ctenophore, whose evolutionary relationship has been clarified recently. Our in silico and PCR-based methods indicate that the HSPG2 gene is conserved in both the placozoan and cnidarian genomes, but not in those of the ctenophores and only partly in poriferan genomes. HSPG2 also is absent from published ctenophore and Capsaspora owczarzaki genomes. The gene in T. adhaerens is encoded as two separate but genetically juxtaposed genes that house all of the constituent pieces of the mammalian HSPG2 gene in tandem. These genetic constituents are found in isolated genes of various poriferan species, indicating a possible intronic recombinatory mechanism for assembly of the HSPG2 gene. Perlecan's expression during wound healing and boundary formation is conserved, as expression of the gene was activated during tissue regeneration and reformation of the basement membrane of N. vectensis. These data indicate that the complex HSPG2 gene evolved concurrently in a common ancestor of placozoans, cnidarians and bilaterians, likely along with the development of differentiated cell types separated by acellular matrices, and is activated to reestablish these tissue borders during wound healing.

  12. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa.

    PubMed Central

    Deretic, V; Gill, J F; Chakrabarty, A M

    1987-01-01

    Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor. Images PMID:3025179

  13. Physical activity-associated gene expression signature in nonhuman primate motor cortex.

    PubMed

    Mitchell, Amanda C; Leak, Rehana K; Garbett, Krassimira; Zigmond, Michael J; Cameron, Judy L; Mirnics, Károly

    2012-03-01

    It has been established that weight gain and weight loss are heavily influenced by activity level. In this study, we hypothesized that the motor cortex exhibits a distinct physical activity-associated gene expression profile, which may underlie changes in weight associated with movement. Using DNA microarrays we profiled gene expression in the motor cortex of a group of 14 female rhesus monkeys (Macaca mulatta) with a wide range of stable physical activity levels. We found that neuronal growth factor signaling and nutrient sensing transcripts in the brain were highly correlated with physical activity. A follow-up of AKT3 expression changes (a gene at the apex of neuronal survival and nutrient sensing) revealed increased protein levels of total AKT, phosphorylated AKT, and forkhead box O3 (FOXO3), one of AKT's main downstream effectors. In addition, we successfully validated three other genes via quantitative polymerase chain reaction (qPCR) (cereblon (CRBN), origin recognition complex subunit 4-like, and pyruvate dehydrogenase 4 (PDK4)). We conclude that these genes are important in the physical activity-associated pathway in the motor cortex, and may be critical for physical activity-associated changes in body weight and neuroprotection.

  14. Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxoplasma gondii

    PubMed Central

    Gissot, Mathieu; Kelly, Krystyna A; Ajioka, James W; Greally, John M; Kim, Kami

    2007-01-01

    Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation “activation” marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code. PMID:17559302

  15. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    PubMed Central

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  16. Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

    NASA Astrophysics Data System (ADS)

    Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

    2016-07-01

    The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

  17. Selenate reductase activity in Escherichia coli requires Isc iron-sulfur cluster biosynthesis genes.

    PubMed

    Yee, Nathan; Choi, Jessica; Porter, Abigail W; Carey, Sean; Rauschenbach, Ines; Harel, Arye

    2014-12-01

    The selenate reductase in Escherichia coli is a multi-subunit enzyme predicted to bind Fe-S clusters. In this study, we examined the iron-sulfur cluster biosynthesis genes that are required for selenate reductase activity. Mutants devoid of either the iscU or hscB gene in the Isc iron-sulfur cluster biosynthesis pathway lost the ability to reduce selenate. Genetic complementation by the wild-type sequences restored selenate reductase activity. The results indicate the Isc biosynthetic system plays a key role in selenate reductase Fe-S cofactor assembly and is essential for enzyme activity.

  18. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    PubMed Central

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M.; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression compendium that lead to alteration of CAR activity. A gene expression biomarker signature of 83 CAR-dependent genes was identified using microarray profiles from the livers of wild-type and CAR-null mice after exposure to three structurally-diverse CAR activators (CITCO, phenobarbital, TCPOBOP). A rank-based algorithm (Running Fisher’s algorithm (p-value ≤ 10-4)) was used to evaluate the similarity between the CAR biomarker signature and a test set of 28 and 32 comparisons positive or negative, respectively, for CAR activation; the test resulted in a balanced accuracy of 97%. The biomarker signature was used to identify chemicals that activate or suppress CAR in an annotated mouse liver/primary hepatocyte gene expression database of ~1850 comparisons. CAR was activated by 1) activators of the aryl hydrocarbon receptor (AhR) in wild-type but not AhR-null mice, 2) pregnane X receptor (PXR) activators in wild-type and to lesser extents in PXR-null mice, and 3) activators of PPARα in wild-type and PPARα-null mice. CAR was consistently activated by five conazole fungicides and four perfluorinated compounds. Comparison of effects in wild-type and CAR-null mice showed that the fungicide propiconazole increased liver weight and hepatocyte proliferation in a CAR-dependent manner, whereas the perfluorinated compound perfluorooctanoic acid (PFOA) increased these endpoints in a CAR-independent manner. A number of compounds suppressed CAR coincident with increases in markers of

  19. Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.

    PubMed Central

    Hanson, R D; Ley, T J

    1990-01-01

    The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined. Images PMID:2233710

  20. Genome-Wide Analysis of Antiviral Signature Genes in Porcine Macrophages at Different Activation Statuses

    PubMed Central

    Sang, Yongming; Brichalli, Wyatt; Rowland, Raymond R. R.; Blecha, Frank

    2014-01-01

    Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons of any two activation statuses revealed more than 20,000 DEGs that were normalized to filter out 153–5,303 significant DEGs [false discovery rate (FDR) ≤0.001, fold change ≥2] in each comparison. The highest 5,303 significant DEGs were found between lipopolysaccharide- (LPS) and interferon (IFN)γ-stimulated M1 cells, whereas only 153 significant DEGs were detected between interleukin (IL)-10-polarized M2 cells and control mock-activated cells. To identify signature genes for antiviral regulation pertaining to each activation status, we identified a set of DEGs that showed significant up-regulation in only one activation state. In addition, pathway analyses defined the top 20–50 significantly regulated pathways at each activation status, and we further analyzed DEGs pertinent to pathways mediated by AMP kinase (AMPK) and epigenetic mechanisms. For the first time in porcine macrophages, our transcriptomic analyses not only compared family-wide differential expression of most known immune genes at different activation statuses, but also revealed transcription evidence of multiple gene families. These findings show that using RNA-Seq transcriptomic analyses in virus-infected and status-synchronized macrophages effectively profiled signature genes and gene response pathways for antiviral regulation, which may provide a framework for optimizing antiviral immunity and immune homeostasis. PMID:24505295

  1. DNA methylation, riboswitches, and transcription factor activity: fundamental mechanisms of gene-nutrient interactions involving vitamins.

    PubMed

    Huang, Janet; Vieira, Amandio

    2006-12-01

    Nutrient-gene interactions occur with a variety of nutrients including some minerals, vitamins, polyunsaturated fatty acids and other lipids. Fundamental molecular mechanisms that underlie many of the effects of nutrients on gene expression are presented herein. Two of the mechanisms described influence gene transcription: DNA methylation and transcription factor activation. Another mechanism, riboswitching, can regulate gene expression at different levels, for example, at the mRNA translation level. The first two mechanisms are widely distributed across animal phyla. Riboswitches are documented primarily in more primitive organisms, but may prove to be of wider relevance. Riboswitches are known for several vitamins; those involving thiamine are presented here. The role of folates and retinoids in DNA methylation and transcriptional factor (nuclear retinoid receptor) activities, respectively, is presented in the context of cell proliferation and differentiation, and related physiological or pathological effects during embryogenesis and cancer.

  2. The regulatory region of the human plasminogen activator inhibitor type-1 (PAI-1) gene.

    PubMed Central

    Riccio, A; Lund, L R; Sartorio, R; Lania, A; Andreasen, P A; Danø, K; Blasi, F

    1988-01-01

    The human gene for plasminogen activator inhibitor type-1 (PAI-1) has been isolated and its promoter region characterized. PAI-1 regulation by glucocorticoids, transforming growth factor-beta (TGF-beta) and the phorbol ester PMA is shown to be exerted at the promoter level. A fragment spanning 805 nucleotides of the 5' flanking and 72 of the 5' untranslated region contain information enough to promote transcription and to respond to glucocorticoids when fused to a reporter gene and transfected into human fibrosarcoma cells. A moderately repetitive DNA sequence, containing a TATA box, a GRE consensus, a Z-DNA forming sequence and two imperfect direct repeats at the extremities, is present a few nucleotides 5' of the human PAI-1 gene transcription start site, raising the possibility that this gene could have been activated by DNA insertion during evolution. Images PMID:3130610

  3. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

    PubMed Central

    Bikard, David; Jiang, Wenyan; Samai, Poulami; Hochschild, Ann; Zhang, Feng; Marraffini, Luciano A.

    2013-01-01

    The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications. PMID:23761437

  4. The circadian Clock gene regulates acrosin activity of sperm through serine protease inhibitor A3K

    PubMed Central

    Cheng, Shuting; Liang, Xin; Wang, Yuhui; Jiang, Zhou; Liu, Yanyou; Hou, Wang; Li, Shiping; Zhang, Jing

    2015-01-01

    Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements. PMID:26264441

  5. Encoding four gene expression programs in the activation dynamics of a single transcription factor.

    PubMed

    Hansen, Anders S; O'Shea, Erin K

    2016-04-01

    Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3-5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs. PMID:27046808

  6. Evolution of the Perlecan/HSPG2 Gene and Its Activation in Regenerating Nematostella vectensis

    PubMed Central

    Warren, Curtis R.; Kassir, Elias; Spurlin, James; Martinez, Jerahme; Putnam, Nicholas H.; Farach-Carson, Mary C.

    2015-01-01

    The heparan sulfate proteoglycan 2 (HSPG2)/perlecan gene is ancient and conserved in all triploblastic species. Its presence maintains critical cell boundaries in tissue and its large (up to ~900 kDa) modular structure has prompted speculation about the evolutionary origin of the gene. The gene’s conservation amongst basal metazoans is unclear. After the recent sequencing of their genomes, the cnidarian Nematostella vectensis and the placozoan Trichoplax adhaerens have become favorite models for studying tissue regeneration and the evolution of multicellularity. More ancient basal metazoan phyla include the poriferan and ctenophore, whose evolutionary relationship has been clarified recently. Our in silico and PCR-based methods indicate that the HSPG2 gene is conserved in both the placozoan and cnidarian genomes, but not in those of the ctenophores and only partly in poriferan genomes. HSPG2 also is absent from published ctenophore and Capsaspora owczarzaki genomes. The gene in T. adhaerens is encoded as two separate but genetically juxtaposed genes that house all of the constituent pieces of the mammalian HSPG2 gene in tandem. These genetic constituents are found in isolated genes of various poriferan species, indicating a possible intronic recombinatory mechanism for assembly of the HSPG2 gene. Perlecan’s expression during wound healing and boundary formation is conserved, as expression of the gene was activated during tissue regeneration and reformation of the basement membrane of N. vectensis. These data indicate that the complex HSPG2 gene evolved concurrently in a common ancestor of placozoans, cnidarians and bilaterians, likely along with the development of differentiated cell types separated by acellular matrices, and is activated to reestablish these tissue borders during wound healing. PMID:25876075

  7. Screening of the Enterocin-Encoding Genes and Antimicrobial Activity in Enterococcus Species.

    PubMed

    Ogaki, Mayara Baptistucci; Rocha, Katia Real; Terra, MÁrcia Regina; Furlaneto, MÁrcia Cristina; Maia, Luciana Furlaneto

    2016-06-28

    In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens. PMID:26907753

  8. SATB1 packages densely-looped, transciptionally-active chromatinfor coordinated expression of cytokine genes

    SciTech Connect

    Cai, Shutao; Lee, Charles C.; Kohwi-Shigematsu, Terumi

    2006-05-23

    SATB1 is an important regulator of nuclear architecture that anchors specialized DNA sequences onto its cage-like network and recruits chromatin remodeling/modifying factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4, and Il13 from the 200kb cytokine gene cluster region of mouse chromosome 11 during T-helper 2 (Th2)-cell activation. We show that upon cell activation, SATB1 is rapidly induced to form a unique transcriptionally-active chromatin structure that includes the cytokine gene region. Chromatin is folded into numerous small loops all anchored by SATB1, is histone H3 acetylated at lysine 9/14, and associated with Th2-specific factors, GATA3, STAT6, c-Maf, the chromatin-remodeling enzyme Brg-1, and RNA polymerase II across the 200kb region. Before activation, the chromatin displays some of these features, such as association with GATA3 and STAT6, but these were insufficient for cytokine gene expression. Using RNA interference (RNAi), we show that upon cell activation, SATB1 is not only required for chromatin folding into dense loops, but also for c-Maf induction and subsequently for Il4, Il5, and Il13 transcription. Our results show that SATB1 is an important determinant for chromatin architecture that constitutes a novel higher-order, transcriptionally-active chromatin structure upon Th2-cell activation.

  9. Functional activation of the egr-1 (early growth response-1) gene by hydrogen peroxide.

    PubMed

    Nose, K; Ohba, M

    1996-06-01

    The redox-based regulation of gene expression is one of the fundamental mechanisms of cellular functions, and hydrogen peroxide seems to act as an intracellular second messenger of signal transduction of cytokines. Hydrogen peroxide at non-toxic doses induced the accumulation of mRNA for the early growth response-1 (egr-1) gene in mouse osteoblastic cells. The Egr-1 protein is a transcription factor that binds the GCGGGGGCG sequence and contains a zinc-finger structure that is essential for DNA binding. Egr-1 protein is sensitive to oxidative stress and loses specific DNA-binding activity when exposed to high levels of oxidative stress. Incubating cells with hydrogen peroxide at about 50 microM, however, increased the accumulation of Egr-1 protein, and the Egr-1 product seemed to be functional, judging by its binding activity to the GCGGGGGCG sequence and its ability to activate the chloramphenicol acetyltransferase reporter gene under the control of the human thymidine kinase enhancer containing the Egr-1 binding sequence. It was reported that the activity of Egr-1 protein as a transcription factor was negatively regulated by active oxygens. However, with appropriate concentrations of active oxygen, its capacity to bind a specific DNA sequence and to enhance the transcriptional activity of target genes is thought to be elevated.

  10. A Synthetic Transcriptional Activator of Genes Associated with the Retina in Human Dermal Fibroblasts.

    PubMed

    Syed, Junetha; Chandran, Anandhakumar; Pandian, Ganesh N; Taniguchi, Junichi; Sato, Shinsuke; Hashiya, Kaori; Kashiwazaki, Gengo; Bando, Toshikazu; Sugiyama, Hiroshi

    2015-07-01

    Small molecules capable of modulating epigenetic signatures can activate the transcription of tissue-restricted genes in a totally unrelated cell type and have potential use in epigenetic therapy. To provide an example for an initial approach, we report here on one synthetic small-molecule compound-termed "SAHA-PIP X"-from our library of conjugates. This compound triggered histone acetylation accompanied by the transcription of retinal-tissue-related genes in human dermal fibroblasts (HDFs).

  11. Residues beyond the selectivity filter of the K+ channel kir2.1 regulate permeation and block by external Rb+ and Cs+.

    PubMed

    Thompson, G A; Leyland, M L; Ashmole, I; Sutcliffe, M J; Stanfield, P R

    2000-07-15

    1. Kir2.1 channels are blocked by Rb+ and Cs+ in a voltage-dependent manner, characteristic of many inward rectifier K+ channels. Mutation of Ser165 in the transmembrane domain M2 to Leu (S165L) abolished Rb+ blockage and lowered Cs+ blocking affinity. At negative voltages Rb+ carried large inward currents. 2. A model of the Kir2.1 channel, built by homology with the structure of the Streptomyces lividans K+ channel KcsA, suggested the existence of an intersubunit hydrogen bond between Ser165 and Thr141 in the channel pore-forming P-region that helps stabilise the structure of this region. However, mutations of Thr141 and Ser165 did not produce effects consistent with a hydrogen bond between these residues being essential for blockage. 3. An alternative alignment between the M2 regions of Kir2.1 and KcsA suggested that Ser165 is itself a pore-lining residue, more directly affecting blockage. We were able to replace Ser165 with a variety of polar and non-polar residues, consistent with this residue being pore lining. Some of these changes affected channel blockage. 4. We tested the hypothesis that Asp172 - a residue implicated in channel gating by polyamines - formed an additional selectivity filter by using the triple mutant T141A/S165L/D172N. Large Rb+ and Cs+ currents were measured in this mutant. 5. We propose that both Thr141 and Ser165 are likely to provide binding sites for monovalent blocking cations in wild-type channels. These residues lie beyond the carbonyl oxygen tunnel thought to form the channel selectivity filter, which the blocking cations must therefore traverse.

  12. Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

    PubMed Central

    Proks, Peter; Capener, Charlotte E.; Jones, Phillippa; Ashcroft, Frances M.

    2001-01-01

    The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings. PMID:11585848

  13. Genes Involved in Interleukin-1 Receptor Type II Activities Are Associated With Asthmatic Phenotypes

    PubMed Central

    Madore, Anne-Marie; Vaillancourt, Vanessa T.; Bouzigon, Emmanuelle; Sarnowski, Chloé; Monier, Florent; Dizier, Marie-Hélène; Demenais, Florence

    2016-01-01

    Purpose Interleukin-1 (IL-1) plays a key role in inflammation and immunity and its decoy receptor, IL-1R2, has been implicated in transcriptomic and genetic studies of asthma. Methods Two large asthma family collections, the French-Canadian Saguenay—Lac-St-Jean (SLSJ) study and the French Epidemiological Study on the Genetics and Environment of Asthma (EGEA), were used to investigate the association of SNPs in 10 genes that modulate IL-1R2 activities with asthma, allergic asthma, and atopy. Gene-gene interactions were also tested. Results One SNP in BACE2 was associated with allergic asthma in the SLSJ study and replicated in the EGEA study before statistical correction for multiple testing. Additionally, two SNPs in the MMP2 gene were replicated in both studies prior to statistical correction and reached significance in the combined analysis. Moreover, three gene-gene interactions also survived statistical correction in the combined analyses (BACE1-IL1RAP in asthma and allergic asthma and IL1R1-IL1RAP in atopy). Conclusions Our results highlight the relevance of genes involved in the IL-1R2 activity in the context of asthma and asthma-related traits. PMID:27334786

  14. Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia.

    PubMed

    Wood, M S; Lory, C; Lessie, T G

    1990-04-01

    We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRP1::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-beta-D-thiogalactopyranoside. Lac+ variants of the pGC91.14-containing strains which formed beta-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria. PMID:2156800

  15. Genome-wide distribution of Auts2 binding localizes with active neurodevelopmental genes

    PubMed Central

    Oksenberg, N; Haliburton, G D E; Eckalbar, W L; Oren, I; Nishizaki, S; Murphy, K; Pollard, K S; Birnbaum, R Y; Ahituv, N

    2014-01-01

    The autism susceptibility candidate 2 gene (AUTS2) has been associated with multiple neurological diseases including autism spectrum disorders (ASDs). Previous studies showed that AUTS2 has an important neurodevelopmental function and is a suspected master regulator of genes implicated in ASD-related pathways. However, the regulatory role and targets of Auts2 are not well known. Here, by using ChIP-seq (chromatin immunoprecipitation followed by deep sequencing) and RNA-seq on mouse embryonic day 16.5 forebrains, we elucidated the gene regulatory networks of Auts2. We find that the majority of promoters bound by Auts2 belong to genes highly expressed in the developing forebrain, suggesting that Auts2 is involved in transcriptional activation. Auts2 non-promoter-bound regions significantly overlap developing brain-associated enhancer marks and are located near genes involved in neurodevelopment. Auts2-marked sequences are enriched for binding site motifs of neurodevelopmental transcription factors, including Pitx3 and TCF3. In addition, we characterized two functional brain enhancers marked by Auts2 near NRXN1 and ATP2B2, both ASD-implicated genes. Our results implicate Auts2 as an active regulator of important neurodevelopmental genes and pathways and identify novel genomic regions that could be associated with ASD and other neurodevelopmental diseases. PMID:25180570

  16. Epidermal patterning genes are active during embryogenesis in Arabidopsis.

    PubMed

    Costa, Silvia; Dolan, Liam

    2003-07-01

    Epidermal cells in the root of Arabidopsis seedling differentiate either as hair or non-hair cells, while in the hypocotyl they become either stomatal or elongated cells. WEREWOLF (WER) and GLABRA2 (GL2) are positive regulators of non-hair and elongated cell development. CAPRICE (CPC) is a positive regulator of hair cell development in the root. We show that WER, GL2 and CPC are expressed and active during the stages of embryogenesis when the pattern of cells in the epidermis of the root-hypocotyl axis forms. GL2 is first expressed in the future epidermis in the heart stage embryo and its expression is progressively restricted to those cells that will acquire a non-hair identity in the transition between torpedo and mature stage. The expression of GL2 at the heart stage requires WER function. WER and CPC are transiently expressed throughout the root epidermal layer in the torpedo stage embryo when the cell-specific pattern of GL2 expression is being established in the epidermis. We also show that WER positively regulates CPC transcription and GL2 negatively regulates WER transcription in the mature embryo. We propose that the restriction of GL2 to the future non-hair cells in the root epidermis can be correlated with the activities of WER and CPC during torpedo stage. In the embryonic hypocotyl we show that WER controls GL2 expression. We also provide evidence indicating that CPC may also regulate GL2 expression in the hypocotyl.

  17. TAP1, a yeast gene that activates the expression of a tRNA gene with a defective internal promoter.

    PubMed Central

    Di Segni, G; McConaughy, B L; Shapiro, R A; Aldrich, T L; Hall, B D

    1993-01-01

    We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNA(Tyr) internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAP1 (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap1-1 mutant than in the wild type. The tap1-1 mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap1-1 mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts- growth defect, we cloned the wild-type TAP1 gene. It is essential for yeast growth. We show in the accompanying report (T. L. Aldrich, G. Di Segni, B. L. McConaughy, N. J. Keen, S. Whelen, and B. D. Hall, Mol. Cell. Biol. 13:3434-3444, 1993) that TAP1 is identical to RAT1, a yeast gene implicated in poly(A)+ RNA export and that the TAP1/RAT1 gene product has extensive sequence similarity to the protein encoded by another yeast gene (variously named DST2, KEM1, RAR5, SEP1, or XRN1) having exonuclease and DNA strand transfer activity (reviewed by Kearsey and Kipling [Trends Cell Biol. 1:110-112, 1991]). Images PMID:8497259

  18. Quantitative structure-activity relationships and docking studies of calcitonin gene-related peptide antagonists.

    PubMed

    Kyani, Anahita; Mehrabian, Mohadeseh; Jenssen, Håvard

    2012-02-01

    Defining the role of calcitonin gene-related peptide in migraine pathogenesis could lead to the application of calcitonin gene-related peptide antagonists as novel migraine therapeutics. In this work, quantitative structure-activity relationship modeling of biological activities of a large range of calcitonin gene-related peptide antagonists was performed using a panel of physicochemical descriptors. The computational studies evaluated different variable selection techniques and demonstrated shuffling stepwise multiple linear regression to be superior over genetic algorithm-multiple linear regression. The linear quantitative structure-activity relationship model revealed better statistical parameters of cross-validation in comparison with the non-linear support vector regression technique. Implementing only five peptide descriptors into this linear quantitative structure-activity relationship model resulted in an extremely robust and highly predictive model with calibration, leave-one-out and leave-20-out validation R(2) of 0.9194, 0.9103, and 0.9214, respectively. We performed docking of the most potent calcitonin gene-related peptide antagonists with the calcitonin gene-related peptide receptor and demonstrated that peptide antagonists act by blocking access to the peptide-binding cleft. We also demonstrated the direct contact of residues 28-37 of the calcitonin gene-related peptide antagonists with the receptor. These results are in agreement with the conclusions drawn from the quantitative structure-activity relationship model, indicating that both electrostatic and steric factors should be taken into account when designing novel calcitonin gene-related peptide antagonists. PMID:21974743

  19. Combinatorial Control of Light Induced Chromatin Remodeling and Gene Activation in Neurospora

    PubMed Central

    Sancar, Cigdem; Ha, Nati; Yilmaz, Rüstem; Tesorero, Rafael; Fisher, Tamas; Brunner, Michael; Sancar, Gencer

    2015-01-01

    Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression. PMID:25822411

  20. Involvement of Trichoderma Trichothecenes in the Biocontrol Activity and Induction of Plant Defense-Related Genes

    PubMed Central

    Malmierca, M. G.; Cardoza, R. E.; Alexander, N. J.; McCormick, S. P.; Hermosa, R.; Monte, E.

    2012-01-01

    Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied. PMID:22562989

  1. Characterisation of a Trichoderma hamatum monooxygenase gene involved in antagonistic activity against fungal plant pathogens.

    PubMed

    Carpenter, Margaret A; Ridgway, Hayley J; Stringer, Alison M; Hay, Amanda J; Stewart, Alison

    2008-04-01

    A monooxygenase gene was isolated from a biocontrol strain of Trichoderma hamatum and its role in biocontrol was investigated. The gene had homologues in other fungal genomes, but was not closely related to any fully characterised gene. The T. hamatum monooxygenase gene was expressed specifically in response to the plant pathogens Sclerotinia sclerotiorum, Sclerotinia minor and Sclerotium cepivorum, but not in response to Botrytis cinerea or T. hamatum. Expression of the gene did not occur until contact had been made between the two fungal species. Homologues in T. atroviride and T. virens showed similar expression patterns. Expression of the gene in response to S. sclerotiorum was influenced by pH, with a peak of expression at pH 4, and was subject to nitrogen catabolite repression. Disruption of the monooxygenase gene did not affect the growth or morphology of T. hamatum, but caused a decrease in its ability to inhibit the growth and sclerotial production of S. sclerotiorum. The monooxygenase gene had a role in the antagonistic activity of Trichoderma species against specific fungal plant pathogens and is therefore a potentially important factor in biocontrol by Trichoderma species. PMID:18231791

  2. Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor.

    PubMed Central

    Henriksson, A; Almlöf, T; Ford, J; McEwan, I J; Gustafsson, J A; Wright, A P

    1997-01-01

    We have shown that the Ada adaptor complex is important for the gene activation capacity of the glucocorticoid receptor in yeast. The recently isolated human Ada2 protein also increases the potency of the receptor protein in mammalian cells. The Ada pathway is of key significance for the tau1 core transactivation domain (tau1c) of the receptor, which requires Ada for activity in vivo and in vitro. Ada2 can be precipitated from nuclear extracts by a glutathione S-transferase-tau1 fusion protein coupled to agarose beads, and a direct interaction between Ada2 and tau1c can be shown by using purified proteins. This interaction is strongly reduced by a mutation in tau1c that reduces transactivation activity. Mutations affecting the Ada complex do not reverse transcriptional squelching by the tau1 domain, as they do for the VP16 transactivation domain, and thus these powerful acidic activators differ in at least some important aspects of gene activation. Mutations that reduce the activity of the tau1c domain in wild-type yeast strains cause similar reductions in ada mutants that contain little or no Ada activity. Thus, gene activation mechanisms, in addition to the Ada pathway, are involved in the activity of the tau1c domain. PMID:9154805

  3. Measurement of immediate-early gene activation- c-fos and beyond.

    PubMed

    Kovács, K J

    2008-06-01

    Immediate-early genes (IEG) are powerful tools for identifying activated neurosecretory neurones and extended circuits that affect neuroendocrine functions. The generally acknowledged scenario is when cells became activated, IEGs expressed and IEG-encoded transcription factors affect target gene expression. However, there are several examples in which: (i) neuronal activation occurs without induction of IEGs; (ii) IEG induction is not related to challenge-induced neuropeptide expression; and (iii) markers of neuronal activation are not expressed in chronically activated neurones. In spite of these limitations, the use of c-Fos and other regulatory- or effector transcription factors as markers of neuronal activation will continue to be an extremely powerful technique. Recently-developed models, including transgenic mice expressing different marker genes under the regulation of IEG promoters, will help to monitor neuronal activity in vivo or ex vivo and to reveal connection between activated neurones. Furthermore, combinations between novel imaging techniques, such as magnetic resonance and IEG-based mapping strategies, will open new means with which to study functional activity in the neurosecretory systems.

  4. Identification of Alpha Interferon-Induced Genes Associated with Antiviral Activity in Daudi Cells and Characterization of IFIT3 as a Novel Antiviral Gene

    PubMed Central

    Schmeisser, H.; Mejido, J.; Balinsky, C. A.; Morrow, A. N.; Clark, C. R.; Zhao, T.; Zoon, K. C.

    2010-01-01

    A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products. PMID:20686046

  5. SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression.

    PubMed

    Marathe, Himangi G; Mehta, Gaurav; Zhang, Xiaolu; Datar, Ila; Mehrotra, Aanchal; Yeung, Kam C; de la Serna, Ivana L

    2013-01-01

    SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to

  6. Absence of canonical marks of active chromatin in developmentally regulated genes.

    PubMed

    Pérez-Lluch, Sílvia; Blanco, Enrique; Tilgner, Hagen; Curado, Joao; Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-10-01

    The interplay of active and repressive histone modifications is assumed to have a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that the transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated with the stable production of RNA, whereas unmarked chromatin would permit rapid gene activation and deactivation during development. In the latter case, regulation by transcription factors would have a comparatively more important regulatory role than chromatin marks.

  7. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    SciTech Connect

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  8. Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

    PubMed

    Hom, S S; Graham, L A; Maier, R J

    1985-03-01

    Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.

  9. Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia.

    PubMed

    Knopov, V; Leach, R M; Barak-Shalom, T; Hurwitz, S; Pines, M

    1995-04-01

    Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.

  10. Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    PubMed Central

    Nitulescu, Ioana I.; Tangpeerachaikul, Anupong; Poss, Zachary C.; Da Silva, Diogo H.; Caruso, Brittany T.; Arefolov, Alexander; Fadeyi, Olugbeminiyi; Christie, Amanda L.; Du, Karrie; Banka, Deepti; Schneider, Elisabeth V.; Jestel, Anja; Zou, Ge; Si, Chong; Ebmeier, Christopher C.; Bronson, Roderick T.; Krivtsov, Andrei V.; Myers, Andrew G.; Kohl, Nancy E.; Kung, Andrew L.; Armstrong, Scott A.; Lemieux, Madeleine E.; Taatjes, Dylan J.; Shair, Matthew D.

    2015-01-01

    Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML. PMID:26416749

  11. A Homeodomain Transcription Factor Gene, PfMSX, Activates Expression of Pif Gene in the Pearl Oyster Pinctada fucata

    PubMed Central

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5′ flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster. PMID:25099698

  12. A homeodomain transcription factor gene, PfMSX, activates expression of Pif gene in the pearl oyster Pinctada fucata.

    PubMed

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi

    2014-01-01

    We reported pearl oyster Pinctada fucata cDNA and genomic characterization of a new homeobox-containing protein, PfMSX. The PfMSX gene encodes a transcription factor that was localized to the nucleus. Analyses of PfMSX mRNA in tissues and developmental stages showed high expressions in mantle or D-shaped larvae. In electrophoretic mobility shift assays (EMSAs) PfMSX binded to MSX consensus binding sites in the 5' flanking region of the Pif promoter. In co-transfection experiment PfMSX transactivated reporter constructs containing Pif promoter sequences, and mutation of the MSX-binding sites attenuated transactivation. A knockdown experiment using PfMSX dsRNA showed decreased Pif mRNA and unregular crystallization of the nacreous layer using scanning electron microscopy. Our results suggested that PfMSX was a conserved homeodomain transcription factor gene, which can activate Pif gene expression through MSX binding site, and was then involved in the mineralization process in pearl oyster Pinctada fucata. Our data provided important clues about mechanisms regulating biomineralization in pearl oyster.

  13. [Construction of the flavinogenic yeast Candida famata strains with high riboflavin kinase activity using gene engineering].

    PubMed

    Ishchuk, O P; Iatsyshyn, V Iu; Dmytruk, K V; Voronovs'kyĭ, A Ia; Fedorovych, D V; Sybirnyĭ, A A

    2006-01-01

    The recombinant strains of the flavinogenic yeast Candida famata, which contain the DNA fragment consisting of the FMN1 gene (encoding the riboflavin kinase, enzyme that converts riboflavin to flavinmononucleotide) driven by the strong promoters (the regulated RIB1 or constitutive TEF1 promoter) were isolated. Riboflavin kinase activity in the isolated transformants was tested. The 6-8-fold increase of the riboflavin kinase activity was shown in the recombinant strains containing the integrated Debaryomyces hansenii FMN1 gene under the strong constitutive TEF1 promoter. The recombinant strains can be used for the following construction of flavinmononucleotide overproducers. PMID:17290783

  14. Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements.

    PubMed

    Ochi, Kozo; Tanaka, Yukinori; Tojo, Shigeo

    2014-02-01

    Since bacteria were found to contain genes encoding enzymes that synthesize a plethora of potential secondary metabolites, interest has grown in the activation of these cryptic pathways. Homologous and heterologous expression of these cryptic secondary metabolite-biosynthetic genes, often "silent" under ordinary laboratory fermentation conditions, may lead to the discovery of novel secondary metabolites. We review current progress on this topic, describing concepts for activating silent genes. We especially focus on genetic manipulation of transcription and translation, as well as the utilization of rare earth elements as a novel method to activate the silent genes. The possible roles of silent genes in bacterial physiology are also discussed. PMID:24127067

  15. Computer-aided design of modular protein devices: Boolean AND gene activation

    NASA Astrophysics Data System (ADS)

    Salis, H.; Kaznessis, Y. N.

    2006-12-01

    Many potentially useful synthetic gene networks require the expression of an engineered gene if and only if two different DNA-binding proteins exist in sufficient concentration. While some natural and engineered systems activate gene expression according to a logical AND-like behavior, they often utilize allosteric or cooperative protein-protein interactions, rendering their components unsuitable for a toolbox of modular parts for use in multiple applications. Here, we develop a quantitative model to demonstrate that a small system of interacting fusion proteins, called a protein device, can activate an engineered gene according to the Boolean AND behavior while using only modular protein domains and DNA sites. The fusion proteins are created from transactivating, DNA-binding, non-DNA binding, and protein-protein interaction domains along with the corresponding peptide ligands. Using a combined kinetic and thermodynamic model, we identify the characteristics of the molecular components and their rates of constitutive production that maximize the fidelity of AND behavior. These AND protein devices facilitate the creation of complex genetic programs and may be used to create gene therapies, biosensors and other biomedical and biotechnological applications that turn on gene expression only when multiple DNA-binding proteins are simultaneously present.

  16. Identification and Diversity of Killer Cell Ig-Like Receptors in Aotus vociferans, a New World Monkey

    PubMed Central

    Garzón-Ospina, Diego; López, Carolina; Cadavid, Luis F.; Patarroyo, Manuel E.; Patarroyo, Manuel A.

    2013-01-01

    Previous BAC clone analysis of the Platyrrhini owl monkey KIRs have shown an unusual genetic structure in some loci. Therefore, cDNAs encoding KIR molecules from eleven Aotus vociferans monkeys were characterized here; ten putative KIR loci were found, some of which encoded atypical proteins such as KIR4DL and transcripts predicted to encode a D0+D1 configuration (AOTVOKIR2DL1*01v1) which appear to be unique in the Aotus genus. Furthermore, alternative splicing was found as a likely mechanism for producing activator receptors in A. vociferans species. KIR proteins from New World monkeys may be split into three new lineages according to domain by domain phylogenetic analysis. Although the A. vociferans KIR family displayed a high divergence among paralogous genes, individual loci were limited in their genetic polymorphism. Selection analysis showed that both constrained and rapid evolution may operate within the AvKIR family. The frequent alternative splicing (as a likely mechanism generating activator receptors), the presence of KIR4DL and KIR2DL1 (D0+D1) molecules and other data reported here suggest that the KIR family in Aotus has had a rapid evolution, independent from its Catarrhini counterparts. PMID:24223188

  17. CRISPR-on system for the activation of the endogenous human INS gene.

    PubMed

    Giménez, C A; Ielpi, M; Mutto, A; Grosembacher, L; Argibay, P; Pereyra-Bonnet, F

    2016-06-01

    Advances in the field of epigenetics have allowed the design of new therapeutic strategies to address complex diseases such as type 1 diabetes (T1D). Clustered regularly interspaced short palindromic repeats (CRISPR)-on is a novel and powerful RNA-guided transcriptional activator system that can turn on specific gene expression; however, it remains unclear whether this system can be widely used or whether its use will be restricted depending on cell types, methylation promoter statuses or the capacity to modulate chromatin state. Our results revealed that the CRISPR-on system fused with transcriptional activators (dCas9-VP160) activated endogenous human INS, which is a silenced gene with a fully methylated promoter. Similarly, we observed a synergistic effect on gene activation when multiple single guide RNAs were used, and the transcriptional activation was maintained until day 21. Regarding the epigenetic profile, the targeted promoter gene did not exhibit alteration in its methylation status but rather exhibited altered levels of H3K9ac following treatment. Importantly, we showed that dCas9-VP160 acts on patients' cells in vitro, particularly the fibroblasts of patients with T1D. PMID:27052801

  18. Timing and Variability of Galactose Metabolic Gene Activation Depend on the Rate of Environmental Change

    PubMed Central

    Ma, Bo; Ott, William; Josić, Krešimir; Bennett, Matthew R.

    2015-01-01

    Modulation of gene network activity allows cells to respond to changes in environmental conditions. For example, the galactose utilization network in Saccharomyces cerevisiae is activated by the presence of galactose but repressed by glucose. If both sugars are present, the yeast will first metabolize glucose, depleting it from the extracellular environment. Upon depletion of glucose, the genes encoding galactose metabolic proteins will activate. Here, we show that the rate at which glucose levels are depleted determines the timing and variability of galactose gene activation. Paradoxically, we find that Gal1p, an enzyme needed for galactose metabolism, accumulates more quickly if glucose is depleted slowly rather than taken away quickly. Furthermore, the variability of induction times in individual cells depends non-monotonically on the rate of glucose depletion and exhibits a minimum at intermediate depletion rates. Our mathematical modeling suggests that the dynamics of the metabolic transition from glucose to galactose are responsible for the variability in galactose gene activation. These findings demonstrate that environmental dynamics can determine the phenotypic outcome at both the single-cell and population levels. PMID:26200924

  19. Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

    PubMed Central

    Chiaramello, A; Neuman, K; Palm, K; Metsis, M; Neuman, T

    1995-01-01

    Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression. PMID:7565756

  20. Preferential Repair of DNA Double-strand Break at the Active Gene in Vivo*

    PubMed Central

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K.; Bhaumik, Sukesh R.

    2012-01-01

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3′ end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells. PMID:22910905

  1. Cooperative activation of tissue-specific genes by pRB and E2F1.

    PubMed

    Flowers, Stephen; Xu, Fuhua; Moran, Elizabeth

    2013-04-01

    The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to cooccupy and repress Alpl and Bglap in proliferating osteoblast precursors before differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be cotargeting genes for activation. Chromatin immunoprecipitation analysis in an osteoblast differentiation model shows that Alpl and Bglap are indeed targeted by an activator E2F, i.e., is E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte- and adipocyte-specific genes are also cotargeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

  2. Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

    PubMed Central

    Trimbur, D E; Gutshall, K R; Prema, P; Brenchley, J E

    1994-01-01

    Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme. Images PMID:7811090

  3. Genetic Characterization of the Homeodomain-Independent Activity of the Drosophila Fushi Tarazu Gene Product

    PubMed Central

    Hyduk, D.; Percival-Smith, A.

    1996-01-01

    The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results. PMID:8852847

  4. Gene expression profiling of peripheral blood mononuclear cells from children with active hemophagocytic lymphohistiocytosis.

    PubMed

    Sumegi, Janos; Barnes, Michael G; Nestheide, Shawnagay V; Molleran-Lee, Susan; Villanueva, Joyce; Zhang, Kejian; Risma, Kimberly A; Grom, Alexei A; Filipovich, Alexandra H

    2011-04-14

    Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, genetically heterogeneous autosomal recessive immune disorder that results when the critical regulatory pathways that mediate immune defense mechanisms and the natural termination of immune/inflammatory responses are disrupted or overwhelmed. To advance the understanding of FHL, we performed gene expression profiling of peripheral blood mononuclear cells from 11 children with untreated FHL. Total RNA was isolated and gene expression levels were determined using microarray analysis. Comparisons between patients with FHL and normal pediatric controls (n = 30) identified 915 down-regulated and 550 up-regulated genes with more than or equal to 2.5-fold difference in expression (P ≤ .05). The expression of genes associated with natural killer cell functions, innate and adaptive immune responses, proapoptotic proteins, and B- and T-cell differentiation were down-regulated in patients with FHL. Genes associated with the canonical pathways of interleukin-6 (IL-6), IL-10 IL-1, IL-8, TREM1, LXR/RXR activation, and PPAR signaling and genes encoding of antiapoptotic proteins were overexpressed in patients with FHL. This first study of genome-wide expression profiling in children with FHL demonstrates the complexity of gene expression patterns, which underlie the immunobiology of FHL.

  5. TFRC and ACTB as the best reference genes to quantify Urokinase Plasminogen Activator in breast cancer

    PubMed Central

    2011-01-01

    Background Biomedical researchers have long looked for ways to diagnose and treat cancer patients at the early stages through biomarkers. Although conventional techniques are routinely applied in the detection of biomarkers, attitudes towards using Real-Time PCR techniques in detection of many biomarkers are increasing. Normalization of quantitative Real-Time PCR is necessary to validate non-biological alteration occurring during the steps of RNA quantification. Selection of variably expressed housekeeping genes (HKs) will affect the validity of the data. The aim of the present study was to identify uniformly expressed housekeeping genes in order to use in the breast cancer gene expression studies. Urokinase Plasminogen Activator was used as a gene of interest. Findings The expression of six HKs (TFRC, GUSB, GAPDH, ACTB, HPRT1 and RPLP0) was investigated using geNorm and NormFinder softwares in forty breast tumor, four normal and eight adjacent tissues. RPLP0 and GAPDH revealed maximum M value, while TFRC demonstrated lowest M value. Conclusions In the present study the most and the least stable genes were TFRC and RPLP0 respectively. TFRC and ACTB were verified as the best combination of two genes for breast cancer quantification. The result of this study shows that in each gene expression analysis HKs selection should be done based on experiment conditions. PMID:21702980

  6. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    PubMed

    Book, Adam J; Lewin, Gina R; McDonald, Bradon R; Takasuka, Taichi E; Wendt-Pienkowski, Evelyn; Doering, Drew T; Suh, Steven; Raffa, Kenneth F; Fox, Brian G; Currie, Cameron R

    2016-06-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  7. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

    DOE PAGES

    Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Suh, Steven; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-06-08

    In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less

  8. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    PubMed

    Book, Adam J; Lewin, Gina R; McDonald, Bradon R; Takasuka, Taichi E; Wendt-Pienkowski, Evelyn; Doering, Drew T; Suh, Steven; Raffa, Kenneth F; Fox, Brian G; Currie, Cameron R

    2016-06-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

  9. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

    PubMed Central

    McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-01-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

  10. DNA Topoisomerases Are Required for Preinitiation Complex Assembly during GAL Gene Activation

    PubMed Central

    Pedersen, Jakob Madsen; Bjergbaek, Lotte; Andersen, Anni Hangaard

    2015-01-01

    To investigate the importance of topoisomerases for transcription of the galactose induced genes, we have studied the expression of GAL1, GAL2, GAL7 and GAL10 in Saccharomyces cerevisiae cells deficient for topoisomerases I and II. We find that topoisomerases are required for transcriptional activation of the GAL genes, but are dispensable for ongoing transcription, eliminating a role of the enzymes in transcriptional elongation. Furthermore, we demonstrate that promoter chromatin remodeling of the GAL genes is unaffected in the topoisomerase deficient strain. However, the cells fail to successfully recruit RNA polymerase II due to an inability of the TATA-binding protein (TBP) to bind to the TATA box in these promoters. We therefore argue that topoisomerases are required for accurate assembly of the preinitiation complex at the promoters of the GAL genes. PMID:26173127

  11. Stem cell-based gene therapy activated using magnetic hyperthermia to enhance the treatment of cancer.

    PubMed

    Yin, Perry T; Shah, Shreyas; Pasquale, Nicholas J; Garbuzenko, Olga B; Minko, Tamara; Lee, Ki-Bum

    2016-03-01

    Stem cell-based gene therapies, wherein stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential for cancer applications owing to their innate ability to home to tumors. However, traditional stem cell-based gene therapies are hampered by our current inability to control when the therapeutic genes are actually turned on, thereby resulting in detrimental side effects. Here, we report the novel application of magnetic core-shell nanoparticles for the dual purpose of delivering and activating a heat-inducible gene vector that encodes TNF-related apoptosis-inducing ligand (TRAIL) in adipose-derived mesenchymal stem cells (AD-MSCs). By combining the tumor tropism of the AD-MSCs with the spatiotemporal MCNP-based delivery and activation of TRAIL expression, this platform provides an attractive means with which to enhance our control over the activation of stem cell-based gene therapies. In particular, we found that these engineered AD-MSCs retained their innate ability to proliferate, differentiate, and, most importantly, home to tumors, making them ideal cellular carriers. Moreover, exposure of the engineered AD-MSCS to mild magnetic hyperthermia resulted in the selective expression of TRAIL from the engineered AD-MSCs and, as a result, induced significant ovarian cancer cell death in vitro and in vivo.

  12. Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation.

    PubMed

    Li, Zhenfei; Nie, Fen; Wang, Sheng; Li, Lin

    2011-02-22

    Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 monomethylation (H4K20me-1) in transcriptional regulation remains unclear. Here, we show that Wnt3a specifically stimulates H4K20 monomethylation at the T cell factor (TCF)-binding element through the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with lymphoid enhancing factor-1 (LEF1)/TCF4 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly through H4K20 monomethylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. PMID:21282610

  13. Acetohydroxyacid synthase activity and transcripts profiling reveal tissue-specific regulation of ahas genes in sunflower.

    PubMed

    Ochogavía, Ana C; Breccia, Gabriela; Vega, Tatiana; Felitti, Silvina A; Picardi, Liliana A; Nestares, Graciela

    2014-07-01

    Acetohydroxyacid synthase (AHAS) is the target site of several herbicides and catalyses the first step in the biosynthesis of branched chain amino acid. Three genes coding for AHAS catalytic subunit (ahas1, ahas2 and ahas3) have been reported for sunflower. The aim of this work was to study the expression pattern of ahas genes family and AHAS activity in sunflower (Helianthus annuus L.). Different organs (leaves, hypocotyls, roots, flowers and embryos) were evaluated at several developmental stages. The transcriptional profile was studied through RT-qPCR. The highest expression for ahas1 was shown in leaves, where all the induced and natural gene mutations conferring herbicide resistance were found. The maximal expression of ahas2 and ahas3 occurred in immature flowers and embryos. The highest AHAS activity was found in leaves and immature embryos. Correlation analysis among ahas gene expression and AHAS activity was discussed. Our results show that differences in ahas genes expression are tissue-specific and temporally regulated. Moreover, the conservation of multiple AHAS isoforms in sunflower seems to result from different expression requirements controlled by tissue-specific regulatory mechanisms at different developmental stages. PMID:24908515

  14. Keeping the blood flowing-plasminogen activator genes and feeding behavior in vampire bats.

    PubMed

    Tellgren-Roth, Asa; Dittmar, Katharina; Massey, Steven E; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A; Liberles, David A

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  15. Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

    NASA Astrophysics Data System (ADS)

    Tellgren-Roth, Åsa; Dittmar, Katharina; Massey, Steven E.; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A.; Liberles, David A.

    2009-01-01

    The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

  16. Persistent STAT5 activation in myeloid neoplasms recruits p53 into gene regulation.

    PubMed

    Girardot, M; Pecquet, C; Chachoua, I; Van Hees, J; Guibert, S; Ferrant, A; Knoops, L; Baxter, E J; Beer, P A; Giraudier, S; Moriggl, R; Vainchenker, W; Green, A R; Constantinescu, S N

    2015-03-01

    STAT (Signal Transducer and Activator of Transcription) transcription factors are constitutively activated in most hematopoietic cancers. We previously identified a target gene, LPP/miR-28 (LIM domain containing preferred translocation partner in lipoma), induced by constitutive activation of STAT5, but not by transient cytokine-activated STAT5. miR-28 exerts negative effects on thrombopoietin receptor signaling and platelet formation. Here, we demonstrate that, in transformed hematopoietic cells, STAT5 and p53 must be synergistically bound to chromatin for induction of LPP/miR-28 transcription. Genome-wide association studies show that both STAT5 and p53 are co-localized on the chromatin at 463 genomic positions in proximal promoters. Chromatin binding of p53 is dependent on persistent STAT5 activation at these proximal promoters. The transcriptional activity of selected promoters bound by STAT5 and p53 was significantly changed upon STAT5 or p53 inhibition. Abnormal expression of several STAT5-p53 target genes (LEP, ATP5J, GTF2A2, VEGFC, NPY1R and NPY5R) is frequently detected in platelets of myeloproliferative neoplasm (MPN) patients, but not in platelets from healthy controls. In conclusion, persistently active STAT5 can recruit normal p53, like in the case of MPN cells, but also p53 mutants, such as p53 M133K in human erythroleukemia cells, leading to pathologic gene expression that differs from canonical STAT5 or p53 transcriptional programs.

  17. The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

    PubMed

    Scholthof, H B; Gowda, S; Wu, F C; Shepherd, R J

    1992-05-01

    Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

  18. Role of peroxisome proliferator-activated receptors gene polymorphisms in type 2 diabetes and metabolic syndrome

    PubMed Central

    Dong, Chen; Zhou, Hui; Shen, Chong; Yu, Lu-Gang; Ding, Yi; Zhang, Yong-Hong; Guo, Zhi-Rong

    2015-01-01

    Metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) are the serious public health problems worldwide. Moreover, it is estimated that MetS patients have about five-fold greater risk of the T2DM development compared with people without the syndrome. Peroxisome proliferator-activated receptors are a subgroup of the nuclear hormone receptor superfamily of ligand-activated transcription factors which play an important role in the pathogenesis of MetS and T2DM. All three members of the peroxisome proliferator-activated receptor (PPAR) nuclear receptor subfamily, PPARα, PPARβ/δ and PPARγ are critical in regulating insulin sensitivity, adipogenesis, lipid metabolism, and blood pressure. Recently, more and more studies indicated that the gene polymorphism of PPARs, such as Leu162Val and Val227Ala of PPARα, +294T > C of PPARβ/δ, Pro12Ala and C1431T of PPARγ, are significantly associated with the onset and progressing of MetS and T2DM in different population worldwide. Furthermore, a large body of evidence demonstrated that the glucose metabolism and lipid metabolism were influenced by gene-gene interaction among PPARs genes. However, given the complexity pathogenesis of metabolic disease, it is unlikely that genetic variation of a single locus would provide an adequate explanation of inter-individual differences which results in diverse clinical syndromes. Thus, gene-gene interactions and gene-environment interactions associated with T2DM and MetS need future comprehensive studies. PMID:25987964

  19. Characterization of the Biocontrol Activity of Pseudomonas fluorescens Strain X Reveals Novel Genes Regulated by Glucose

    PubMed Central

    Kremmydas, Gerasimos F.; Tampakaki, Anastasia P.; Georgakopoulos, Dimitrios G.

    2013-01-01

    Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ), and two genes (sup5 and sup6) which seem to be organized in a putative operon. This operon (named supX) consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon. PMID:23596526

  20. Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

    PubMed

    Kremmydas, Gerasimos F; Tampakaki, Anastasia P; Georgakopoulos, Dimitrios G

    2013-01-01

    Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ), and two genes (sup5 and sup6) which seem to be organized in a putative operon. This operon (named supX) consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon.

  1. Activation of enhancer elements by the homeobox gene Cdx2 is cell line specific.

    PubMed Central

    Taylor, J K; Levy, T; Suh, E R; Traber, P G

    1997-01-01

    Cdx2 is a caudal-related homeodomain transcription factor that is expressed in complex patterns during mouse development and at high levels in the intestinal epithelium of adult mice. Cdx2 activates transcription of intestinal gene promoters containing specific binding sites. Moreover, Cdx2 has been shown to induce intestinal differentiation in cell lines. In this study, we show that Cdx2 is able to bind to two well defined enhancer elements in the HoxC8 gene. We then demonstrate that Cdx2 is able to activate transcription of heterologous promoters when its DNA binding element is placed in an enhancer context. Furthermore, the ability to activate enhancer elements is cell-line dependent. When the Cdx2 activation domain was linked to the Gal4 DNA binding domain, the chimeric protein was able to activate Gal4 enhancer constructs in an intestinal cell line, but was unable to activate transcription in NIH3T3 cells. These data suggest that there are cell-specific factors that allow the Cdx2 activation domain to function in the activation of enhancer elements. We hypothesize that either a co-activator protein or differential phosphorylation of the activation domain may be the mechanism for intestinal cell line-specific function of Cdx2 and possibly in other tissues in early development. PMID:9171078

  2. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment

    PubMed Central

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  3. Driving force-dependent block by internal Ba(2+) on the Kir2.1 channel: Mechanistic insight into inward rectification.

    PubMed

    Hsieh, Chi-Pan; Kuo, Chung-Chin; Huang, Chiung-Wei

    2015-07-01

    The Kir2.1 channel is characterized by strong inward rectification; however, the mechanism of the steep voltage dependence near the equilibrium potential remains to be investigated. Here, we studied the internal Ba(2+) block of the Kir2.1 channel expressed in Xenopus oocytes. We showed that the driving force and thus the K(+) ion flux significantly influenced the apparent affinity of the block by internal Ba(2+). Kinetic analysis revealed that the binding rate shifted with the driving force and changed steeply near the equilibrium point, either in the presence or absence of the transmembrane electrical field. The unbinding rate was determined by the intrinsic affinity of the site. Mutagenesis studies revealed that the high-affinity binding site for Ba(2+) was located near T141 at the internal entrance of the selectivity filter. The steep change of the blocking affinity near the equilibrium potential may result from the flux-coupling effect in the single-file, multi-ion cytoplasmic pore.

  4. Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus, Medicago, and Phaseolus

    PubMed Central

    Neupane, Achal; Nepal, Madhav P; Benson, Benjamin V; MacArthur, Kenton J; Piya, Sarbottam

    2013-01-01

    Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution. PMID:24317362

  5. Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

    PubMed Central

    Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie

    2016-01-01

    The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern. PMID:27723822

  6. Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes.

    PubMed

    Zhou, Xuguo; Smith, Joseph A; Oi, Faith M; Koehler, Philip G; Bennett, Gary W; Scharf, Michael E

    2007-06-15

    Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it (e.g., wood and humus). Lower termites, which are equipped with both endogenous (i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut+salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.

  7. DNA sequences that activate isocitrate lyase gene expression during late embryogenesis and during postgerminative growth.

    PubMed Central

    Zhang, J Z; Santes, C M; Engel, M L; Gasser, C S; Harada, J J

    1996-01-01

    We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants. PMID:8934622

  8. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  9. Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

  10. Effects of novelty stress on hippocampal gene expression, corticosterone and motor activity in mice.

    PubMed

    Kurumaji, Akeo; Umino, Masakazu; Nishikawa, Toru

    2011-10-01

    Exposure to novelty, a mild psychological stressor, induces neuronal activations in the hippocampus of rodents, which may play an important role in the adaptation to stress. We examined the changes in three parameters, i.e., gene expression in the hippocampus using a RT-PCR method, corticosterone and motor activity, in mice exposed to a new environment for 120min. A sharp and short-lasting increase in the gene expression of a set of stress-related genes previously reported, e.g., Fos and Nr4a1, was observed during the stress, with a similar pattern of changes in corticosterone. The motor activity gradually decreased during the novelty stress, indicating a process of adaptation to the new environment. In addition, in order to minimize the effects of elevated adrenal hormones by the stress, we carried out experiments on adrenalectomized (ADX) mice. However, the adrenalectomy produced minimal changes in the pattern and the magnitude of the gene response after the stress, while the motor activity showed a relatively slower pattern of adaptation in the ADX mice. Hence, the present study suggests that there was a coordinated adaptation process to the new environment in mice, and that the transcriptional response was mediated by neuronal networks rather than by adrenal hormones.

  11. Diagnostic value of blood gene expression signatures in active tuberculosis in Thais: a pilot study.

    PubMed

    Satproedprai, N; Wichukchinda, N; Suphankong, S; Inunchot, W; Kuntima, T; Kumpeerasart, S; Wattanapokayakit, S; Nedsuwan, S; Yanai, H; Higuchi, K; Harada, N; Mahasirimongkol, S

    2015-06-01

    Tuberculosis (TB) is a major global health problem. Routine laboratory tests or newly developed molecular detection are limited to the quality of sputum sample. Here we selected genes specific to TB by a minimum redundancy-maximum relevancy package using publicly available microarray data and determine level of selected genes in blood collected from a Thai TB cohort of 40 active TB patients, 38 healthy controls and 18 previous TB patients using quantitative real-time PCR. FCGR1A, FCGR1B variant 1, FCGR1B variant 2, APOL1, GBP5, PSTPIP2, STAT1, KCNJ15, MAFB and KAZN had significantly higher expression level in active TB individuals as compared with healthy controls and previous TB cases (P<0.01). A mathematical method was applied to calculate TB predictive score, which contains the level of expression of seven genes and this score can identify active TB cases with 82.5% sensitivity and 100% specificity as compared with conventional culture confirmation. In addition, TB predictive scores in active TB patients were reduced to normal after completion of standard short-course therapy, which was mostly in concordant with the disease outcome. These finding suggested that blood gene expression measurement and TB Sick Score could have potential value in terms of diagnosis of TB and anti-TB treatment monitoring.

  12. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    EPA Science Inventory

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  13. ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription

    PubMed Central

    Al-Kandari, Wafa; Jambunathan, Srikarthika; Navalgund, Vandana; Koneni, Rupa; Freer, Margot; Parimi, Neeta; Mudhasani, Rajini; Fontes, Joseph D.

    2006-01-01

    The class II trans-activator (CIITA) is recognized as the master regulator of major histocompatibility complex (MHC) class II gene transcription and contributes to the transcription of MHC class I genes. To better understand the function of CIITA, we performed yeast two-hybrid with the C-terminal 807 amino acids of CIITA, and cloned a novel human cDNA named zinc finger, X-linked, duplicated family member C (ZXDC). The 858 amino acid ZXDC protein contains 10 zinc fingers and a transcriptional activation domain, and was found to interact with the region of CIITA containing leucine-rich repeats. Over-expression of ZXDC in human cell lines resulted in super-activation of MHC class I and class II promoters by CIITA. Conversely, silencing of ZXDC expression reduced the ability of CIITA to activate transcription of MHC class II genes. Given the specific interaction between the ZXDC and CIITA proteins, as well as the effect of ZXDC on MHC gene transcription, it appears that ZXDC is an important regulator of both MHC class I and class II transcription. PMID:16600381

  14. T-bet Activates Th1 Genes through Mediator and the Super Elongation Complex.

    PubMed

    Hertweck, Arnulf; Evans, Catherine M; Eskandarpour, Malihe; Lau, Jonathan C H; Oleinika, Kristine; Jackson, Ian; Kelly, Audrey; Ambrose, John; Adamson, Peter; Cousins, David J; Lavender, Paul; Calder, Virginia L; Lord, Graham M; Jenner, Richard G

    2016-06-21

    The transcription factor T-bet directs Th1 cell differentiation, but the molecular mechanisms that underlie this lineage-specific gene regulation are not completely understood. Here, we show that T-bet acts through enhancers to allow the recruitment of Mediator and P-TEFb in the form of the super elongation complex (SEC). Th1 genes are occupied by H3K4me3 and RNA polymerase II in Th2 cells, while T-bet-mediated recruitment of P-TEFb in Th1 cells activates transcriptional elongation. P-TEFb is recruited to both genes and enhancers, where it activates enhancer RNA transcription. P-TEFb inhibition and Mediator and SEC knockdown selectively block activation of T-bet target genes, and P-TEFb inhibition abrogates Th1-associated experimental autoimmune uveitis. T-bet activity is independent of changes in NF-κB RelA and Brd4 binding, with T-bet- and NF-κB-mediated pathways instead converging to allow P-TEFb recruitment. These data provide insight into the mechanism through which lineage-specifying factors promote differentiation of alternative T cell fates. PMID:27292648

  15. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    PubMed Central

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  16. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression.

    PubMed

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  17. Interaction between immunoglobulin allotypes and NK receptor genes in diabetes post-hepatitis C virus infection.

    PubMed

    Granados-Montiel, Julio; Zúñiga, Joaquin; Azocar, Jose; Feris, Edmond J; Terreros, Daniel; Larsen, Charles E; Clavijo, Olga P; Cruz-Lagunas, Alfredo; Middleton, Derek; Alper, Chester A; Pandey, Janardan P; Yunis, Edmond J

    2011-06-01

    Genetic interactions between natural killer (NK) cells immunoglobulin-like receptor (KIR) genes and immunoglobulin allotypes have been previously reported in type 2 diabetes mellitus (DM) patients. Puerto Rican Americans with a history of intravenous drug use who developed DM following HCV infection (n=32) were compared to individuals infected with HCV without diabetes (n=121) and to DM non-infected individuals (n=95). Subjects were genotyped for KIRs and immunoglobulin allotypes. We found interactions of immunoglobulin allotypes KM3/KM3 with NK inhibitory receptors 2DL3/2DL3, 2DL1 in the absence of 2DS4 associated with susceptibility to DM in HCV infected individuals. These data suggest the possibility that a subset of patients with HCV could have an immune-mediated component contributing to the development of DM.

  18. Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms.

    PubMed

    Fang, Liang; Li, Dimin; Welling, Paul A

    2010-12-01

    The renal outer medullary K(+) (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592-599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP(2)) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP(2) depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP(2) hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure. PMID:20926634

  19. Characterization of transcriptional activation and inserted-into-gene preference of various transposable elements in the Brassica species.

    PubMed

    Gao, Caihua; Xiao, Meili; Jiang, Lingyan; Li, Jiana; Yin, Jiaming; Ren, Xiaodong; Qian, Wei; Oscar, Ortegón; Fu, Donghui; Tang, Zhanglin

    2012-07-01

    Transposable elements (TEs) have attracted increasing attention because of their tremendous contributions to genome reorganization and gene variation through dramatic proliferation and excision via transposition. However, less known are the transcriptional activation of various TEs and the characteristics of TE insertion into genomes at the genome-wide level. In the present study, we focused on TE genes for transposition and gene disruption by insertion of TEs in expression sequences of Brassica, to investigate the transcriptional activation of TEs, the biased insertion of TEs into genes, and their salient characteristics. Long terminal repeat (LTR-retrotransposon) accounted for the majority of these active TE genes (70.8%), suggesting that transposition activation varied with TE type. 6.1% genes were interrupted by LTR-retrotransposons, which indicated their preference for insertion into genes. TEs were preferentially inserted into cellular component-specific genes acted as "binding" elements and involved in metabolic processes. TEs have a biased insertion into some host genes that were involved with important molecular functions and TE genes exhibited spatiotemporal expression. These results suggested that various types of transposons differentially contributed to gene variation and affected gene function.

  20. Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

    PubMed Central

    Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B.; Wu, Chia-Chin; Akdemir, Kadir C.; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T.; Welch, Heidi C. E.; Garraway, Levi A.; Chin, Lynda

    2016-01-01

    PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2E824*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57KIP2). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator. PMID:26884185

  1. The Association of the Immune Response Genes to Human Papillomavirus-Related Cervical Disease in a Brazilian Population

    PubMed Central

    Marangon, Amanda Vansan; Guelsin, Gláucia Andreia Soares; Visentainer, Jeane Eliete Laguila; Borelli, Sueli Donizete; Watanabe, Maria Angélica Ehara; Consolaro, Márcia Edilaine Lopes; Caleffi-Ferracioli, Katiany Rizzieri; Rudnick, Cristiane Conceição Chagas; Sell, Ana Maria

    2013-01-01

    The genetic variability of the host contributes to the risk of human papillomavirus (HPV)-related cervical disease. Immune response genes to HPV must be investigated to define patients with the highest risk of developing malignant disease. The aim of this study was to investigate the association of polymorphic immune response genes, namely KIR, HLA class I and II, and single-nucleotide polymorphisms (SNPs) of cytokines with HPV-related cervical disease. We selected 79 non-related, admixed Brazilian women from the state of Paraná, southern region of Brazil, who were infected with high carcinogenic risk HPV and present cervical intraepithelial neoplasia grade 3 (CIN3), and 150 HPV-negative women from the same region matched for ethnicity. KIR genes were genotyped using an in-house PCR-SSP. HLA alleles were typed using a reverse sequence-specific oligonucleotide technique. SNPs of TNF −308G>A, IL6 −174G>C, IFNG +874T>A, TGFB1 +869T>C +915G>C, and IL10 −592C>A −819C>T −1082G>A were evaluated using PCR-SSP. The KIR genes were not associated with HPV, although some pairs of i(inhibitory)KIR-ligands occurred more frequently in patients, supporting a role for NK in detrimental chronic inflammatory and carcinogenesis. Some HLA haplotypes were associated with HPV. The associations of INFG and IL10 SNPs potentially reflect impaired or invalid responses in advanced lesions. PMID:23936772

  2. Association of peroxisome proliferator-activated receptor-gamma gene polymorphisms and gene-gene interaction with asthma risk in a Chinese adults population

    PubMed Central

    Li, Wancheng; Dai, Wenjing; Sun, Jian; Zhang, Wei; Jiang, Yi; Ma, Chunlan; Wang, Chunmao; He, Jie

    2015-01-01

    Aims: To investigate the association between single nucleotide polymorphism (SNP) of peroxisome proliferator-activated receptors γ (PPAR γ) and additional gene-gene interactions on asthma risk. Methods: A total of 882 subjects (602 males, 280 females), with a mean age of 61.3±14.8 years old, including 430 asthma patients and 452 normal subjects were selected in this study, including the genotyping of polymorphisms. Logistic regression was performed to investigate association between SNP and asthma. Generalized MDR (GMDR) was used to analysis the interaction among four SNP. Results: Asthma risk was significantly lower in carriers of Ala allele of the rs1805192 polymorphism than those with Pro/Pro (Pro/Ala+ Ala/Ala versus Pro/Pro, adjusted OR (95% CI)=0.70 (0.51-0.94). In addition, we also found a significant association between rs10865710 and asthma, asthma risk was significantly lower in carriers of G allele of the rs10865710 polymorphism than those with CC (CG+ GG versus CC, adjusted OR (95% CI)=0.68 (0.55-0.95). There was a significant three-locus model (P=0.0107) involving rs1805192, rs10865710 and rs709158, indicating a potential gene-gene interaction among rs1805192, rs10865710 and rs709158. Overall, the three-locus models had a cross-validation consistency of 10 of 10, and had the testing accuracy of 60.72% after covariates adjustment. Conclusions: Our results support an important association of rs1805192 and rs10865710 with asthma, and additional interaction among rs1805192, rs10865710 and rs709158. PMID:26770574

  3. Gene activation-associated long noncoding RNAs function in mouse preimplantation development

    PubMed Central

    Hamazaki, Nobuhiko; Uesaka, Masahiro; Nakashima, Kinichi; Agata, Kiyokazu; Imamura, Takuya

    2015-01-01

    In mice, zygotic activation occurs for a wide variety of genes, mainly at the 2-cell stage. Long noncoding RNAs (lncRNAs) are increasingly being recognized as modulators of gene expression. In this study, directional RNA-seq of MII oocytes and 2-cell embryos identified more than 1000 divergently transcribed lncRNA/mRNA gene pairs. Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes. Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation. In particular, microinjection of siRNA against the abundant pancRNA partner of interleukin 17d (Il17d) mRNA at the 1-cell stage caused embryonic lethality, which was rescued by supplying IL17D protein in vitro at the 4-cell stage. Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development. PMID:25633350

  4. Quorum activation at a distance: spatiotemporal patterns of gene regulation from diffusion of an autoinducer signal

    NASA Astrophysics Data System (ADS)

    Dilanji, Gabriel; Langebrake, Jessica; Deleenheer, Patrick; Hagen, Stephen J.

    2012-02-01

    Bacteria in colonies coordinate gene regulation through the exchange of diffusible signal molecules known as autoinducers (AI). This ``quorum signaling'' often occurs in physically heterogeneous and spatially extended environments such as biofilms. Under these conditions the space and time scales for diffusion of the signal limit the range and timing of effective gene regulation. We expect that spatial and temporal patterns of gene expression will reflect physical environmental constraints as well as nonlinear transcriptional activation and feedback within the gene regulatory system. We have combined experiments and modeling to investigate how these spatiotemporal patterns develop. We embed engineered plasmid/GFP quorum sensor strains or wild type strains in a long narrow agar lane, and then introduce AI signal at one terminus of the lane. Diffusion of the AI initiates reporter expression along the length of the lane, extending to macroscopic distances of mm-cm. Resulting patterns are captured quantitatively by a mathematical model that incorporates logistic growth of the population, diffusion of AI, and nonlinear transcriptional activation. Our results show that a diffusing quorum signal can coordinate gene expression over distances of order 1cm on time scales of order 10 hrs.

  5. Integrating Circadian Activity and Gene Expression Profiles to Predict Chronotoxicity of Drosophila suzukii Response to Insecticides

    PubMed Central

    Hamby, Kelly A.; Kwok, Rosanna S.; Zalom, Frank G.; Chiu, Joanna C.

    2013-01-01

    Native to Southeast Asia, Drosophila suzukii (Matsumura) is a recent invader that infests intact ripe and ripening fruit, leading to significant crop losses in the U.S., Canada, and Europe. Since current D. suzukii management strategies rely heavily on insecticide usage and insecticide detoxification gene expression is under circadian regulation in the closely related Drosophila melanogaster, we set out to determine if integrative analysis of daily activity patterns and detoxification gene expression can predict chronotoxicity of D. suzukii to insecticides. Locomotor assays were performed under conditions that approximate a typical summer or winter day in Watsonville, California, where D. suzukii was first detected in North America. As expected, daily activity patterns of D. suzukii appeared quite different between ‘summer’ and ‘winter’ conditions due to differences in photoperiod and temperature. In the ‘summer’, D. suzukii assumed a more bimodal activity pattern, with maximum activity occurring at dawn and dusk. In the ‘winter’, activity was unimodal and restricted to the warmest part of the circadian cycle. Expression analysis of six detoxification genes and acute contact bioassays were performed at multiple circadian times, but only in conditions approximating Watsonville summer, the cropping season, when most insecticide applications occur. Five of the genes tested exhibited rhythmic expression, with the majority showing peak expression at dawn (ZT0, 6am). We observed significant differences in the chronotoxicity of D. suzukii towards malathion, with highest susceptibility at ZT0 (6am), corresponding to peak expression of cytochrome P450s that may be involved in bioactivation of malathion. High activity levels were not found to correlate with high insecticide susceptibility as initially hypothesized. Chronobiology and chronotoxicity of D. suzukii provide valuable insights for monitoring and control efforts, because insect activity as well as

  6. SNORD116 and SNORD115 change expression of multiple genes and modify each other's activity.

    PubMed

    Falaleeva, Marina; Surface, Justin; Shen, Manli; de la Grange, Pierre; Stamm, Stefan

    2015-11-10

    The loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116 contribute to Prader-Willi syndrome (PWS), the most common syndromic form of obesity in humans. SNORD115 and SNORD116 are considered to be orphan C/D box snoRNAs (SNORDs) as they do not target rRNAs or snRNAs. SNORD115 exhibits sequence complementarity towards the serotonin receptor 2C, but SNORD116 shows no extended complementarities to known RNAs. To identify molecular targets, we performed genome-wide array analysis after overexpressing SNORD115 and SNORD116 in HEK 293T cells, either alone or together. We found that SNORD116 changes the expression of over 200 genes. SNORD116 mainly changed mRNA expression levels. Surprisingly, we found that SNORD115 changes SNORD116's influence on gene expression. In similar experiments, we compared gene expression in post-mortem hypothalamus between individuals with PWS and aged-matched controls. The synopsis of these experiments resulted in 23 genes whose expression levels were influenced by SNORD116. Together our results indicate that SNORD115 and SNORD116 influence expression levels of multiple genes and modify each other activity. PMID:26220404

  7. Lactobacillus acidophilus L-92 Cells Activate Expression of Immunomodulatory Genes in THP-1 Cells

    PubMed Central

    YANAGIHARA, Sae; GOTO, Hiroaki; HIROTA, Tatsuhiko; FUKUDA, Shinji; OHNO, Hiroshi; YAMAMOTO, Naoyuki

    2014-01-01

    To understand the immunomodulatory effects of Lactobacillus acidophilus L-92 cells suggested from our previous study of in vivo anti-allergy and anti-virus effects, host immune responses in macrophage-like THP-1 cells after 4 h (the early phase) and 24 h (the late phase) of cocultivation with L-92 cells were investigated by transcriptome analysis. In the early phase of L-92 treatment, various transcription regulator genes, such as, NFkB1, NFkB2, JUN, HIVEP2 and RELB, and genes encoding chemokines and cytokines, such as CCL4, CXCL11, CCL3 and TNF, were upregulated. Two transmembrane receptor genes, TLR7 and ICAM1, were also upregulated in the early phase of treatment. In contrast, many transmembrane receptor genes, such as IL7R, CD80, CRLF2, CD86, CD5, HLA-DQA1, IL2RA, IL15RA and CSF2RA, and some cytokine genes, including IL6, IL23A and CCL22, were significantly upregulated in the late phase after L-92 exposure. Some genes encoding cytokines, such as IL1A, IL1B and IL8, and the enzyme IDO1 were upregulated at both the early and the late phases of treatment. These results suggest that probiotic L-92 might promote Th1 and regulatory T-cell responses by activation of the MAPK signaling pathway, followed by the NOD-like receptor signaling pathway in THP-1 cells. PMID:25379363

  8. CXXC5 plays a role as a transcription activator for myelin genes on oligodendrocyte differentiation.

    PubMed

    Kim, Mi-Yeon; Kim, Hyun-Yi; Hong, Jiso; Kim, Daesoo; Lee, Hyojung; Cheong, Eunji; Lee, Yangsin; Roth, Jürgen; Kim, Dong Goo; Min, Do Sik; Choi, Kang-Yell

    2016-03-01

    Myelination in corpus callosum plays important role for normal brain functions by transferring neurological information between various brain regions. However, the factors controlling expression of myelin genes in myelination are poorly understood. Here, CXXC5, a recently identified protein with CXXC-type zinc finger DNA binding motif, was characterized as a transcriptional activator of major myelin genes. We identified expression of CXXC5 expression was increased by Wnt/β-catenin signaling. CXXC5 specifically expressed in the white matter induced expression of myelin genes through the direct binding of CXXC DNA-binding motif of CXXC5 on the MBP promoter. During the differentiation of neural stem cells (NSCs) of CXXC5(-/-) mice, the expressions of myelin genes were simultaneously reduced. The CXXC5(-/-) mice exhibited severely reduction of myelin genes expression in corpus callosum as well as abnormalities in myelin structure. The disrupted structural integrity of myelin in the CXXC5(-/-) mice resulted in reduced electrical conduction amplitudes at corpus callosum. These findings indicate that the regulation of myelin genes expression by CXXC5 is important for forming myelin structure involved with axonal electrical signal transfer in the corpus callosum.

  9. In vitro secondary activation (memory effect) of avian vitellogenin II gene in isolated liver nuclei.

    PubMed Central

    Jost, J P; Moncharmont, B; Jiricny, J; Saluz, H; Hertner, T

    1986-01-01

    The vitellogenin II gene is specifically reactivated in vitro (secondary stimulation, memory effect) in purified liver nuclei that had ceased to express the gene in vivo a month after the roosters had received a single injection of estradiol (primary stimulation). The in vitro reactivation depends on the addition to the nuclei of nuclear and cytoplasmic extracts from estradiol-stimulated livers, polyamines (0.1-1.0 mM), and calmodulin (0.1 mM). Under identical incubation conditions the vitellogenin gene could not be reactivated in oviduct, embryonic, and immature chicken liver nuclei. Two other genes, those for ovalbumin and lysozyme, which are regulated by estradiol in the oviduct, could not be activated in the liver nuclei. The correct initiation of vitellogenin gene transcription in the liver nuclei was tested by primer extension studies. Addition of the antiestrogen tamoxifen (0.1 microM) to the system decreased vitellogenin mRNA synthesis by about 45% without affecting total RNA synthesis. Addition of quercetin (0.1 mM) and trans-flupenthixol (0.2 mM), inhibitors of nuclear protein kinase II and calmodulin-dependent kinase, respectively, inhibited the synthesis of vitellogenin mRNA by about 55% without affecting total RNA synthesis. The inhibitory effects of the antiestrogen and the kinase inhibitors were not additive, suggesting that both classes of inhibitor act on the same target or related targets. Depleting the estradiol receptors from the cell and nuclear extracts by means of estradiol-receptor antibodies covalently bound to Matrex beads reduced the stimulation of the vitellogenin gene by 40%. We conclude that in addition to the estradiol receptor and phosphorylation of nuclear protein(s) there are additional factors responsible for the in vitro secondary activation of the avian vitellogenin II gene. Images PMID:3455757

  10. Gene expression profiling of potential peroxisome proliferator-activated receptor (PPAR) target genes in human hepatoblastoma cell lines inducibly expressing different PPAR isoforms

    PubMed Central

    Tachibana, Keisuke; Kobayashi, Yumi; Tanaka, Toshiya; Tagami, Masayuki; Sugiyama, Akira; Katayama, Tatsuya; Ueda, Chihiro; Yamasaki, Daisuke; Ishimoto, Kenji; Sumitomo, Mikako; Uchiyama, Yasutoshi; Kohro, Takahide; Sakai, Juro; Hamakubo, Takao; Kodama, Tatsuhiko; Doi, Takefumi

    2005-01-01

    Background Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis. To identify human PPARs-responsive genes, we established tetracycline-regulated human hepatoblastoma cell lines that can be induced to express each human PPAR and investigated the gene expression profiles of these cells. Results The expression of each introduced PPAR gene was investigated using the various concentrations of doxycycline in the culture media. We found that the expression of each PPAR subtype was tightly controlled by the concentration of doxycycline in these established cell lines. DNA microarray analyses using these cell lines were performed with or without adding each subtype ligand and provided much important information on the PPAR target genes involved in lipid metabolism, transport, storage and other activities. Interestingly, it was noted that while ligand-activated PPARδ induced target gene expression, unliganded PPARδ repressed these genes. The real-time RT-PCR was used to verify the altered expression of selected genes by PPARs and we found that these genes were induced to express in the same pattern as detected in the microarray analyses. Furthermore, we analysed the 5'-flanking region of the human adipose differentiation-related protein (adrp) gene that responded to all subtypes of PPARs. From the detailed analyses by reporter assays, the EMSAs, and ChIP assays, we determined the functional PPRE of the human adrp gene. Conclusion The results suggest that these cell lines are important tools used to identify the human PPARs-responsive genes. PMID:16197558

  11. Activation protein 1-dependent transcriptional activation of interleukin 2 gene by Ca2+/calmodulin kinase type IV/Gr

    PubMed Central

    1996-01-01

    The Ca2+/calmodulin-dependent protein kinase (CaMK) type IV/Gr is selectively expressed in T lymphocytes and is activated after signaling via the T cell antigen receptor (TCR), indicating that it mediates some of the Ca(2+)-dependent transcriptional events that follow TCR engagement. Here we show that CaMKIV/Gr induces the transcription factor activation protein 1 (AP-1) alone or in synergy with T cell mitogens and with the p21ras oncoprotein. CaMKIV/ Gr signaling is associated with transcriptional activation of c-fos but is independent of p21ras or calcineurin. AP-1 is an integral component of the nuclear factor of activated T cells (NFAT) transcriptional complex, which is required for interleukin 2 gene expression in T cells. We demonstrate that CaMKIV/Gr reconstitutes the capacity of the cytosolic component of NFAT to direct transcription from NFAT sites in non-T cells. These results reveal a central role for CaMKIV/Gr as a Ca(2+)-regulated activator of gene transcription in T lymphocytes. PMID:8691123

  12. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

    PubMed

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G

    2016-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We