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Sample records for human beta satellite

  1. Satellite cells in human skeletal muscle plasticity.

    PubMed

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  2. Satellite cells in human skeletal muscle plasticity

    PubMed Central

    Snijders, Tim; Nederveen, Joshua P.; McKay, Bryon R.; Joanisse, Sophie; Verdijk, Lex B.; van Loon, Luc J. C.; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models. PMID:26557092

  3. Human erythrocytes have binding sites for beta-endorphin.

    PubMed Central

    Simpkins, C. O.; Chenet, B. P.; Kang, Y. H.; Mazorow, D. L.; Millar, D. B.; Hollis, V. W.

    1989-01-01

    Monoiodinated human beta-endorphin was found to bind specifically to human erythrocytes. Unlabeled beta-endorphin and beta-endorphin inhibited binding, but (-)naloxone, [D-Ala2, D-Leu5]-enkephalin, and leu- and met-enkephalin did not. Immunoelectron microscopy, using rabbit anti-beta-endorphin antibody, an antirabbit IgG secondary antibody, and complexed horseradish peroxidase, revealed that at low concentrations beta-endorphin binds to the cell surface. Electron spin resonance spectroscopy showed no effect of beta-endorphin on membrane fluidity. This receptor does not appear to conform to the characteristics of an opiate receptor. Images Figures 2 and 3 PMID:2560064

  4. Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.

    PubMed Central

    Ottolenghi, S; Lanyon, W G; Williamson, R; Weatherall, D J; Clegg, J B; Pitcher, C S

    1975-01-01

    Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene. PMID:49057

  5. Cytosine methylation of an ancient satellite family in the wild beet Beta procumbens.

    PubMed

    Schmidt, Martin; Hense, Sarah; Minoche, André E; Dohm, Juliane C; Himmelbauer, Heinz; Schmidt, Thomas; Zakrzewski, Falk

    2014-01-01

    DNA methylation is an essential epigenetic feature for the regulation and maintenance of heterochromatin. Satellite DNA is a repetitive sequence component that often occurs in large arrays in heterochromatin of subtelomeric, intercalary and centromeric regions. Knowledge about the methylation status of satellite DNA is important for understanding the role of repetitive DNA in heterochromatization. In this study, we investigated the cytosine methylation of the ancient satellite family pEV in the wild beet Beta procumbens. The pEV satellite is widespread in species-specific pEV subfamilies in the genus Beta and most likely originated before the radiation of the Betoideae and Chenopodioideae. In B. procumbens, the pEV subfamily occurs abundantly and spans intercalary and centromeric regions. To uncover its cytosine methylation, we performed chromosome-wide immunostaining and bisulfite sequencing of pEV satellite repeats. We found that CG and CHG sites are highly methylated while CHH sites show only low levels of methylation. As a consequence of the low frequency of CG and CHG sites and the preferential occurrence of most cytosines in the CHH motif in pEV monomers, this satellite family displays only low levels of total cytosine methylation.

  6. Cytosine methylation of an ancient satellite family in the wild beet Beta procumbens.

    PubMed

    Schmidt, Martin; Hense, Sarah; Minoche, André E; Dohm, Juliane C; Himmelbauer, Heinz; Schmidt, Thomas; Zakrzewski, Falk

    2014-01-01

    DNA methylation is an essential epigenetic feature for the regulation and maintenance of heterochromatin. Satellite DNA is a repetitive sequence component that often occurs in large arrays in heterochromatin of subtelomeric, intercalary and centromeric regions. Knowledge about the methylation status of satellite DNA is important for understanding the role of repetitive DNA in heterochromatization. In this study, we investigated the cytosine methylation of the ancient satellite family pEV in the wild beet Beta procumbens. The pEV satellite is widespread in species-specific pEV subfamilies in the genus Beta and most likely originated before the radiation of the Betoideae and Chenopodioideae. In B. procumbens, the pEV subfamily occurs abundantly and spans intercalary and centromeric regions. To uncover its cytosine methylation, we performed chromosome-wide immunostaining and bisulfite sequencing of pEV satellite repeats. We found that CG and CHG sites are highly methylated while CHH sites show only low levels of methylation. As a consequence of the low frequency of CG and CHG sites and the preferential occurrence of most cytosines in the CHH motif in pEV monomers, this satellite family displays only low levels of total cytosine methylation. PMID:24994030

  7. Identification of two human beta-tubulin isotypes.

    PubMed Central

    Hall, J L; Dudley, L; Dobner, P R; Lewis, S A; Cowan, N J

    1983-01-01

    The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs. Images PMID:6865944

  8. A transforming growth factor. beta. (TGF-. beta. ) receptor from human placenta exhibits greater affinity for TGF-. beta. 2 than for TGF-. beta. 1

    SciTech Connect

    Mitchell, E.J.; O'Connor-McCourt, M.D. )

    1991-04-30

    Affinity-labeling techniques have been used to identify three types of high-affinity receptors for transforming growth factor {beta} (TGF-{beta}) on the surface of many cells in culture. Here the authors demonstrate that membrane preparations from tissue sources may also be used as an alternative system for studying the binding properties of TGF-{beta} receptors. Using a chemical cross-linking technique with {sup 125}I-TGF-{beta}1 and {sup 125}I-TGF-{beta}2 and bis(sulfosuccinimidyl)suberate (BS{sup 3}), they have identified and characterized two high-affinity binding components in membrane preparations derived from human term placenta. The larger species, which migrates as a diffuse band of molecular mass 250-350 kDa on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, is characteristic of the TGF-{beta} receptor type III, a proteoglycan containing glycosaminoglycan (GAG) chains of chondroitin and heparan sulfate. The smaller species of molecular mass 140 kDa was identified as the core glycoprotein of this type III receptor by using the techniques of enzymatic deglycosylation and peptide mapping. Competition experiments, using {sup 125}I-TGF-{beta}1 or {sup 125}I-TGF-{beta}2 and varying amounts of competing unlabeled TGF-{beta}1 or TGF-{beta}2, revealed that both the placental type III proteoglycan and its core glycoprotein belong to a novel class of type III receptors that exhibit a greater affinity for TGF-{beta}2 than for TGF-{beta}1. This preferential binding of TGF-{beta}2 to placental type III receptors suggests differential roles for TGF-{beta}2 and TGF-{beta} 1 in placental function.

  9. Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

    PubMed Central

    Carlier, Géraldine; Maugein, Alicia; Cordier, Corinne; Pechberty, Séverine; Garfa-Traoré, Meriem; Martin, Patrick; Scharfmann, Raphaël; Albagli, Olivier

    2014-01-01

    Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation. PMID:25259951

  10. Diversity of a Complex Centromeric Satellite and Molecular Characterization of Dispersed Sequence Families in Sugar Beet (Beta vulgaris)

    PubMed Central

    Menzel, Gerhard; Dechyeva, Daryna; Wenke, Torsten; Holtgräwe, Daniela; Weisshaar, Bernd; Schmidt, Thomas

    2008-01-01

    Background and Aims The aim of this work was the identification and molecular characterization of novel sugar beet (Beta vulgaris) repetitive sequences to unravel the impact of repetitive DNA on size and evolution of Beta genomes via amplification and diversification. Methods Genomic DNA and a pool of B. vulgaris repetitive sequences were separately used as probes for a screening of high-density filters from a B. vulgaris plasmid library. Novel repetitive motifs were identified by sequencing and further used as probes for Southern analyses in the genus Beta. Chromosomal localization of the repeats was analysed by fluorescent in situ hybridization on chromosomes of B. vulgaris and two other species of the section Beta. Key Results Two dispersed repetitive families pDvul1 and pDvul2 and the tandemly arranged repeat family pRv1 were isolated from a sugar beet plasmid library. The dispersed repetitive families pDvul1 and pDvul2 were identified in all four sections of the genus Beta. The members of the pDvul1 and pDvul2 family are scattered over all B. vulgaris chromosomes, although amplified to a different extent. The pRv1 satellite repeat is exclusively present in species of the section Beta. The centromeric satellite pBV1 by structural variations of the monomer and interspersion of pRv1 units forms complex satellite structures, which are amplified in different degrees on the centromeres of 12 chromosomes of the three species of the Beta section. Conclusions The complexity of the pBV1 satellite family observed in the section Beta of the genus Beta and, in particular, the strong amplification of the pBV1/pRv1 satellite in the domesticated B. vulgaris indicates the dynamics of centromeric satellite evolution during species radiation within the genus. The dispersed repeat families pDvul1 and pDvul2 might represent derivatives of transposable elements. PMID:18682437

  11. Human liver alcohol dehydrogenase. 1. The primary structure of the beta 1 beta 1 isoenzyme.

    PubMed

    Hempel, J; Bühler, R; Kaiser, R; Holmquist, B; de Zalenski, C; von Wartburg, J P; Vallee, B; Jörnvall, H

    1984-12-17

    Determination of the amino acid sequence of the beta 1 subunit from the class I (pyrazole-sensitive) human liver alcohol dehydrogenase isoenzyme beta 1 beta 1 revealed a 373-residue structure differing at 48 positions (including a gap) from that of the subunit of the well studied horse liver alcohol dehydrogenase EE isoenzyme. The structure deduced is compatible with known differences in composition, ultraviolet absorbance, electrophoretic mobility and catalytic properties between the horse and human enzymes. All zinc-liganding residues of the horse E subunit are strictly conserved in the human beta 1 subunit, despite an earlier report of a mutation involving Cys-46. This residue therefore remains conserved in all known alcohol dehydrogenase structures. However, the total cysteine content of the beta 1 structure is raised from 14 in the subunit of the horse enzyme to 15 by a Tyr----Cys exchange. Most exchanges are on the surface of the molecule and of a well conserved nature. Substitutions close to the catalytic centre are of interest to explain the altered substrate specificity and different catalytic activity of the beta 1 homodimer. Functionally, a Ser----Thr exchange at position 48 appears to be of special importance, since Thr-48 in beta 1 instead of Ser-48 in the horse enzyme can restrict available space. Four other substitutions also line the active-site pocket, and appear to constitute partly compensated exchanges. PMID:6391920

  12. Nucleotide sequence of HS-beta satellite DNA from kangaroo rat Dipodomys ordii.

    PubMed

    Fry, K; Poon, R; Whitcome, P; Idriss, J; Salser, W; Mazrimas, J; Hatch, F

    1973-09-01

    The sequence of the highly repetitive satellite HS-beta DNA fraction from kangaroo rat Dipodomys ordii was determined independently by RNA and DNA sequencing techniques. A basic iterated sequence of 10 nucleotides with several mutational variations was found. Base-composition data are consistent with the proposed sequence and revealed a high content of 5-methylcytosine. DNA and RNA sequencing techniques used gave identical results, showing that the fidelity of synthesis of riboguanidine-substituted DNA under our conditions is adequate for nucleotide sequence studies.

  13. Cysteines beta93 and beta112 as probes of conformational and functional events at the human hemoglobin subunit interfaces.

    PubMed Central

    Vásquez, G B; Karavitis, M; Ji, X; Pechik, I; Brinigar, W S; Gilliland, G L; Fronticelli, C

    1999-01-01

    Three variants of tetrameric human hemoglobin, with changes at the alpha1beta2/alpha2beta1-interface, at the alpha1beta1/alpha2beta2-interface, and at both interfaces, have been constructed. At alpha1beta2/alpha2beta1-interface the beta93 cysteine was replaced by alanine (betaC93A), and at the alpha1beta1/alpha2beta2-interface the beta112 cysteine was replaced by glycine (betaC112G). The alpha1beta2 interface variant, betaC93A, and the alpha1beta1/alpha1beta2 double mutant, beta(C93A+C112G), were crystallized in the T-state, and the structures determined at 2. 0 and 1.8 A resolution, respectively. A comparison of the structures with that of natural hemoglobin A shows the absence of detectable changes in the tertiary folding of the protein or in the T-state quaternary assembly. At the beta112 site, the void left by the removal of the cysteine side chain is filled by a water molecule, and the functional characteristics of betaC112G are essentially those of human hemoglobin A. At the beta93 site, water molecules do not replace the cysteine side chain, and the alanine substitution increases the conformational freedom of beta146His, weakening the important interaction of this residue with beta94Asp. As a result, when Cl- is present in the solution, at a concentration 100 mM, the Bohr effect of the two mutants carrying the beta93Cys-->Ala substitution, betaC93A and beta(C93A+C112G), is significantly modified being practically absent below pH 7.4. Based on the crystallographic data, we attribute these effects to the competition between beta94Asp and Cl- in the salt link with beta146His in T-state hemoglobin. These results point to an interplay between the betaHis146-betaAsp94 salt bridge and the Cl- in solution regulated by the Cys present at position beta93, indicating yet another role of beta93 Cys in the regulation of hemoglobin function. PMID:9876125

  14. Cloning and sequencing of the gene for human. beta. -casein

    SciTech Connect

    Loennerdal, B.; Bergstroem, S.; Andersson, Y.; Hialmarsson, K.; Sundgyist, A.; Hernell, O. )

    1990-02-26

    Human {beta}-casein is a major protein in human milk. This protein is part of the casein micelle and has been suggested to have several physiological functions in the newborn. Since there is limited information on {beta}casein and the factors that affect its concentration in human milk, the authors have isolated and sequenced the gene for this protein. A human mammary gland cDNA library (Clontech) in gt 11 was screened by plaque hy-hybridization using a 42-mer synthetic {sup 32}p-labelled oligo-nucleotide. Positive clones were identified and isolated, DNA was prepared and the gene isolated by cleavage with EcoR1. Following subcloning (PUC18), restriction mapping and Southern blotting, DNA for sequencing was prepared. The gene was sequenced by the dideoxy method. Human {beta}-casein has 212 amino acids and the amino acid sequence deducted from the nucleotide sequence is to 91% identical to the published sequence for human {beta}-casein show a high degree of conservation at the leader peptide and the highly phosphorylated sequences, but also deletions and divergence at several positions. These results provide insight into the structure of the human {beta}-casein gene and will facilitate studies on factors affecting its expression.

  15. On the association of human beta 2 microglobulin with cell culture-grown human cytomegalovirus (HCMV).

    PubMed

    Yamashita, Y; Shimokata, K; Nishiyama, Y

    1992-09-01

    We studied the production of human beta 2 microglobulin (beta 2m) in mock-infected or human cytomegalovirus (HCMV) infected human embryonic lung fibroblasts (HEL) and the association of human beta 2m with HCMV virions. Titration of beta 2m by two-step sandwich enzyme immunoassay revealed that HEL released considerable amounts of human beta 2m into the culture medium and that the production of beta 2m was significantly enhanced by HCMV infection. The concentration of human beta 2m in the culture medium of HCMV-infected HEL reached 500 to 600 ng/ml, which corresponded to 7- to 12-fold of levels found in healthy adult urine. Immunoprecipitation assays showed that HEL-grown HCMV bound a significant amount of endogenous beta 2m, but the viruses were efficiently neutralized by either human hyperimmune anti-HCMV globulin or anti-HCMV monoclonal antibody even when treated with a large amount of human beta 2m or with dialysed urine. Thus it seems unlikely that the binding of beta 2m by HCMV is involved in masking the viral antigenic site necessary for neutralization.

  16. Release of salusin-beta from human monocytes/macrophages.

    PubMed

    Sato, Kengo; Fujimoto, Kazumi; Koyama, Takatoshi; Shichiri, Masayoshi

    2010-06-01

    Salusin-alpha and salusin-beta are related bioactive peptides biosynthesized from the same precursor, prosalusin. Despite the potent hemodynamic and proatherosclerotic activities of salusin-beta, its exact distribution and biological functions remain largely undetermined because of technical difficulties associated with its unique physicochemical characteristics, such as marked adhesiveness to polypropylene and polystyrene. By circumventing these problems, we recently established a specific radioimmunoassay for detecting immunoreactive human salusin-beta. In the current study, we demonstrated the release of salusin-beta from the human monoblastic leukemia cell lines, THP-1 and U937. Dilution curves of extracted conditioned media from both cells were parallel with those of standard human salusin-beta by radioimmunoassay. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection of the culture supernatants revealed a major immunoreactive component that co-eluted with authentic salusin-beta. Both cell lines secreted salusin-beta-like immunoreactivity (LI) into serum-free media as a function of time (1234.3 + or - 122.7 and 186.7 + or - 9.1 fmol/10(5) cells per 24h). When THP-1 and U937 cells differentiated into macrophages after incubation with 2-O-tetradecanoylphorbol-13-acetate (TPA), they secreted far greater amounts of salusin-beta-LI into the culture supernatant (3351.9 + or - 899.3 and 1545.8 + or - 183.3 fmol/10(5) cells per 24h). TPA treatment accelerated the processing of prosalusin into its cleaved fragments, suggesting that the increased secretion of salusin-beta-LI in THP-1-derived macrophages was caused by the enhanced intracellular processing of prosalusin. Stimulation with the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS), resulted in increased secretion of salusin-beta without inducing expression of the gene for preprosalusin, suggesting that TNF-alpha and LPS stimulated

  17. Genetic linkage studies of the human glycosphingolipid beta-galactosidases.

    PubMed

    Rushton, A R; Dawson, G

    1977-12-01

    The genetic linkage relationships of the human glycosphingolipid beta-galactosidases were determined using human--mouse somatic cell hybrids. A new method was devised for the estimation of human galactosylceramide, lactosylceramide, and GMI-ganglioside beta-galactosidase activities in the presence of their mouse counterparts, which takes advantage of the reproducible specific activity of lysosomal hydrolases under a given set of culture conditions and is based on differences in both pH optima and sensitivity to chloride ion. Human and mouse chromosomes were identified by their characteristic banding patterns obtained after quinacrine staining, and the optimum glycolipid beta-galactosidase activity was determined for three different substrates. A ratio was defined for each activity which was the specific activity at the human pH optimum divided by the specific activity at the mouse pH optimum. Linear regression analysis was used to test for concordant segregation between pH ratios for each enzyme and the frequency of occurrence of different human chromosomes in the man--mouse somatic hybrid clones. The results obtained from two independent series of hybrid clones indicated that human beta-galactosidase activities consistently segregated with human chromosome 12 in these somatic cell hybrids.

  18. Human satellite cells have regenerative capacity and are genetically manipulable.

    PubMed

    Marg, Andreas; Escobar, Helena; Gloy, Sina; Kufeld, Markus; Zacher, Joseph; Spuler, Andreas; Birchmeier, Carmen; Izsvák, Zsuzsanna; Spuler, Simone

    2014-10-01

    Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon-mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.

  19. Human satellite cells have regenerative capacity and are genetically manipulable

    PubMed Central

    Marg, Andreas; Escobar, Helena; Gloy, Sina; Kufeld, Markus; Zacher, Joseph; Spuler, Andreas; Birchmeier, Carmen; Izsvák, Zsuzsanna; Spuler, Simone

    2014-01-01

    Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies. PMID:25157816

  20. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: immunolocalization according to the odontogenesis phases.

    PubMed

    Sassá Benedete, Ana Paula; Sobral, Ana Paula Veras; Lima, Dirce Mary Correia; Kamibeppu, Leonardo; Soares, Fernando Augusto; Lourenço, Silvia Vanessa

    2008-01-01

    Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo, including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunohistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3-were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

  1. Radiosensitization of human bronchogenic carcinoma cells by interferon beta

    SciTech Connect

    Gould, M.N.; Kakria, R.C.; Olson, S.; Borden, E.C.

    1984-01-01

    The effects of interferons on the radiosensitivity of in vitro human bronchogenic carcinoma cells was investigated. Human fibroblast-derived interferon (IFN-beta) was found to sensitize cells to gamma irradiation while either HuIFN-alpha or mouse IFN-alpha/beta did not. The observed radiosensitization was supra-additive and resulted in a decrease in the shoulder width of the radiation dose-cell survival curve but did not affect the slope. The degree of radiosensitization of the various IFNs tested paralleled the antiproliferative effects of these IFNs on this cell line.

  2. Genetic basis of human complement C8[beta] deficiency

    SciTech Connect

    Kaufmann, T.; Rittner, C.; Schneider, P.M. ); Haensch, G. ); Spaeth, P. ); Tedesco, F. )

    1993-06-01

    The eighth component of human complement (c8) is a serum protein consisting of three chains ([alpha], [beta], and [gamma]) and encoded by three different genes, C8A, C8B, and C8G. C8A and C8B are closely linked on chromosome 1p, whereas C8G is located on chromosome 9q. In the serum the [beta] subunit is non-covalently bound to the disulfide-linked [alpha]-[gamma] subunit. Patients with C8[beta] deficiency suffer from recurrent neisserial infections such as meningitis. Exon-specific polymerase chain reaction (PCR) amplification with primer pairs from the flanking intron sequences was used to amplify all 12 C8B exons separately. No difference regarding the exon sizes was observed in a C8[beta]-deficient patient compared with a normal person. Therefore, direct sequence analysis of all exon-specific PCR products from normal and C8[beta]-deficient individuals was carried out. As a cause for C8[beta] deficiency, we found a single C-T exchange in exon 9 leading to a stop codon. An allele-specific PCR system was designed to detect the normal and the deficiency allele simultaneously. Using this approach as well as PCR typing of the Taql polymorphism located in intron 11, five families with 7 C8[beta]-deficient members were investigated. The mutation was not found to be restricted to one of the two Taql RFLP alleles. The mutant allele was observed in all families investigated and can therefore be regarded as a major cause of C8[beta] deficiency in the Caucasian population. In addition, two C8[beta]-deficient patients were found to be heterozygous for the C-T exchange. The molecular basis of the alleles without this point mutation also causing deficiency has not yet been defined. 23 refs., 4 figs., 3 tabs.

  3. Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles.

    PubMed

    Xu, Xiaoti; Wilschut, Karlijn J; Kouklis, Gayle; Tian, Hua; Hesse, Robert; Garland, Catharine; Sbitany, Hani; Hansen, Scott; Seth, Rahul; Knott, P Daniel; Hoffman, William Y; Pomerantz, Jason H

    2015-09-01

    Identification of human satellite cells that fulfill muscle stem cell criteria is an unmet need in regenerative medicine. This hurdle limits understanding how closely muscle stem cell properties are conserved among mice and humans and hampers translational efforts in muscle regeneration. Here, we report that PAX7 satellite cells exist at a consistent frequency of 2-4 cells/mm of fiber in muscles of the human trunk, limbs, and head. Xenotransplantation into mice of 50-70 fiber-associated, or 1,000-5,000 FACS-enriched CD56(+)/CD29(+) human satellite cells led to stable engraftment and formation of human-derived myofibers. Human cells with characteristic PAX7, CD56, and CD29 expression patterns populated the satellite cell niche beneath the basal lamina on the periphery of regenerated fibers. After additional injury, transplanted satellite cells robustly regenerated to form hundreds of human-derived fibers. Together, these findings conclusively delineate a source of bona-fide endogenous human muscle stem cells that will aid development of clinical applications.

  4. Biotinylated human. beta. -endorphins as probes for the opioid receptor

    SciTech Connect

    Hochhaus, G.; Gibson, B.W.; Sadee, W.

    1988-01-05

    The reaction of human ..beta..-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH/sup +/), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human ..beta..-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Try-1. Three HPLC fractions were isolated for receptor binding studies monobiotinylation of Lys-9, Lys-19, and a mixture of Lys-24, Lys-28, and Lys-29 derivatives. IC/sub 50/ values for binding to ..mu.. and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human ..beta..-endorphin. Association with avidin decreased opioid receptor affinities for the C/sub 6/ spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to ..mu.. and delta sites for derivatives biotinylated at the ..cap alpha..-helical part of the molecule (Lys-19, -24, -28, and -29). Biotinylated human ..beta..-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human ..beta..-endorphin to cross-link the ..mu.. and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.

  5. Human Beta-papillomavirus infection and keratinocyte carcinomas.

    PubMed

    Quint, Koen D; Genders, Roel E; de Koning, Maurits N C; Borgogna, Cinzia; Gariglio, Marisa; Bouwes Bavinck, Jan Nico; Doorbar, John; Feltkamp, Mariet C

    2015-01-01

    Although the role of oncogenic human Alpha-papillomaviruses (HPVs) in the development of mucosal carcinomas at different body sites (eg cervix, anus, oropharynx) is fully recognized, a role for HPV in keratinocyte carcinomas (KCs; basal and squamous cell carcinomas) of the skin is not yet clear. KCs are the most common cancers in Caucasians, with the major risk factor being ultraviolet (UV) light exposure. A possible role for Beta-HPV types (BetaPV) in the development of KC was suggested several decades ago, supported by a number of epidemiological studies. Our current review summarizes the recent molecular and histopathological evidence in support of a causal association between BetaPV and the development of KC, and outlines the suspected synergistic effect of viral gene expression with UV radiation and immune suppression. Further insights into the molecular pathways and protein interactions used by BetaPV and the host cell is likely to extend our understanding of the role of BetaPV in KC. PMID:25131163

  6. Natural human antibodies to amyloid beta peptide.

    PubMed

    Szabo, Paul; Relkin, Norman; Weksler, Marc E

    2008-06-01

    Properties of human, natural anti-Abeta antibodies and commercially available intravenous immunoglobulin (IVIg) have been examined in light of the beneficial effects of passive immunotherapy with IVIg for patients with mild to moderate Alzheimer's disease (AD). Anti-Abeta antibodies in IVIg recognize conformation-specific epitopes as well as linear epitopes from different regions of the Abeta peptide. Anti-Abeta antibodies in circulation, especially those with high avidity, are often masked by ligands and the avidity of these antibodies increases upon dissociation of the bound ligands from the antibodies. Such natural anti-Abeta antibodies have the capacity to prevent Abeta oligomer-induced neurotoxicity in N2A neuroblastoma cells. This neuro-protective effect may reflect the therapeutic potential of the natural anti-Abeta antibodies found in IVIg for the treatment of patients with AD.

  7. Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens.

    PubMed

    Dechyeva, Daryna; Gindullis, Frank; Schmidt, Thomas

    2003-01-01

    Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences. The tandem repeats are 229-246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin. Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed

  8. Specific binding of beta-endorphin to normal human erythrocytes

    SciTech Connect

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  9. Satellites

    SciTech Connect

    Burns, J.A.; Matthews, M.S.

    1986-01-01

    The present work is based on a conference: Natural Satellites, Colloquium 77 of the IAU, held at Cornell University from July 5 to 9, 1983. Attention is given to the background and origins of satellites, protosatellite swarms, the tectonics of icy satellites, the physical characteristics of satellite surfaces, and the interactions of planetary magnetospheres with icy satellite surfaces. Other topics include the surface composition of natural satellites, the cratering of planetary satellites, the moon, Io, and Europa. Consideration is also given to Ganymede and Callisto, the satellites of Saturn, small satellites, satellites of Uranus and Neptune, and the Pluto-Charon system.

  10. Human-Centered Design for the Personal Satellite Assistant

    NASA Technical Reports Server (NTRS)

    Bradshaw, Jeffrey M.; Sierhuis, Maarten; Gawdiak, Yuri; Thomas, Hans; Greaves, Mark; Clancey, William J.; Swanson, Keith (Technical Monitor)

    2000-01-01

    The Personal Satellite Assistant (PSA) is a softball-sized flying robot designed to operate autonomously onboard manned spacecraft in pressurized micro-gravity environments. We describe how the Brahms multi-agent modeling and simulation environment in conjunction with a KAoS agent teamwork approach can be used to support human-centered design for the PSA.

  11. A human factors approach to range scheduling for satellite control

    NASA Technical Reports Server (NTRS)

    Wright, Cameron H. G.; Aitken, Donald J.

    1991-01-01

    Range scheduling for satellite control presents a classical problem: supervisory control of a large-scale dynamic system, with unwieldy amounts of interrelated data used as inputs to the decision process. Increased automation of the task, with the appropriate human-computer interface, is highly desirable. The development and user evaluation of a semi-automated network range scheduling system is described. The system incorporates a synergistic human-computer interface consisting of a large screen color display, voice input/output, a 'sonic pen' pointing device, a touchscreen color CRT, and a standard keyboard. From a human factors standpoint, this development represents the first major improvement in almost 30 years to the satellite control network scheduling task.

  12. Human pancreatic beta-like cells converted from fibroblasts

    PubMed Central

    Zhu, Saiyong; Russ, Holger A.; Wang, Xiaojing; Zhang, Mingliang; Ma, Tianhua; Xu, Tao; Tang, Shibing; Hebrok, Matthias; Ding, Sheng

    2016-01-01

    Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes. PMID:26733021

  13. Human alpha and beta papillomaviruses use different synonymous codon profiles.

    PubMed

    Cladel, Nancy M; Bertotto, Alex; Christensen, Neil D

    2010-06-01

    Human papillomaviruses use rare codons relative to their hosts. It has been theorized that this is a mechanism to allow the virus to escape immune surveillance. In the present study, we examined the codings of four major genes of 21 human alpha (mucosatropic) viruses and 16 human beta (cutaneous-tropic) viruses. We compared the codon usage of different genes from a given papillomavirus and also the same genes from different papillomaviruses. Our data showed that codon usage was not always uniform between two genes of a given papillomavirus or between the same genes of papillomaviruses from different genera. We speculate as to why this might be and conclude that codon usage in the papillomaviruses may not only play a role in facilitating escape from immune surveillance but may also underlie some of the unanswered questions in the papillomavirus field.

  14. The immunomodulatory activity of human amniotic fluid can be correlated with transforming growth factor-beta 1 (TGF-beta 1) and beta 2 activity.

    PubMed Central

    Lang, A K; Searle, R F

    1994-01-01

    The role of alphafetoprotein (AFP) in the immunomodulatory activity of amniotic fluids (AF) from normally progressing human pregnancy (weeks 14-16) was investigated. A panel of 42 AF (25% v/v) reduced significantly phytohaemagglutinin (PHA)-induced peripheral blood mononuclear cell (PBMC) proliferation in serum-free cultures with a mean per cent inhibition of 68.4 +/- 5.5%. In contrast, AFP preparations, with one exception (U.AFP), failed to display inhibitory activity. Pretreatment of AF with anti-TGF-beta 1 and beta 2 antibodies used alone resulted in the mean per cent loss of inhibition of 33.1 +/- 3.9% and 52.3 +/- 7.5%, respectively. A summative loss of AF-mediated inhibition was detected when anti-TGF-beta 1 and beta 2 antibodies were used in combination, but immunomodulation was rarely abolished 100% by this treatment. Anti-TGF-beta 2 antibody treatment, unlike anti-TGF-beta 1 antibody treatment, reversed the inhibitory activity of U.AFP. The amount of TGF-beta 1 and beta 2 contained in human AF was studied by growth inhibition of Mv1 Lu cells. The mean levels of TGF-beta 1 and beta 2 in AF were 11 +/- 0.9 U/ml and 2.3 +/- 0.4 U/ml, respectively, which corresponds with a mean per cent inhibition of 49 +/- 4.7%. U.AFP also significantly inhibited Mv1 Lu cell growth. To investigate the mechanism of AF-mediated inhibition, the effect of AF and AFP on IL-2 production by concanavalin A (Con A)-stimulated PBMC blasts was determined by the CTLL-2 cell bioassay. IL-2 production was reduced 55.5% in AF-treated blasts and 61% in U.AFP-treated blasts compared with controls. Our findings indicate that the immunomodulatory activity of human AF can be correlated with TGF-beta 1 and beta 2 and not with AFP, the inhibitory activity of U.AFP preparation reflecting copurifying TGF-beta 2 activity. PMID:7518368

  15. Neural mechanisms of transient neocortical beta rhythms: Converging evidence from humans, computational modeling, monkeys, and mice.

    PubMed

    Sherman, Maxwell A; Lee, Shane; Law, Robert; Haegens, Saskia; Thorn, Catherine A; Hämäläinen, Matti S; Moore, Christopher I; Jones, Stephanie R

    2016-08-16

    Human neocortical 15-29-Hz beta oscillations are strong predictors of perceptual and motor performance. However, the mechanistic origin of beta in vivo is unknown, hindering understanding of its functional role. Combining human magnetoencephalography (MEG), computational modeling, and laminar recordings in animals, we present a new theory that accounts for the origin of spontaneous neocortical beta. In our MEG data, spontaneous beta activity from somatosensory and frontal cortex emerged as noncontinuous beta events typically lasting <150 ms with a stereotypical waveform. Computational modeling uniquely designed to infer the electrical currents underlying these signals showed that beta events could emerge from the integration of nearly synchronous bursts of excitatory synaptic drive targeting proximal and distal dendrites of pyramidal neurons, where the defining feature of a beta event was a strong distal drive that lasted one beta period (∼50 ms). This beta mechanism rigorously accounted for the beta event profiles; several other mechanisms did not. The spatial location of synaptic drive in the model to supragranular and infragranular layers was critical to the emergence of beta events and led to the prediction that beta events should be associated with a specific laminar current profile. Laminar recordings in somatosensory neocortex from anesthetized mice and awake monkeys supported these predictions, suggesting this beta mechanism is conserved across species and recording modalities. These findings make several predictions about optimal states for perceptual and motor performance and guide causal interventions to modulate beta for optimal function. PMID:27469163

  16. Neural mechanisms of transient neocortical beta rhythms: Converging evidence from humans, computational modeling, monkeys, and mice.

    PubMed

    Sherman, Maxwell A; Lee, Shane; Law, Robert; Haegens, Saskia; Thorn, Catherine A; Hämäläinen, Matti S; Moore, Christopher I; Jones, Stephanie R

    2016-08-16

    Human neocortical 15-29-Hz beta oscillations are strong predictors of perceptual and motor performance. However, the mechanistic origin of beta in vivo is unknown, hindering understanding of its functional role. Combining human magnetoencephalography (MEG), computational modeling, and laminar recordings in animals, we present a new theory that accounts for the origin of spontaneous neocortical beta. In our MEG data, spontaneous beta activity from somatosensory and frontal cortex emerged as noncontinuous beta events typically lasting <150 ms with a stereotypical waveform. Computational modeling uniquely designed to infer the electrical currents underlying these signals showed that beta events could emerge from the integration of nearly synchronous bursts of excitatory synaptic drive targeting proximal and distal dendrites of pyramidal neurons, where the defining feature of a beta event was a strong distal drive that lasted one beta period (∼50 ms). This beta mechanism rigorously accounted for the beta event profiles; several other mechanisms did not. The spatial location of synaptic drive in the model to supragranular and infragranular layers was critical to the emergence of beta events and led to the prediction that beta events should be associated with a specific laminar current profile. Laminar recordings in somatosensory neocortex from anesthetized mice and awake monkeys supported these predictions, suggesting this beta mechanism is conserved across species and recording modalities. These findings make several predictions about optimal states for perceptual and motor performance and guide causal interventions to modulate beta for optimal function.

  17. Neural mechanisms of transient neocortical beta rhythms: Converging evidence from humans, computational modeling, monkeys, and mice

    PubMed Central

    Sherman, Maxwell A.; Lee, Shane; Law, Robert; Haegens, Saskia; Thorn, Catherine A.; Hämäläinen, Matti S.; Moore, Christopher I.; Jones, Stephanie R.

    2016-01-01

    Human neocortical 15–29-Hz beta oscillations are strong predictors of perceptual and motor performance. However, the mechanistic origin of beta in vivo is unknown, hindering understanding of its functional role. Combining human magnetoencephalography (MEG), computational modeling, and laminar recordings in animals, we present a new theory that accounts for the origin of spontaneous neocortical beta. In our MEG data, spontaneous beta activity from somatosensory and frontal cortex emerged as noncontinuous beta events typically lasting <150 ms with a stereotypical waveform. Computational modeling uniquely designed to infer the electrical currents underlying these signals showed that beta events could emerge from the integration of nearly synchronous bursts of excitatory synaptic drive targeting proximal and distal dendrites of pyramidal neurons, where the defining feature of a beta event was a strong distal drive that lasted one beta period (∼50 ms). This beta mechanism rigorously accounted for the beta event profiles; several other mechanisms did not. The spatial location of synaptic drive in the model to supragranular and infragranular layers was critical to the emergence of beta events and led to the prediction that beta events should be associated with a specific laminar current profile. Laminar recordings in somatosensory neocortex from anesthetized mice and awake monkeys supported these predictions, suggesting this beta mechanism is conserved across species and recording modalities. These findings make several predictions about optimal states for perceptual and motor performance and guide causal interventions to modulate beta for optimal function. PMID:27469163

  18. Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor.

    PubMed

    Kim, Jaewon; Blaber, Sachiko I; Blaber, Michael

    2002-03-01

    Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.

  19. Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines.

    PubMed

    Hao, H N; Zhao, J; Lotoczky, G; Grever, W E; Lyman, W D

    2001-05-01

    Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.

  20. Beta human chorionic gonadotropin (beta-hCG) expression in pituitary adenomas: relationship to endocrine function and tumour recurrence.

    PubMed

    Doyle, Paul M; Thiryayi, Waziq A; Joshi, Abhijit; du Plessis, Daniel; Kearney, Tara; Gnanalingham, Kanna K

    2009-01-01

    The beta subunit of human chorionic gonadotropin (beta-hCG) is a marker of malignancies. Recent studies have also reported its expression in pituitary adenomas, although its significance is unclear. In this retrospective study, the authors quantitatively investigated the immunohistochemical expression of beta-hCG in 123 patients undergoing surgery for pituitary adenomas and explored its relationship to the rest of the endocrine function, tumour recurrence and Ki-67 nuclear labelling. Based on the endocrine profile and immunohistochemistry, the pituitary adenomas were grouped into non-functioning (NFPA; N = 78) and functioning pituitary adenomas (N = 45). The latter included, 20 growth hormone (GH), 12 prolactin (PRL), 8 adreno-corticotrophin hormone (ACTH) and 5 mixed GH-PRL-producing adenomas. Ninety-three (76%) tumours were classified as primary and 30 (24%) tumours classified as recurrent adenomas. Immunohistochemically, 107 (87%) of pituitary adenomas expressed beta-hCG, which was more common in NFPA (91%) than functioning pituitary adenomas (80%). beta-hCG expression was not different between primary (86%) and recurrent pituitary adenomas (90%) and it was also not related to raised Ki-67 labelling. But, Ki-67 labelling was raised in recurrent pituitary adenomas (33%), compared to primary pituitary adenomas (11%). Although, beta-hCG is expressed in the majority of pituitary adenomas, more especially in NFPA, it is un-related to the risk of tumour recurrence or cellular proliferation as measured by Ki-67 nuclear labelling. The high incidence of beta-hCG expression in pituitary adenomas may provide a target for specific beta-hCG-directed tumour therapies in the future. PMID:19005764

  1. Assignment of the {beta}-arrestin 1 gene (ARRB1) to human chromosome 11q13

    SciTech Connect

    Calabrese, G.; Morizio, E.; Palka, G.

    1994-11-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor, and its functional cofactor, {beta}-arrestin. {beta}ARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1 to 6) due to the apparently unique functional association of such kinases with this receptor family. The gene for {beta}ARK1 has been localized to human chromosome 11q13. The four members of the arrestin/{beta}-arrestin gene family identified so far are arrestin, X-arrestin, {beta}-arrestin 1, and {beta}-arrestin 2. Here the authors report the chromosome mapping of the human gene for {beta}-arrestin 1 (ARRB1) to chromosome 11q13 by fluorescence in situ hybridization (FISH). Two-color FISH confirmed that the two genes coding for the functionally related proteins {beta}ARK1 and {beta}arrestin 1 both map to 11q13. 16 refs., 1 fig., 1 tab.

  2. The role of EKLF in human beta-globin gene competition.

    PubMed

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  3. Adrenergic lipolysis in guinea pig is not a beta 3-adrenergic response: comparison with human adipocytes.

    PubMed

    Carpéné, C; Castan, I; Collon, P; Galitzky, J; Moratinos, J; Lafontan, M

    1994-03-01

    beta 3-Adrenoceptor agonists are potent lipolytic activators in rats, but they are only weak stimulators in human adipocytes, indicating interspecies differences in the adrenergic regulation of lipid mobilization. Like human but not rat adipocytes, guinea pig fat cells were poorly responsive to the beta 3-agonists BRL-37344, CGP-12177, SR-58611, and ICI-215001, acid metabolite of ICI-D7114. In guinea pigs, the beta 1-agonist dobutamine was more lipolytic than the beta 2-agonist procaterol. Anatomic location of fat deposits was without major influence on the beta-adrenergic responsiveness. Weak responses to beta 3-agonists were found whatever the sex or the age (from 2 days to 16 mo) of the animals. Even in the interscapular brown adipose tissue, which is well known in rats for its beta 3-adrenergic responsiveness, a blunted response to BRL-37344 was observed. The alpha 2-adrenergic antilipolytic effect and receptor number were smaller in guinea pig than in human adipocytes, but the beta-adrenergic receptor number was similar in the two species. Thus guinea pig adipocytes resemble human fat cells when their weak beta 3-adrenergic responsiveness is considered. PMID:7909205

  4. Expression of human beta defensin 4 in genetically modified keratinocytes enhances antimicrobial activity.

    PubMed

    Smiley, Andrea K; Gardner, Jason; Klingenberg, Jennifer M; Neely, Alice N; Supp, Dorothy M

    2007-01-01

    Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.

  5. Molecular cloning and characterization of human pyruvate dehydrogenase. beta. subunit gene

    SciTech Connect

    Koike, Kichiko; Urata, Yoshishige; Koike, Masahiko )

    1990-08-01

    A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase {beta} subunit (PDH{beta}) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH{beta} cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH{beta} structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5{prime} flanking region of the PDH{beta} gene contains a CAAT consensus promoter sequence but no TATA sequence. Primer-extension analysis indicated the PDH{beta} gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

  6. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  7. No evidence of the human chorionic gonadotropin-beta gene 5 betaV79M polymorphism in Mexican women.

    PubMed

    Dominguez-Lopez, Pablo; Diaz-Cueto, Laura; Ulloa-Aguirre, Alfredo; Lopez-Valle, Miguel Angel; Arechavaleta-Velasco, Fabian

    2008-01-01

    Human chorionic gonadotropin (hCG) is a placental hormone essential for the maintenance of pregnancy. Previous studies have shown a G to A transition in exon 3 of the hCGbeta gene 5, which changes the naturally occurring valine to methionine in codon 79. The frequency of this transition varies among different ethnic groups, being high in USA women, and less common, or absent, in various European populations. The purpose of the present study was to determine the frequency of the betaV79M allelic variant of the beta-subunit of hCG in a Mexican population, and to compare this frequency with those found in other ethnic groups. Placental DNA from 161 pregnant Mexican women was genotyped for the betaV79M by polymerase chain reaction (PCR)-restriction fragments length polymorphism analysis. No polymorphic betaV79M alleles were identified in the population studied. The allele and genotypic frequencies of betaV79M polymorphism in Mexican Mestizo women were significantly different from those reported for the US population, but not from five different European populations. In contrast to what has been found in women from the USA, it seems that the hCGbeta V79M polymorphism is absent or extremely rare in Mexican Mestizo women.

  8. Hyperhomocysteinemia inhibits satellite cell regenerative capacity through p38 alpha/beta MAPK signaling.

    PubMed

    Veeranki, Sudhakar; Lominadze, David; Tyagi, Suresh C

    2015-07-15

    Chronic failure in maintenance and regeneration of skeletal muscles leads to lower muscle mass (sarcopenia), muscle weakness, and poor response to injury. Evidence suggests that aberrant p38 MAPK signaling undermines the repair process after injury in aged mice. Previous studies have shown that hyperhomocysteinemia (HHcy) has been associated with muscle weakness and lower than normal body weights. However, whether or not HHcy condition also compromises skeletal muscle regenerative capabilities is not clear. In the current study, we show that CBS-/+ mice, a model for HHcy condition, exhibited compromised regenerative function and cell proliferation upon injury. However, there was no significant difference in Pax7 expression levels in the satellite cells from CBS-/+ mouse skeletal muscles. Interestingly, the satellite cells from CBS-/+ mice not only exhibited diminished in vitro proliferative capabilities, but also there was heightened oxidative stress. In addition, there was enhanced p38 MAPK activation as well as p16 and p21 expression in the CBS-/+ mouse satellite cells. Moreover, the C2C12 myoblasts also exhibited higher p38 MAPK activation and p16 expression upon treatment with homocysteine in addition to enhanced ROS presence. Tissue engraftment potential and regeneration after injury were restored to some extent upon treatment with the p38-MAPK inhibitor, SB203580, in the CBS-/+ mice. These results together suggest that HHcy-induced diminished satellite cell proliferation involves excessive oxidative stress and p38 MAPK signaling. Our study further proposes that HHcy is a potential risk factor for elderly frailty, and need to be considered as a therapeutic target while designing the alleviation interventions/postinjury rehabilitation measures for adults with HHcy.

  9. Two different satellite DNAs in Beta vulgaris L.: evolution, quantification and distribution in the genus.

    PubMed

    Santoni, S; Bervillé, A

    1992-09-01

    Two highly repeated EcoRI (0.45 × 10(6)) and BamHI (0.17 × 10(6)) fragments per haploid genome were found in sugar beet genomic DNA. Both fragments were located by 6% acrylamide-gel electrophoresis, purified and cloned in pUC18. Four of the inserts corresponding to each family were chosen for further study. Both fragment families display the main characteristics of the satellite DNA of animals and plants. The EcoRI and BamHI fragment families are arranged in long tandem arrays. Fragments of the EcoRI family (pBVE) were analyzed. They vary both in sequence and in length (158-160 nt) in comparison with the consensus sequence of 159nt. Both families are A-T rich; pBVE is 59% rich while pBVB is 69% rich. The BVESAT family is present in all the members of the section Vulgares. It is conserved in the section Procumbentes with 80% homology and the same length, but is not detectable in the Corollinae. The sequence variation rate and the variation in length (330±5 nt) are of the same order in comparison with those of the BVESAT family. However, the BVBSAT family is present in species of the section Vulgares only. As regards other plant satellite DNAs, the BVESAT family shares homology with Allium cepa satellite DNA, with three of the yeast centromeric sequences, and with three Arabidopsis thaliana sequences. The BVBSAT family is unique to the Vulgares and does not share any homology with other plant or animal satellite DNAs sequences so far.

  10. [The mechanism of beta-receptor desensitization in human myometrial culture cell].

    PubMed

    Okamura, Y; Oku, M; Fujii, E; Otsuki, T; Adachi, S; Morimoto, K

    1995-01-01

    Beta-adrenoceptor desensitization is considered to be primarily due to phosphorylation of receptors by protein kinase A (PKA) and beta-adrenaline receptor kinase (beta-ARK) and sequestration of receptors themselves. But in the human uterine muscle, the desensitization mechanism has been evaluated only as a phenomenon, and there are few studies on its mechanism. We evaluated cAMP production by beta-agonist and changes in the number of beta-receptors in cultured human myometrial cells. Uterine muscle cell were obtained from patients with benign disease before menopause and cultured. 1) At the confluent stage, dl-Isoproterenol Hydrochloride (ISP) was added under various conditions, and the intracellular cAMP concentration was determined by EIA. 2) After the addition of ISP (10(-6) M), plates were incubated at 37 degrees C, and beta-AR on the cell membrane surface (S beta-AR) and total beta-AR (T beta-AR) was measured in a binding assay with 125I-pindolol. The production of cAMP dose-dependently increased 30 minutes after the addition of ISP at 10(-6) M or higher, but rapidly decreased thereafter. T beta-AR was similar in the cells treated with ISP (10(-6) M) and the untreated cells. On the other hand, S beta-AR decreased by about 50% in the ISP treated cells. These result suggest the desensitization of beta-AR in human uterine muscle, and the involvement of the sequestration mechanism as its cause.

  11. Reduced expression of immunoreactive beta2-adrenergic receptor protein in human myometrium with labor.

    PubMed

    Chanrachakul, Boonsri; Matharoo-Ball, Balwir; Turner, Anita; Robinson, Graham; Broughton-Pipkin, Fiona; Arulkumaran, Sabaratnam; Khan, Raheela N

    2003-10-01

    A considerable body of evidence exists suggesting that the beta(2)-adrenergic receptor (beta(2)-AR) mediates uterine relaxation. However, little information exists on the localization, distribution, or expression of beta(2)-ARs in the human myometrium during the nonpregnant to labor transition. We have used immunochemical methods to investigate beta(2)-AR localization and expression in the nonpregnant, term pregnant, and term parturient uterus. Myometrial biopsies were obtained from 1) nonpregnant, menstruating women undergoing hysterectomy; 2) singleton term pregnant women undergoing elective cesarean section before the onset of labor; or 3) singleton term pregnant women undergoing emergency cesarean section after spontaneous labor. Tissues were processed for immunohistochemistry, immunofluorescence, and Western blotting and a primary polyclonal antibody specific to the human beta(2)-AR to identify immunoreactive myometrial beta(2)-AR. Protein levels were subsequently quantified by densitometry relative to rat brain protein. Immunohistochemistry and immunofluorescence demonstrated the presence of beta(2)-AR predominantly at the plasma membrane and also in the cytosol of myometrial cells. A 2-fold decrease in protein levels of the beta(2)-AR was apparent in the myometrium of labor compared with that of nonpregnant and pregnant nonlaboring women (P < 0.05). These results demonstrate that down-regulation of beta(2)-AR protein with labor may constitute a contributory mechanism by which uterine quiescence is removed at term.

  12. Coronal magnetic field and the plasma beta determined from radio and multiple satellite observations

    NASA Astrophysics Data System (ADS)

    Iwai, Kazumasa; Shibasaki, Kiyoto; Nozawa, Satoshi; Takahashi, Takuya; Sawada, Shinpei; Kitagawa, Jun; Miyawaki, Shun; Kashiwagi, Hirotaka

    2014-12-01

    We derived the coronal magnetic field, plasma density, and temperature from the observation of polarization and intensity of radio thermal free-free emission using the Nobeyama Radioheliograph (NoRH) and extreme ultraviolet (EUV) observations. We observed a post-flare loop on the west limb on 11 April 2013. The line-of-sight magnetic field was derived from the circularly polarized free-free emission observed by NoRH. The emission measure and temperature were derived from the Atmospheric Imaging Assembly (AIA) onboard Solar Dynamics Observatory (SDO). The derived temperature was used to estimate the emission measure from the NoRH radio free-free emission observations. The derived density from NoRH was larger than that determined using AIA, which can be explained by the fact that the low-temperature plasma is not within the temperature coverage of the AIA filters used in this study. We also discuss the other observation of the post-flare loops by the EUV Imager onboard the Solar Terrestrial Relations Observatory (STEREO), which can be used in future studies to reconstruct the coronal magnetic field strength. The derived plasma parameters and magnetic field were used to derive the plasma beta, which is a ratio between the magnetic pressure and the plasma pressure. The derived plasma beta is about 5.7 × 10-4 to 7.6 × 10-4 at the loop top region.

  13. Binding of radioiodinated human. beta. -endorphin to serum proteins from rats and humans, determined by several methods

    SciTech Connect

    Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

    1985-10-07

    Binding of immunoreactive radioiodinated human ..beta..-endorphin (/sup 125/I-..beta..-EP) to rat serum was demonstrated by gel filtration of /sup 125/I-..beta..-EP in pooled rat serum on Sephadex G-200. Two radioactive peaks associated with proteins eluted from the column. The first peak eluted at the void volume containing lipoproteins, ..cap alpha../sub 2/- and ..beta../sub 2/-macroglobulins, and the second peak at the fraction of albumin. Binding of /sup 125/I-..beta..-EP to albumin was directly proved by gel filtration of /sup 125/I-..beta..-EP in buffer containing 4% human serum albumin on Sephadex G-200. Equilibrium dialysis was not applicable to investigating the interaction of /sup 125/I-..beta..-EP with serum proteins, because of the intense nonspecific adsorption to the semi-permeable membrane and the degradation of the peptide during dialysis. Therefore, in order to quantitatively evaluate the binding of /sup 125/I-..beta..-EP in sera from rats and humans, the authors utilized four other methods (ultrafiltration, charcoal adsorption, polyethylene glycol precipitation and equilibrium gel filtration). These methods corresponded well with each other and indicated 35-44% binding of /sup 125/I-..beta..-EP in rat serum. Binding of /sup 125/I-..beta..-EP in normal human serum was 36%, determined by ultrafiltration. Serum protein binding of /sup 125/I-..beta..-EP was concentration independent over the concentration range studied (1-1000 nM). 23 references, 4 figures, 1 table.

  14. Nonsense mutations in the human. beta. -globin gene affect mRNA metabolism

    SciTech Connect

    Baserga, S.J.; Benz, E.J. Jr. )

    1988-04-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human {alpha}- and {beta}-globin genes. Studies on mRNA isolated from patients with {beta}{sup 0}-thalassemia have shown that for both the {beta}-17 and the {beta}-39 mutations less than normal levels of {beta}-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human {beta}-globin mRNA). In vitro studies using the cloned {beta}-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human {beta}-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation.

  15. Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

    PubMed Central

    Chalmers, J J; Kim, E; Telford, J N; Wong, E Y; Tacon, W C; Shuler, M L; Wilson, D B

    1990-01-01

    The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble. Images PMID:2155574

  16. Mouse model to study human A beta2M amyloidosis: generation of a transgenic mouse with excessive expression of human beta2-microglobulin.

    PubMed

    Zhang, Pengyao; Fu, Xiaoying; Sawashita, Jinko; Yao, Junjie; Zhang, Beiru; Qian, Jinze; Tomozawa, Hiroshi; Mori, Masayuki; Ando, Yukio; Naiki, Hironobu; Higuchi, Keiichi

    2010-06-01

    Patients on long-term hemodialysis can develop dialysis-related amyloidosis (DRA) due to deposition of beta(2)-microglobulin (beta(2)m) into amyloid fibrils (Abeta(2)M). Despite intensive biochemical studies, the pathogenesis of amyloid deposition in DRA patients remains poorly understood. To elucidate the mechanisms that underlie Abeta(2)M fibril formation in DRA, we generated transgenic mice that overexpress human beta(2)m protein in a mouse beta(2)m gene knockout background (hB2MTg(+/+) mB2m(+/+)). The hB2MTg(+/+)mB2m(-/-) mice express a high level of human beta(2)m protein in many tissues as well as a high plasma beta(2)m concentration (192.8 mg/L). This concentration is >100 times higher than that observed in healthy humans and >4 times higher than that detected in patients on dialysis. We examined spontaneous and amyloid fibril-induced amyloid deposition in these mice. Amyloid deposition of beta(2)m protein was not observed in aged or amyloid fibril injected animals. However, mouse senile apolipoprotein A-II amyloidosis (AApoAII) was detected, particularly in the joints of mice that were injected with AApoAII amyloid fibrils. This study demonstrates that this mouse model could be valuable in studying the components and conditions that promote DRA, and indicates that high plasma concentrations of hbeta(2)m as well as seeding with pre-existing amyloid fibrils may not be sufficient to induce Abeta(2)M.

  17. A monopartite begomovirus-associated DNA beta satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection.

    PubMed

    Saeed, Muhammad; Zafar, Yusuf; Randles, John W; Rezaian, M Ali

    2007-10-01

    DNA beta is a circular single-stranded satellite DNA which co-infects with certain monopartite helper begomoviruses to cause economically important diseases, such as cotton leaf curl disease (CLCuD). DNA beta encodes a single protein, betaC1. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection, whereas co-inoculation with DNA A and the DNA beta associated with CLCuD resulted in systemic infection. DNA beta containing a disrupted betaC1 open reading frame (ORF) did not mobilize DNA A systemically. Co-inoculation of plants with DNA A and a construct of the betaC1 ORF, under the control of the cauliflower mosaic virus 35S promoter, resulted in the systemic movement of DNA A. In inoculated tobacco and onion epidermal cells, betaC1 fused to GFP was localized at the cell periphery in association with punctate bodies, around and within the cell nucleus and with the endoplasmic reticulum. It is concluded that heterologous betaC1 protein can replace the movement function of the DNA B of a bipartite begomovirus. Evidence is also provided that tomato leaf curl virus-encoded C4 protein confers the same movement function to ToLCNDV DNA A. The intracellular distribution of betaC1 is consistent with the hypothesis that it has a role in transporting the DNA A from the nuclear site of replication to the plasmodesmatal exit sites of the infected cell. PMID:17872543

  18. Transactivation of vimentin by beta-catenin in human breast cancer cells.

    PubMed

    Gilles, Christine; Polette, Myriam; Mestdagt, Mélanie; Nawrocki-Raby, Béatrice; Ruggeri, Philippe; Birembaut, Philippe; Foidart, Jean-Michel

    2003-05-15

    The cytoplasmic and nuclear redistribution of beta-catenin and the de novo expression of vimentin are frequently involved in the epithelial-to-mesenchymal transition associated with increased invasive/migratory properties of epithelial cells. Because beta-catenin can act as a coactivator of transcription through its binding to the T-cell factor (TCF)/lymphoid enhancer factor 1 transcription factor family, we have explored the possibility that beta-catenin/TCF could directly transactivate vimentin. We first compared vimentin expression in relation with the localization of beta-catenin in eight breast cancer cell lines displaying various degrees of invasiveness and in a model of cell migration using human mammary MCF10A cells. We could thus show a cytoplasmic and/or nuclear distribution of beta-catenin in invasive/migratory cells expressing vimentin, but not in noninvasive/stationary vimentin-negative cell lines. In addition, the human vimentin promoter was found to be up-regulated by beta-catenin and TCF-4 cotransfection. Varying with the cellular background, a diminution of this up-regulation was observed when the putative beta-catenin/TCF binding site of the vimentin promoter was mutated. Our results therefore demonstrate that the vimentin promoter is a target of the beta-catenin/TCF pathway and strongly suggest an implication of this regulation in epithelial cell migration/invasion.

  19. Role of human GABA(A) receptor beta3 subunit in insecticide toxicity.

    PubMed

    Ratra, G S; Kamita, S G; Casida, J E

    2001-05-01

    The gamma-aminobutyric acid type A (GABA(A)) receptor is the target for the major insecticides alpha-endosulfan, lindane, and fipronil and for many analogs. Their action as chloride channel blockers is directly measured by binding studies with [(3)H]ethynylbicycloorthobenzoate ([(3)H]EBOB). This study tests the hypothesis that GABA(A) receptor subunit composition determines the sensitivity and selectivity of insecticide toxicity. Human receptor subtypes were expressed individually (alpha1, alpha6, beta1, beta3, and gamma2) and in combination in insect Sf9 cells. Binding parameters were similar for [(3)H]EBOB in the beta3 homooligomer, alpha1beta3gamma2 heterooligomer, and native brain membranes, but toxicological profiles were very different. Surprisingly, alpha-endosulfan, lindane, and fipronil were all remarkably potent on the recombinant beta3 homooligomeric receptor (IC50 values of 0.5-2.4 nM), whereas they were similar in potency on the alpha1beta3gamma2 subtype (IC50 values of 16-33 nM) and highly selective on the native receptor (IC50 values of 7.3, 306, and 2470 nM, respectively). The selectivity order for 29 insecticides and convulsants as IC50 ratios for native/beta3 or alpha1beta3gamma2/beta3 was as follows: fipronil > lindane > 19 other insecticides including alpha-endosulfan and picrotoxinin > 4 trioxabicyclooctanes and dithianes (almost nonselective) > tetramethylenedisulfotetramine, 4-chlorophenylsilatrane, or alpha-thujone. Specificity between mammals and insects at the target site (fipronil > lindane > alpha-endosulfan) paralleled that for toxicity. Potency at the native receptor is more predictive for inhibition of GABA-stimulated chloride uptake than that at the beta3 or alpha1beta3gamma2 receptors. Therefore, the beta3 subunit contains the insecticide target and other subunits differentially modulate the binding to confer compound-dependent specificity and selective toxicity.

  20. Correlation between beta- and alpha-adrenergic receptor concentrations in human placenta.

    PubMed

    Falkay, G; Melis, K; Kovács, L

    1994-05-01

    alpha 2- and beta-adrenergic receptors in human placental membranes have been investigated using the radioligands [3H]-RX 821002 and [3H]-dihydroalprenolol, respectively. The specific binding of the alpha 2-adrenoceptor antagonist RX 821002 confirms the presence of an alpha 2-adrenoceptor in the human placenta, which has been characterized previously with [3H]-rauwolscine. The major finding presented here is a correlation between the alpha 2- and beta-adrenergic receptor concentrations (r = 0.765) in the human placenta at term. It is suggested that the alpha 2/beta adrenoceptor balance may play an important role in regulation of the vascular bed of the placenta. Determination of the alpha 2/beta ratio may help towards an understanding of the contractility of the placental vascular muscles.

  1. Isolation and characterization of cDNA clones for human erythrocyte. beta. -spectrin

    SciTech Connect

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-11-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical ..cap alpha.. (M/sub r/ 240,000) and ..beta.. (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific ..beta..-spectrin cDNA clone that encodes parts of the ..beta..-9 through ..beta..-12 repeat segments. This cDNA was used as a hybridization probe to assign the ..beta..-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte ..beta..-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the ..beta..-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

  2. Human {beta}2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    SciTech Connect

    Wewer, U.M.; Durkin, M.E.; Albrechtsen, R.

    1994-11-15

    Overlapping cDNA clones that encode the full-length human laminin {beta}2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature {beta}2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human {beta}2 chain is predicted to have all of the seven structural domains typical of the {beta} chains of laminin, including the short cysteine-rich {alpha} region. The amino acid sequence of human {beta}2 chain showed 86.1% sequence identity to the rat {beta}2 chain, 50.0% to human {beta}1 chain, and 36.3% to the human {beta}3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the {beta}2 chain of laminin purified from human amniotic basement membrane matched the sequence predicted from the cDNA, confirming that the cDNA encodes human {beta}2 laminin. The cDNA was used to assign the gene (LAMB2) to human chromosome 3p21 by in situ hybridization. It is not linked to genes for human laminin chains {alpha}1, {beta}1, and {gamma}1 or other known laminin genes. Immunostaining showed that the {beta}2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous {beta}1 chain is confined to the subendothelial basement membranes. The {beta}2 chain was found in the basement membranes of ovarian carcinomas but not colon carcinomas. These results indicate that the expression of the {beta}2 chain gene is tightly regulated in normal human tissues and in disease. 43 refs., 6 figs., 1 tab.

  3. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    SciTech Connect

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  4. Transgenic mice over-expressing human beta-amyloid have functional nicotinic alpha 7 receptors.

    PubMed

    Spencer, J P; Weil, A; Hill, K; Hussain, I; Richardson, J C; Cusdin, F S; Chen, Y H; Randall, A D

    2006-02-01

    A potentially major factor in the development of Alzheimer's disease is the enhanced production of soluble beta-amyloid peptide fragments amyloid beta peptide(1-40) and amyloid beta peptide(1-42). These amyloid peptides are generated by cleavage of the amyloid-precursor protein and aggregate spontaneously to form amyloid plaques, which are a classical pathological hallmark in Alzheimer's disease. Although the precise mechanisms are unknown, it is widely believed that amyloid peptides initiate the degenerative process, resulting in subsequent cognitive decline. One interaction of amyloid beta peptide that may contribute to an impairment of cognition is its high affinity binding to the alpha 7 nicotinic receptor; a receptor shown to be important for cognition in a number of studies. There is some controversy, however, whether amyloid beta peptide inhibits or activates this receptor. We have cloned and stably expressed the human alpha 7 receptor and investigated its interaction with amyloid beta peptide using patch clamp electrophysiology. Human alpha 7 was activated in a concentration-dependent fashion by nicotine, acetylcholine and choline and potently inhibited by methyllycaconitine citrate. The responses were inwardly rectifying and exhibited rapid activation, desensitization and deactivation. Amyloid beta peptide(1-42) antagonized human alpha7 responses in a partially reversible fashion; no agonist effects of amyloid beta peptide(1-42) were detected. A similar inhibition of mouse alpha 7 was also observed. In addition, we have assessed the function of native alpha 7 receptors in hippocampal slices prepared from transgenic mice that over-express human amyloid. Despite this clear inhibition of recombinant receptors, hippocampal GABAergic interneurones in slices from beta-amyloid over-expressing mice still possess alpha 7 receptor-mediated currents.

  5. Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.

    PubMed

    Baroni, M G; Cavallo, M G; Mark, M; Monetini, L; Stoehrer, B; Pozzilli, P

    1999-04-01

    Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.

  6. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  7. Beta- and gamma-range human lower limb corticomuscular coherence

    PubMed Central

    Gwin, Joseph T.; Ferris, Daniel P.

    2012-01-01

    Coherence between electroencephalography (EEG) recorded on the scalp above the motor cortex and electromyography (EMG) recorded on the skin of the limbs is thought to reflect corticospinal coupling between motor cortex and muscle motor units. Beta-range (13–30 Hz) corticomuscular coherence has been extensively documented during static force output while gamma-range (31–45 Hz) coherence has been linked to dynamic force output. However, the explanation for this beta-to-gamma coherence shift remains unclear. We recorded 264-channel EEG and 8-channel lower limb EMG while eight healthy subjects performed isometric and isotonic, knee, and ankle exercises. Adaptive mixture independent component analysis (AMICA) parsed EEG into models of underlying source signals. We computed magnitude squared coherence between electrocortical source signals and EMG. Significant coherence between contralateral motor cortex electrocortical signals and lower limb EMG was observed in the beta- and gamma-range for all exercise types. Gamma-range coherence was significantly greater for isotonic exercises than for isometric exercises. We conclude that active muscle movement modulates the speed of corticospinal oscillations. Specifically, isotonic contractions shift corticospinal oscillations toward the gamma-range while isometric contractions favor beta-range oscillations. Prior research has suggested that tasks requiring increased integration of visual and somatosensory information may shift corticomuscular coherence to the gamma-range. The isometric and isotonic tasks studied here likely required similar amounts of visual and somatosensory integration. This suggests that muscle dynamics, including the amount and type of proprioception, may play a role in the beta-to-gamma shift. PMID:22973219

  8. Augmentation of endotoxin fever by recombinant human beta interferon in rabbits.

    PubMed Central

    Kawasaki, H; Moriyama, M; Tanaka, A

    1987-01-01

    Nonpyrogenic amounts of endotoxin (0.1 to 1 ng/kg), hardly detectable by conventional Limulus amoebocyte lysate tests, could produce a fever of around 1 degree C when injected with a nonpyrogenic dose (6 X 10(5) U/kg) of recombinant human beta interferon (IFN-beta) in rabbits. Release of endogenous IFN and tumor necrosis factor by endotoxin was also dramatically increased by recombinant human IFN-beta, and their levels in the blood were closely correlated with the increase of body temperature. These data suggest, if the synergism between IFN and endotoxin also operates in the homologous system (human IFN-human cells), that contaminating endotoxin in IFNs, even if not detectable by Limulus amoebocyte lysate test, can contribute to IFN fever to a considerable extent in humans. PMID:2437032

  9. Mapping the human footprint from satellite measurements in Japan

    NASA Astrophysics Data System (ADS)

    Yang, Fan; Matsushita, Bunkei; Yang, Wei; Fukushima, Takehiko

    2014-02-01

    Due to increasing global urbanization and climate change, the quantification of "human footprints" has become an urgent goal in the fields of biodiversity conservation and regional environment management. A human footprint is defined as the impact of a particular human activity on the Earth's surface, which can be represented mainly by impervious surfaces (related to industry and urbanization) and cropland (related to agriculture). Here we present a method called sorted temporal mixture analysis with post-classification (STMAP) for mapping impervious surfaces and cropland simultaneously at the subpixel level to fill the demand for precise human footprint information on a national scale. The STMAP method applies a four-endmember sorted temporal mixture analysis to provide the initial fractions of evergreen forests, deciduous forests, cropland, and impervious surfaces as a first step. Endmembers are selected from the sorted temporal profiles of the MODIS-normalized difference vegetation index (NDVI), as guided by a principal component analysis. The yearly maximum land surface temperatures and averaged stable nighttime light are then statistically analyzed to provide the thresholds for post-classification to further separate cropland from deciduous forest and bare land from impervious surface. As the four outputs of STMAP, the fractions of forest, cropland, impervious surfaces and bare land are derived. We used the reference maps of impervious surfaces and cropland obtained from the Landsat/TM and ALOS precise land-use/land-cover map at the subpixel level to evaluate the performance of the proposed method, respectively. Historical satellite images with high spatial resolution were used to further evaluate the cropland results derived with the STMAP method. The results showed that the STMAP method has promising accuracy for estimating impervious surfaces and cropland in Japan. The root mean square errors obtained with the STMAP method were 6.3% for the estimation of

  10. beta-Sitosterol inhibits HT-29 human colon cancer cell growth and alters membrane lipids.

    PubMed

    Awad, A B; Chen, Y C; Fink, C S; Hennessey, T

    1996-01-01

    The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary phytosterol on the growth of HT-29 cells, a human colon cancer cell line. In addition, the incorporation of this phytosterol into cellular membranes and how this might influence the lipid composition of the membranes were investigated. Tumor cells were grown in DMEM containing 10% FBS and supplemented with sterols (cholesterol or beta-sitosterol) at final concentrations up to 16 microM. The sterols were supplied to the media in the form of sterol cyclodextrin complexes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin. The sterol to cyclodextrin molar ratio was maintained at 1:300. The study indicated that 8 and 16 microM beta-sitosterol were effective at cel growth inhibition as compared to cholesterol or to the control (no sterol supplementation). After supplementation with 16 microM beta-sitosterol for 9 days, cell growth was only one-third that of cells supplemented with equimolar concentration of cholesterol. No effect was observed on total membrane phospholipid concentration. At 16 microM beta-sitosterol supplementation, membrane cholesterol was reduced by 26%. Cholesterol supplementation resulted in a significant increase in the cholesterol/phospholipid ratio compared to either beta-sitosterol supplemented cells or controls. There was a 50% reduction in membrane sphingomyelin (SM) of cells grown in 16 microM beta-sitosterol. Additional changes were observed in the fatty acid composition of minor phospholipids of beta-sitosterol supplemented cells, such as SM, phosphatidylserine (PS), and phosphatidylinositol (PI). Only in the case of PI, was there an effect of these fatty acid changes on the unsaturation index, beta-sitosterol incorporation resulted in an increase in the U.I. It is possible that the observed growth inhibition by beta-sitosterol may be mediated through the influence of signal transduction pathways that involve membrane phospholipids.

  11. Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human. beta. -hexosaminidase

    SciTech Connect

    O'Dowd, B.F.; Cumming, D.A.; Gravel, R.A.; Mahuran, D.

    1988-07-12

    Human ..beta..-hexosaminidase is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM/sub 2/ ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure ..cap alpha..(..beta../sub a/..beta../sub b/), and hexosaminidase B, 2(..beta../sub a/..beta../sub b/). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the ..beta../sub b/f chain, and one non concanavalin A binding glycopeptide, localized to the ..beta../sub a/ chain, were found associated with the ..beta..-subunit in both hexosaminidase A and hexosaminidase B. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. The unique glycopeptide associated with the ..beta../sub a/ chain contained a single GlcNAc residue. Thus all three mature polypeptides comprising the ..cap alpha.. and ..beta.. subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the ..cap alpha.. chain they found only one possible site for in vivo phosphorylation. In the ..beta.. it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an ..cap

  12. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil.

    PubMed

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan

    2009-09-01

    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil.

  13. The exon-intron organization of the human erythroid [beta]-spectrin gene

    SciTech Connect

    Amin, K.M.; Forget, B.G. ); Scarpa, A.L.; Curtis, P.J. ); Winkelmann, J.C. )

    1993-10-01

    The human erythrocyte [beta]-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid [beta]-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the [beta]-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present [beta]-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat [beta]10 is encoded by 4 exons. No single position of an intron in the [beta]-spectrin gene is conserved between any of the 17 [beta]-spectrin and 22 [alpha]-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5[prime] donor splice-site sequence begins with GC. The [beta]-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid [beta]-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp. 55 refs., 3 figs., 3 tabs.

  14. Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Hashiro, M.; Yoshimasa, H.; Yoshikawa, K. )

    1990-10-01

    The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of (3H)thymidine incorporation. The decrease of (3H)thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions.

  15. Mechanosensitivity of human osteosarcoma cells and phospholipase C {beta}2 expression

    SciTech Connect

    Hoberg, M. . E-mail: Maik.Hoberg@med.uni-tuebingen.de; Gratz, H.-H.; Noll, M.; Jones, D.B.

    2005-07-22

    Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 {mu}strains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC {beta}1, {beta}3, {gamma}1, {gamma}2, and {delta}1 in all cells, but PLC {beta}2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC {beta}2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC {beta}1 in U-2/OS cells by 35% and PLC {beta}2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC {beta}2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

  16. Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.

    PubMed Central

    Ruddon, R. W.; Sherman, S. A.; Bedows, E.

    1996-01-01

    There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects. PMID:8844836

  17. Human pulmonary acinar aplasia: reduction of transforming growth factor-beta ligands and receptors.

    PubMed

    Chen, M F; Gray, K D; Prentice, M A; Mariano, J M; Jakowlew, S B

    1999-07-01

    Pulmonary hypoplasia has been found in the human neonatal autopsy population and has been attributed to an alteration in epithelial-mesenchymal interactions during development of the lung. Pulmonary acinar aplasia is a very rare and severe form of pulmonary hypoplasia. The transforming growth factor-betas (TGF-beta) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells and are expressed in developing organs including the lung; their tissue distribution patterns have possible significance for signaling roles in many epithelial-mesenchymal interactions. Here, we report our examination of TGF-beta in the lungs of a term female infant diagnosed with pulmonary acinar aplasia whose autopsy revealed extremely hypoplastic lungs with complete absence of alveolar ducts and alveoli. Immunohistochemical and in situ hybridization analyses were used to localize and measure the proteins and mRNA, respectively, for TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta type I and type II receptors (TGF-beta RI and RII) in formalin-fixed and paraffin-embedded sections of these hypoplastic lungs and normal lungs. Immunostaining for TGF-beta1, TGF-beta2, and TGF-beta RI and RII was significantly lower in the bronchial epithelium and muscle of the hypoplastic lungs than in normal lungs, whereas no difference was detected in staining for other proteins including Clara cell 10-kD protein, adrenomedullin, hepatocyte growth factor/scatter factor, and hepatocyte growth factor receptor/Met in the hypoplastic and normal lungs or in the liver and kidneys of this infant compared with normal liver and kidney. In addition, in situ hybridization showed that TGF-beta1 and TGF-beta RI transcripts were considerably reduced in the bronchial epithelium of the hypoplastic lung compared with normal lung. These results show that there is a selective reduction of TGF-beta in pulmonary acinar aplasia and suggest that the signaling action of TGF-beta in epithelial

  18. Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

    SciTech Connect

    Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A. )

    1990-08-01

    Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC.

  19. No evidence of the existence of beta adrenoceptors in human sperm using radioligand binding technique.

    PubMed

    Falkay, G; Bozóki, Z; Szöllösi, J; Kovács, L

    1989-01-01

    The investigations were carried out on 20 freshly ejaculated and pooled samples of human semen with normozoospermia and motility of 70-80%. In the present study no difference was found between total and non-specific binding to semen membranes using [3H]-dihydroalprenolol (DHA) as a radioligand. Our results suggest that beta adrenoceptors could not be found in human sperm.

  20. Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming human beta cell function

    PubMed Central

    Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M. Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat; Ricordi, Camillo; Roper, Stephen D.; Berggren, Per-Olof; Caicedo, Alejandro

    2011-01-01

    Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. PMID:21685896

  1. MISR Satellite Observations of Aerosol Types Affecting Human Health

    NASA Astrophysics Data System (ADS)

    Kalashnikova, O. V.; Franklin, M.; Garay, M. J.; Diner, D. J.

    2015-12-01

    Ground-based observations of pollutants and concentrations of particulate matter (PM), that includes small particles designated PM2.5 and dust-dominated PM10, are the gold standard in studies of environmental impacts on human health. However, because monitoring stations are costly, they typically provide only limited spatial coverage, especially in rural and remote areas. We will demonstrate how data from the Multi-angle Imaging SpectroRadiometer (MISR) instrument that has been flying on NASA's Terra Earth Observing System satellite since early 2000 can be used to provide estimates of surface PM types. The current MISR operational aerosol retrieval uses a combination of multi-spectral and multi-angle data to retrieve aerosol optical depth (AOD) and particle property information (including dust AOD) globally at 17.6 km spatial resolution. Using the same algorithm with data collected in all 36-channels at 275 m resolution (Local Mode), which is available over greater Los Angeles area, and also was activated during 2013 DISCOVER-AQ California field campaign, high-resolution 4.4 km aerosol retrievals were performed in addition to the standard 17.6 km retrievals. The 4.4 km spatial resolution of the PM information data is fine enough to be able to resolve local differences in PM loading that may be important for understanding regional health effects of pollution in the region. In particular, we demonstrate that MISR high-resolution AOD retrievals are in better agreement with ground-based aerosol observations and reveal more details about the aerosol spatial variability compared to the MISR standard 17.6 km product. Then we will discuss techniques and show examples of the application of high-resolution MISR data to provide estimates of surface PM for the greater Los Angeles area in 2008 and for California San Joaquin Valley during the 2013 DISCOVER-AQ field campaign. Finally, we will discuss future NASA instruments that will provide new information allowing for better

  2. Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines.

    PubMed Central

    Damstrup, L.; Rygaard, K.; Spang-Thomsen, M.; Skovgaard Poulsen, H.

    1993-01-01

    A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGF beta-receptors and also produce TGF beta mRNAs. Images Figure 3 Figure 4 PMID:8388229

  3. Human T cells expressing V beta 8 do not predominantly recognize DR2 alloantigen.

    PubMed

    Christmas, S E; Brew, R; Crosby, I; Gecim, I E; Sells, R A

    1992-06-01

    A panel of seven monoclonal antibodies recognizing human T-cell antigen receptor (TcR) V alpha or V beta subsets has been used to measure TcR gene expression in peripheral blood lymphocytes and mixed lymphocyte culture responses (MLR) between DR2- and DR2+ (DRw15+) donors. There were no significant differences between DR2- and DR2+ donors in per cent T cells in fresh peripheral blood labelled with any of these antibodies, which included an antibody recognizing V beta 8. This indicates strongly that increased negative selection of V beta 8+ T cells does not occur in DR2+ compared with DR2- individuals. In MLR between DR2- and DR2+ donors the only significant change compared with fresh peripheral lymphocytes was that T cells expressing V beta 5.1 were decreased in DR2- lymphocyte populations responding to DR2 alloantigen. No changes in levels of V beta 8+ T cells were detected in MLR between DR2- and DR2+ donors. This suggests that V beta 8+ T cells are not predominantly reactive against DR2 (DRw15). The data support the concept that alloreactivity against a single class II major histocompatibility complex (MHC) mismatch is mediated by T cells expressing a range of different TcR V beta molecules.

  4. Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

    PubMed

    Rao, M J; Malavalli, A; Manjula, B N; Kumar, R; Prabhakaran, M; Sun, D P; Ho, N T; Ho, C; Nagel, R L; Acharya, A S

    2000-07-28

    Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of

  5. Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

    SciTech Connect

    Kyle, J.W.; Vogler, C.; Hoffmann, J.W.; Sly, W.S. ); Birkenmeier, E.H.; Gwynn, B. )

    1990-05-01

    The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{sup mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.

  6. Comparison of the canine and human acid {beta}-galactosidase gene

    SciTech Connect

    Ahern-Rindell, A.J.; Kretz, K.A.; O`Brien, J.S.

    1996-05-17

    Several canine cDNA libraries were screened with human {beta}-galactosidase cDNA as probe. Seven positive clones were isolated and sequenced yielding a partial (2060 bp) canine {beta}-galactosidase cDNA with 86% identity to the human {beta}-galactosidase cDNA. Preliminary analysis of a canine genomic library indicated conservation of exon number and size. Analysis by Northern blotting disclosed a single mRNA of 2.4 kb in fibroblasts and liver from normal dogs and dogs affected with GM1 gangliosidosis. Although incomplete, these results indicate canine GM1 gangliosidosis is a suitable animal model of the human disease and should further efforts to devise a gene therapy strategy for its treatment. 20 refs., 2 figs., 1 tab.

  7. CCAAT/enhancer binding protein beta protects muscle satellite cells from apoptosis after injury and in cancer cachexia

    PubMed Central

    Marchildon, F; Fu, D; Lala-Tabbert, N; Wiper-Bergeron, N

    2016-01-01

    CCAAT/enhancer binding protein beta (C/EBPβ), a transcription factor expressed in muscle satellite cells (SCs), inhibits the myogenic program and is downregulated early in differentiation. In a conditional null model in which C/EBPβ expression is knocked down in paired box protein 7+ (Pax7+) SCs, cardiotoxin (CTX) injury is poorly repaired, although muscle regeneration is efficient in control littermates. While myoblasts lacking C/EBPβ can differentiate efficiently in culture, after CTX injury poor regeneration was attributed to a smaller than normal Pax7+ population, which was not due to a failure of SCs to proliferate. Rather, the percentage of apoptotic SCs was increased in muscle lacking C/EBPβ. Given that an injury induced by BaCl2 is repaired with greater efficiency than controls in the absence of C/EBPβ, we investigated the inflammatory response following BaCl2 and CTX injury and found that the levels of interleukin-1β (IL-1β), a proinflammatory cytokine, were robustly elevated following CTX injury and could induce C/EBPβ expression in myoblasts. High levels of C/EBPβ expression in myoblasts correlated with resistance to apoptotic stimuli, while its loss increased sensitivity to thapsigargin-induced cell death. Using cancer cachexia as a model for chronic inflammation, we found that C/EBPβ expression was increased in SCs and myoblasts of tumor-bearing cachectic animals. Further, in cachectic conditional knockout animals lacking C/EBPβ in Pax7+ cells, the SC compartment was reduced because of increased apoptosis, and regeneration was impaired. Our findings indicate that the stimulation of C/EBPβ expression by IL-1β following muscle injury and in cancer cachexia acts to promote SC survival, and is therefore a protective mechanism for SCs and myoblasts in the face of inflammation. PMID:26913600

  8. Identification of a recent recombination event within the human beta-globin gene cluster.

    PubMed Central

    Gerhard, D S; Kidd, K K; Kidd, J R; Egeland, J A; Housman, D E

    1984-01-01

    In a detailed study of inheritance of DNA sequence polymorphism in a large reference pedigree, an individual was identified with an apparent genetic recombination event within the human beta-globin gene cluster. Analysis of the haplotypes of relevant individuals within this pedigree suggested that the meiotic crossing-over event is likely to have occurred within a 19.8-kilobase-pair region of the beta-globin gene cluster. Analysis of other DNA markers closely linked to the beta-globin gene cluster--segment 12 of chromosome 11 (D11S12) and loci for insulin, the cellular oncogene c-Ha-ras, and preproparathyroid hormone--confirmed that a crossover event must have occurred within the region of chromosome 11 between D11S12 and the beta-globin gene cluster. It is suggested that the event observed has occurred within a DNA region compatible with recombinational "hot spots" suggested by population studies. PMID:6096866

  9. Magnetic measurements on human erythrocytes: Normal, beta thalassemia major, and sickle

    NASA Astrophysics Data System (ADS)

    Sakhnini, Lama

    2003-05-01

    In this article magnetic measurements were made on human erythrocytes at different hemoglobin states (normal and reduced hemoglobin). Different blood samples: normal, beta thalassemia major, and sickle were studied. Beta thalassemia major and sickle samples were taken from patients receiving lifelong blood transfusion treatment. All samples examined exhibited diamagnetic behavior. Beta thalassemia major and sickle samples showed higher diamagnetic susceptibilities than that for the normal, which was attributed to the increase of membrane to hemoglobin volume ratio of the abnormal cells. Magnetic measurements showed that the erythrocytes in the reduced state showed less diamagnetic response in comparison with erythrocytes in the normal state. Analysis of the paramagnetic component of magnetization curves gave an effective magnetic moment of μeff=7.6 μB per reduced hemoglobin molecule. The same procedure was applied to sickle and beta thalassemia major samples and values for μeff were found to be comparable to that of the normal erythrocytes.

  10. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  11. Human factors dimensions in the evolution of increasingly automated control rooms for near-earth satellites

    NASA Technical Reports Server (NTRS)

    Mitchell, C. M.

    1982-01-01

    The NASA-Goddard Space Flight Center is responsible for the control and ground support for all of NASA's unmanned near-earth satellites. Traditionally, each satellite had its own dedicated mission operations room. In the mid-seventies, an integration of some of these dedicated facilities was begun with the primary objective to reduce costs. In this connection, the Multi-Satellite Operations Control Center (MSOCC) was designed. MSOCC represents currently a labor intensive operation. Recently, Goddard has become increasingly aware of human factors and human-machine interface issues. A summary is provided of some of the attempts to apply human factors considerations in the design of command and control environments. Current and future activities with respect to human factors and systems design are discussed, giving attention to the allocation of tasks between human and computer, and the interface for the human-computer dialogue.

  12. Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

    NASA Technical Reports Server (NTRS)

    Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

    2003-01-01

    CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

  13. Transcription factor binding and spacing constraints in the human beta-actin proximal promoter.

    PubMed Central

    Danilition, S L; Frederickson, R M; Taylor, C Y; Miyamoto, N G

    1991-01-01

    The human beta-actin promoter, including its 5' flanking region and 5' untranslated region, is ubiquitously active in mammalian cells in culture. In this report we investigated the transcriptional activity of, and the protein-DNA interactions that occur within, the proximal region of the human beta-actin promoter. Efficient beta-actin promoter activity in transfected human HeLa cells requires only 114bp of 5' flanking sequences. Two of the cis-actin regulatory elements within this region of the beta-actin promoter, the CCAAT box and proximal CCArGG box, are specific in vitro binding sites for the transcription factors, nuclear factor Y (NF-Y) and serum response factor (p67SRF), respectively. These two elements are required together to stimulate in vivo transcription from the homologous as well as a heterologous promoter. Finally, a particular spatial alignment between the CCAAT box and proximal CCArGG box is required for trans-activation in vivo. The above provides strong evidence for a functional interaction between NF-Y and p67SRF when bound to their respective binding sites in the beta-actin promoter. Images PMID:1762920

  14. Transforming growth factor-beta 1 (TGF-beta1) prevents the age-dependent decrease in bone formation in human osteoblast/implant cultures.

    PubMed

    Zhang, Hai; Aronow, Michael S; Gronowicz, Gloria A

    2005-10-01

    Titanium implants have been extensively used in orthopedic surgery and dentistry. Most of the patients who receive such implants are elderly with a compromised ability to heal and form new bone. By using an in vitro osteoblast/implant culture system, the potency of TGF-beta1 in enhancing mineralization of human osteoblast cultures from elderly subjects was investigated in this study. Primary human osteoblast (HOB) cells obtained from different age group human subjects [Young (Y), Middle (M), and Old (O)] were cultured on Ti alloy (Ti-6Al-4V) disks with or without continuous administration of 0.2 ng/mL TGF-beta1 in the medium for 2 or 4 weeks. TGF-beta1 significantly (p < 0.05) increased calcium content and the size of calcified nodules on implant disks in the O group, but had no effect on the Y or M groups. The number of calcified nodules was not different with or without TGF-beta1 in all age groups. As measured by Northern blot analysis and RT-PCR, TGF-beta1 significantly increased the expression of bone-specific extracellular matrix proteins, including alkaline phosphatase, Type I collagen, bone sialoprotein and osteocalcin, after both 2 and 4 weeks in the O group but not in the Y group. In conclusion, TGF-beta1 enhances mineralization on implant materials of osteoblast cultures from elderly human subjects.

  15. Enhancement of XPG mRNA transcription by human interferon-beta in Cockayne syndrome cells with complementation group B.

    PubMed

    Suzuki, Y; Sugita, K; Suzuki, N; Kita, K; Higuchi, Y; Yamaura, A; Kohno, Y

    1999-01-01

    Human interferon-beta (HuIFN-beta) confers UV-refractoriness in association with increased DNA repair capacity to human cells. We examined the modulation of XPG gene expression by HuIFN-beta in UV-sensitive cells from Cockayne syndrome complementation B (CSB), xeroderma pigmentosum complementation A (XPA) and normal control cells. Northern blot analysis revealed that XPG mRNA was more extensively transcribed in CSB cells treated with HuIFN-beta than in those without HuIFN-beta treatment. XPG mRNA from XPA cells and normal control cells was not markedly transcribed by HuIFN-beta treatment compared to that from CSB cells. The findings suggested that different mechanisms of UV-refractoriness by HuIFN-beta exist between CS and XP. PMID:9864391

  16. Effects of halothane on the human beta-adrenergic receptor of lymphocyte membranes

    SciTech Connect

    Marty, J.; Nivoche, Y.; Nimier, M.; Rocchiccioli, C.; Luscombe, F.; Henzel, D.; Loiseau, A.; Desmonts, J.M.

    1987-12-01

    The effects of halothane on beta-adrenergic receptor antagonist interaction were studied using the membranes of human lymphocytes as a model. Membrane preparations of lymphocytes were obtained from blood samples withdrawn from seven healthy young volunteers. Beta-receptor studies were performed using (-)/sup 125/I iodocyanopindolol (/sup 125/ICP) binding. Non-specific binding was determined in the presence of (-)isoproterenol. Beta-receptor density (Bmax) and the dissociation constant (KD) for /sup 125/ICP were determined from saturation curves. Beta-receptor affinity for agonists evaluated by the IC50 (the concentration of isoproterenol required to inhibit 50% of specific /sup 125/ICP binding) and the dissociation constant (KL) for isoproterenol was established from competition curves. The effect of halothane 1%, in an air oxygen mixture (oxygen fraction: 0.3) administered by tonometry during ligand membrane incubation, on beta-adrenergic receptor, was compared to that of control experiments not exposed to halothane. Halothane produced a moderate but significant decrease of Bmax (-10%) and a significant increase in non-specific binding (+30%), while KD, IC50, and KL were unchanged. The authors conclude that halothane, in vitro, decreases beta-adrenergic receptor density. This effect could be mediated by an alteration of the receptor in the membrane due to action of halothane on the lipid phase of the membrane.

  17. Mutated human beta3-adrenergic receptor (Trp64Arg) lowers the response to beta3-adrenergic agonists in transfected 3T3-L1 preadipocytes.

    PubMed

    Kimura, K; Sasaki, N; Asano, A; Mizukami, J; Kayahashi, S; Kawada, T; Fushiki, T; Morimatsu, M; Yoshida, T; Saito, M

    2000-03-01

    Wild-type or mutated human beta3-adrenergic receptor (Trp64Arg) cDNAs were stably expressed in mouse 3T3-L1 cells. Saturation binding study using a beta-adrenergic ligand revealed that there was no significant difference in the receptor density and the equilibrium dissociation constant between the two cell lines. However, the ability of the mutant beta3-adrenergic receptor to accumulate cyclic AMP (cAMP) in response to isoproterenol was much reduced and Kact for cAMP accumulation was lowered as compared to the wild type receptor. The amount of alpha subunit of stimulatory GTP-binding protein (GSalpha) and adenylyl cyclase activity in response to forskolin were not different in the two cell lines. The responses of the mutant receptor to epinephrine, norepinephrine and L-755,507, a highly specific agonist for human beta3-adrenergic receptor, were also reduced, but the reduction of Kact for L-755,507 was more evident than other agonists tested. The cAMP accumulation in response to some conventional beta3 agonists was less than 10% of that to isoproterenol even in the cells expressing the wild type receptor. These results suggest that the Trp64Arg mutant beta3-adrenergic receptor has less ability to stimulate adenylyl cyclase, and that lipolytic activity through the beta3-adrenergic receptor by catecholamines in subjects carrying this mutation might be suppressed. PMID:10786926

  18. Effect of transforming growth factor beta on synthesis of glycosaminoglycans by human lung fibroblasts

    SciTech Connect

    Dubaybo, B.A.; Thet, L.A. )

    1990-09-01

    The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either (3H)glucosamine or (35S)sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

  19. Lectin-like characteristics of recombinant human interleukin-1beta recognizing glycans of the glycosylphosphatidylinositol anchor.

    PubMed

    Fukushima, K; Hara-Kuge, S; Ohkura, T; Seko, A; Ideo, H; Inazu, T; Yamashita, K

    1997-04-18

    We found that 35S-labeled recombinant human interleukin-1beta (rhIL-1beta) binds phosphatidylinositol-specific phospholipase C-treated human placental alkaline phosphatase, phosphatidylinositol-specific phospholipase C-treated trypanosome surface variant glycoproteins, and urinary uromodulin immobilized on plates or immobilized on CNBr-activated Sepharose 4B. The interaction between rhIL-1beta and these glycoproteins was lectin-like, since it was inhibited in the presence of specific saccharides, i.e. mannose 6-phosphate or synthetic Ac-NH.CH2.CH2. PO4--->6Manalpha1-->(+/-2Manalpha1-->+/-6Manalpha1-->) propyl at about 1 microM. On the other hand, a wide variety of compounds including biantennary sugar chains derived from these glycoproteins as well as ethanolamine phosphate, inositol phosphate, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate did not show any inhibitory effect at concentrations up to 1 mM. These results indicate that rhIL-1beta interacts with these glycoproteins via the mannose 6-phosphate diester of glycans on the glycosylphosphatidylinositol (GPI) anchor. Furthermore, when monolayers of polarized Madin-Darby canine kidney cells on polycarbonate filter membranes were incubated with 35S-rhIL-1beta in either the apical or basolateral chamber, 35S-interleukin-1beta was found to bind specifically to the apical membranes with a Ka value of 4.6 x 10(7) M-1, and the specific interaction was inhibited by 1 microM mannose 6-phosphate. Since the mannose 6-phosphate diester moiety exists only in the GPI glycans on plasma membranes, it was evident that interleukin-1beta can directly interact with the mannose 6-phosphate diester component of the intact glycan of GPI anchors on plasma membranes.

  20. Provocation of pulmonary vascular endothelial injury in rabbits by human recombinant interleukin-1 beta.

    PubMed Central

    Goldblum, S E; Yoneda, K; Cohen, D A; McClain, C J

    1988-01-01

    Interleukin-1 (IL-1) mediates components of the acute-phase response, stimulates granulocyte metabolism, and induces endothelial cell surface changes. We studied the effects of human recombinant IL-1 beta (rIL-1 beta) or rIL-1 alpha on circulating granulocytes, their sequestration within the pulmonary microvasculature, pulmonary edema formation, and changes in pulmonary vascular permeability to 125I-labeled albumin. rIL-1 beta administration induced significant (P less than 0.03) but transient granulocytopenia followed by significant (P less than 0.04) neutrophilia and significant (P less than 0.04) pulmonary leukostasis compared with saline-infused rabbits. Rabbits preinfused with 125I-labeled rabbit serum albumin and administered saline, rIL-1 beta, or rIL-1 alpha were sacrificed, and lung wet/dry weight ratios and bronchoalveolar lavage fluid and plasma 125I activities determined. Both rIL-1 beta and rIL-1 alpha increased lung wet/dry weight ratios (P less than 0.025 and P less than 0.01, respectively) compared with saline controls. rIL-1 beta increased bronchoalveolar lavage fluid/plasma 125I radioactivity ratios (P less than 0.025). Electron microscopic analysis of lung sections obtained from rIL-1 beta-infused animals demonstrated endothelial injury, perivascular edema, and extravasation of an ultrastructural permeability tracer. The observation that human rIL-1 can evoke acute pulmonary vascular endothelial injury and lung edema in rabbits supports the hypothesis that IL-1 may play a role in the pathogenesis of the adult respiratory distress syndrome. Images PMID:3261716

  1. Using the NCBI Genome Databases to Compare the Genes for Human & Chimpanzee Beta Hemoglobin

    ERIC Educational Resources Information Center

    Offner, Susan

    2010-01-01

    The beta hemoglobin protein is identical in humans and chimpanzees. In this tutorial, students see that even though the proteins are identical, the genes that code for them are not. There are many more differences in the introns than in the exons, which indicates that coding regions of DNA are more highly conserved than non-coding regions.

  2. Fipronil-based photoaffinity probe for Drosophila and human beta 3 GABA receptors.

    PubMed

    Sirisoma, N S; Ratra, G S; Tomizawa, M; Casida, J E

    2001-11-19

    Modification of the major insecticide fipronil (1) by replacing three pyrazole substituents (hydrogen for both cyano and amino and trifluoromethyldiazirinyl for trifluoromethylsulfinyl) gives a candidate photoaffinity probe (3) of high potency (IC(50) 2-28 nM) in blocking the chloride channel of Drosophila and human beta 3 GABA receptors.

  3. Beta-cell metabolic alterations under chronic nutrient overload in rat and human islets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to assess multifactorial Beta-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of ...

  4. Kinetics of Beta-14[14C] Carotene in a Human Subject Using Accelerator Mass Spectrometry

    SciTech Connect

    Dueker, S.R.; Lin, Y.; Follett, J.R.; Clifford, A.J.; Buchholz, B.A.

    2000-01-31

    {beta}-Carotene is a tetraterpenoid distributed widely throughout the plant kingdom. It is a member of a group of pigments referred to as carotenoids that have the distinction of serving as metabolic precursors to vitamin A in humans and many animals [1,2]. We used Accelerator Mass Spectrometry (AMS) [3] to determine the metabolic behavior of a physiologic oral dose of {beta}-[{sup 14}C]carotene (200 nanoCuries; 0.57 {micro}mol) in a healthy human subject. Serial blood specimens were collected for 210-d and complete urine and feces were collected for 17 and 10-d, respectively. Balance data indicated that the dose was 42% bioavailable. The absorbed {beta}-carotene was lost slowly via urine in accord with the slow body turnover of {beta}-carotene and vitamin A [4]. HPLC fractionation of plasma taken at early time points (0-24-h) showed the label was distributed between {beta}-carotene and retinyl esters (vitamin A) derived from intestinal metabolism.

  5. Crystal Structure of the Central Coiled-Coil Domain from Human Liprin-[beta]2

    SciTech Connect

    Stafford, Ryan L.; Tang, Ming-Yun; Sawaya, Michael R.; Phillips, Martin L.; Bowie, James U.

    2012-02-07

    Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-{alpha}s and two liprin-{beta}s which all contain long coiled-coil domains followed by three tandem SAM domains. Complex interactions between the coiled-coil and SAM domains are thought to create liprin scaffolds, but the structural and biochemical properties of these domains remain largely uncharacterized. In this study we find that the human liprin-{beta}2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-{beta}1 and liprin-{beta}2 coiled-coils were also identified. A 2.0 {angstrom} crystal structure of the central, protease-resistant core of the liprin-{beta}2 coiled-coil reveals a parallel helix orientation. These studies represent an initial step toward determining the overall architecture of liprin scaffolds and understanding the molecular basis for their synaptic functions.

  6. The non-ligand binding beta-isoform of the human glucocorticoid receptor (hGR beta): tissue levels, mechanism of action, and potential physiologic role.

    PubMed Central

    de Castro, M.; Elliot, S.; Kino, T.; Bamberger, C.; Karl, M.; Webster, E.; Chrousos, G. P.

    1996-01-01

    BACKGROUND: Alternative splicing of the transcripts of the human glucocorticoid receptor gene results in two mutually exclusive products, the classic, ligand-binding glucocorticoid receptor (hGR alpha), and a dominant negative non-ligand-binding isoform, hGR beta. MATERIALS AND METHODS: We examined the existence of and quantified both hGR alpha and hGR beta isoforms in a panel of human tissues, as well as in intact and fractionated HeLa cells, using specific quantitative Western blots and/or immunocytochemistry. We studied the potential interactions of hGR beta with heat shock protein (hsp) 90 and/or hGR alpha using cross-immunoadsorption/precipitation procedures followed by Western blots. RESULTS: For the first time, we demonstrated the natural existence of the hGR beta protein, which was widely expressed in human tissues. The ratio of immunoreactive hGR alpha to hGR beta varied from 0.2 to 1.0 among different tissues, and was approximately 0.2 in HeLa cells. In the latter, both isoforms were distributed in the cytoplasm and nucleus in the absence of the hormonal ligand, and translocated into the nucleus after addition of dexamethasone. The cytosolic and nuclear hGR alpha-to-hGR beta ratio remained the same before and after dexamethasone exposure, suggesting that upon activation the two isoforms translocated into the nucleus in equal proportions. hGR alpha- and hGR beta-specific antibodies cross-adsorbed and precipitated cytosolic and nuclear glucocorticoid hGR alpha and hGR beta, respectively, as well as hsp90, suggesting that hGR alpha and hGR beta are in complex with hsp90 and/or each other. CONCLUSIONS: The hGR beta protein is widely expressed throughout the human body and present mostly in the cytoplasm of human cells, in complex with hsp90 and other proteins. In the presence of glucocorticoid, hGR beta probably heterodimerizes with ligand-bound hGR alpha and translocates into the nucleus to act as a dominant negative inhibitor of the classic receptor. Images

  7. Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA.

    PubMed

    Meyne, J; Goodwin, E H; Moyzis, R K

    1994-04-01

    The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the -A-B-A-B-A- form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant -B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashion along the DNA strand. PMID:8055716

  8. Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin.

    PubMed

    Takeya, Akira; Hosomi, Osamu; Nishijima, Hironori; Ohe, Yoshihide; Sugahara, Kunio; Sagi, Morihisa; Yamazaki, Kentaro; Hayakawa, Hideyuki; Takeshita, Hiroshi; Sasaki, Chizuko; Kogure, Tadahisa; Mukai, Toshiji

    2007-01-16

    Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.

  9. Human DNA polymerase beta mutations allowing efficient abasic site bypass.

    PubMed

    Gieseking, Sonja; Bergen, Konrad; Di Pasquale, Francesca; Diederichs, Kay; Welte, Wolfram; Marx, Andreas

    2011-02-01

    The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.

  10. Specific inhibition of transforming growth factor-beta2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides.

    PubMed

    Shen, Z J; Kim, S K; Kwon, O S; Lee, Y S; Moon, B J

    2001-04-01

    To elucidate the role of endogenous transforming growth factor (TGF)-beta2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-beta2 were synthesized and examined their effects on TGF-beta2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-beta2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-beta2 demonstrated a selective and strong inhibitory effect on TGF-beta2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-beta2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-beta2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-beta2. Furthermore, the inhibition of TGF-beta2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-beta2 in osteoblast cells.

  11. Effects of beta 2-agonist- and dexamethasone-treatment on relaxation and regulation of beta-adrenoceptors in human bronchi and lung tissue.

    PubMed

    Hauck, R W; Harth, M; Schulz, C; Präuer, H; Böhm, M; Schömig, A

    1997-08-01

    1. Long-term treatment with beta 2-adrenoceptor agonists can lead to a decreased therapeutic efficacy of bronchodilatation in patients with obstructive pulmonary disease. In order to examine whether or not this is due to beta-adrenoceptor desensitization, human bronchial muscle relaxation was studied in isolated bronchial rings after pretreatment with beta 2-adrenoceptor agonists. Additionally, the influence of pretreatment with dexamethasone on desensitization was studied. 2. The effect of beta 2-agonist incubation alone and after coincubation with dexamethasone on density and affinity of beta-adrenoceptors was investigated by radioligand binding experiments. 3. In human isolated bronchi, isoprenaline induces a time- and concentration-dependent beta-adrenoceptor desensitization as judged from maximal reduction in potency by a factor of 7 and reduction of 73 +/- 4% in efficacy of isoprenaline to relax human bronchial smooth muscle. 4. After an incubation period of 60 min with 100 mumol l-1 terbutaline, a significant decline in its relaxing efficacy (81 +/- 8%) and potency (by a factor 5.5) occurred. 5. Incubation with 30 mumol l-1 isoprenaline for 60 min did not impair the maximal effect of a subsequent aminophylline response but led to an increase in potency (factor 4.4). 6. Coincubation of dexamethasone with isoprenaline (120 min; 30 mumol l-1) preserved the effect of isoprenaline on relaxation (129 +/- 15%). 7. In radioligand binding experiments, pretreatment of lung tissue for 60 min with isoprenaline (30 mumol l-1) resulted in a decrease in beta-adrenoceptor binding sites (Bmax) to 64 +/- 1.6% (P < 0.05), while the antagonist affinity (KD) for [3H]-CGP-12177 remained unchanged. 8. In contrast, radioligand binding studies on lung tissue pretreated with either dexamethasone (30 mumol l-1) or isoprenaline (30 mumol l-1) plus dexamethasone (30 mumol l-1) for 120 min did not lead to a significant change of Bmax (160 +/- 22.1% vs 142.3 +/- 28.7%) or KD (5.0 nmol l-1

  12. Altered regulation of Src tyrosine kinase by transforming growth factor beta1 in a human hepatoma cell line.

    PubMed

    Fukuda, K; Kawata, S; Tamura, S; Matsuda, Y; Inui, Y; Igura, T; Inoue, S; Kudara, T; Matsuzawa, Y

    1998-09-01

    Transforming growth factor betas (TGF-betas) are the potent growth inhibitors for various cell types. Certain transformed cells, however, show poor response to TGF-beta-induced growth inhibition, which contributes to their uncontrolled proliferation. Recently, we have reported that TGF-beta1 induces degradation of activated Src tyrosine kinase in rat fibroblasts. To elucidate the alteration in TGF-beta signaling pathway in tumor cells that cannot respond to the cytokine, we compared the effects of TGF-beta1 on Src kinase in two human hepatoma cell lines, TGF-beta1-insensitive Mahlavu cells and TGF-beta1-sensitive HepG2 cells. TGF-beta1 decreased Src kinase activity in HepG2 cells, but increased cellular Src levels and Src kinase activity in Mahlavu cells. Co-incubation of Mahlavu cells with TGF-beta1 and 12-O-tetradecanoyl phorbol 13-acetate (TPA) decreased Src protein levels and Src kinase activity, inducing TGF-beta1 sensitivity. TGF-beta1 induced tyrosine dephosphorylation of Ras guanosine triphosphatase-activating protein (Ras-GAP) and Ras inactivation in HepG2 cells, but induced Ras-GAP phosphorylation and Ras activation in Mahlavu cells. The Src kinase inhibitor abolished the increase of Src kinase activity in TGF-beta1-treated Mahlavu cells, and induced TGF-beta1 sensitivity. These findings suggest that regulation of Src kinase by TGF-beta1 is altered in Mahlavu cells. The altered regulation of Src may contribute to TGF-beta1 insensitivity in this cell line, at least in part through activation of Ras.

  13. Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta.

    PubMed

    Sommerhoff, Christian P; Avrutina, Olga; Schmoldt, Hans-Ulrich; Gabrijelcic-Geiger, Dusica; Diederichsen, Ulf; Kolmar, Harald

    2010-01-01

    Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors. PMID:19852971

  14. Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta.

    PubMed

    Sommerhoff, Christian P; Avrutina, Olga; Schmoldt, Hans-Ulrich; Gabrijelcic-Geiger, Dusica; Diederichsen, Ulf; Kolmar, Harald

    2010-01-01

    Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.

  15. Haem degradation in human haemoglobin in vitro. Separation of the contribution of the alpha- and beta-subunits.

    PubMed Central

    Docherty, J C; Brown, S B

    1984-01-01

    Human haemoglobin was prepared containing [14C]haem in either the alpha- or the beta-subunits. Coupled oxidation of such hybrid haemoglobins with ascorbate and O2 showed that the biliverdin produced by the alpha-subunits contained approx. 55% alpha-isomer and 45% beta-isomer, whereas that produced by the beta-subunits contained approx. 75% alpha-isomer and 25% beta-isomer. Coupled oxidation of isolated alpha- and beta-subunits gave approx. 70% alpha-isomer, 30% beta-isomer and 78% alpha-isomer, 22% beta-isomer respectively. These results are consistent with calculations of differences in the haem environment in the two subunit types. PMID:6477522

  16. Interleukin-1 beta stimulates glucose uptake of human peritoneal mesothelial cells in vitro.

    PubMed

    Kruse, M; Mahiout, A; Kliem, V; Kurz, P; Koch, K M; Brunkhorst, R

    1996-01-01

    To investigate whether the glucose uptake (GU) of human peritoneal mesothelial cells (HPMC) is mediated by glucose transporters and whether this uptake is influenced by interleukin 1-beta (IL-1 beta), we measured 2-deoxy-(3H)-GU of HPMC in vitro, after exposing the cells for different times (two and 12 hours) to increasing concentrations (0.1, 1.0, and 2.0 ng/mL) of IL-1 beta. To exclude a noncarrier-mediated transport, GU was also tested in the presence of cytochalasin B. All experiments were performed in triplicate in the cells of two donors. Cytochalasin B inhibits GU of HPMC almost completely. GU of HPMC is not stimulated by insulin. GU is stimulated by IL-1 beta in a dose-dependent manner. These data indicate a GU of HPMC, which is mediated by a glucose transporter and stimulated by IL-1 beta. The increased uptake of glucose from the dialysate in patients with peritonitis may be mediated by a (cytokine-induced) increased activity of HPMC glucose transporters.

  17. Glucocorticoid receptor beta increases migration of human bladder cancer cells.

    PubMed

    McBeth, Lucien; Nwaneri, Assumpta C; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D

    2016-05-10

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3' untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3'UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease. PMID:27036026

  18. Glucocorticoid receptor beta increases migration of human bladder cancer cells

    PubMed Central

    McBeth, Lucien; Nwaneri, Assumpta C.; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D.

    2016-01-01

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3′ untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3′UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease. PMID:27036026

  19. Activity of proximal promoter of the human beta(1)-integrin gene was increased in Sézary syndrome.

    PubMed

    Paulin, Y; Boukhelifa, M; Derappe, C; Giner, M; Font, J; Aubery, M

    2001-06-01

    Changes in beta1-integrin expression have been involved in abnormal cellular interactions between malignant lymphocytes from Sézary (Sz) patients and keratinocytes. In this paper, we compare the activity of both distal and proximal promoters of the beta1-integrin gene in malignant lymphocytes from Sz patients with human normal lymphocytes. Activity of both beta1-integrin promoters was also analysed in human normal keratinocytes. Northern blot analysis shows that beta1-integrin mRNA expression is higher in malignant Sz lymphocytes than in normal lymphocytes. CAT assays show that the activity of proximal beta1-integrin promoter is markedly increased (up to 6-fold) in malignant lymphocytes from Sz patients, in comparison to normal lymphocytes. These results suggest that changes in activity of the proximal promoter of beta1-integrin subunit could be, in part, responsible for the abnormal cellular interactions between malignant lymphocytes and keratinocytes observed in Sz syndrome.

  20. Reward feedback stimuli elicit high-beta EEG oscillations in human dorsolateral prefrontal cortex

    PubMed Central

    Hosseini, Azadeh Haji; Holroyd, Clay B.

    2015-01-01

    Reward-related feedback stimuli have been observed to elicit a burst of power in the beta frequency range over frontal areas of the human scalp. Recent discussions have suggested possible neural sources for this activity but there is a paucity of empirical evidence on the question. Here we recorded EEG from participants while they navigated a virtual T-maze to find monetary rewards. Consistent with previous studies, we found that the reward feedback stimuli elicited an increase in beta power (20–30 Hz) over a right-frontal area of the scalp. Source analysis indicated that this signal was produced in the right dorsolateral prefrontal cortex (DLPFC). These findings align with previous observations of reward-related beta oscillations in the DLPFC in non-human primates. We speculate that increased power in the beta frequency range following reward receipt reflects the activation of task-related neural assemblies that encode the stimulus-response mapping in working memory. PMID:26278335

  1. Identification of the 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein in human mononuclear leukocytes.

    PubMed

    Fiore, C; Nardi, A; Dalla Valle, L; Pellati, D; Krozowski, Z; Colombo, L; Armanini, D

    2009-10-01

    The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion between inactive 11-ketoglucocorticoids and their active 11beta-hydroxy derivatives, such as cortisol and corticosterone. We have investigated the expression of 11beta-HSD1 in freshly isolated human peripheral mononuclear leukocytes (MNL). The presence of 11beta-HSD1 mRNA was demonstrated in total RNA by RT-PCR using specific primers designed on the 4th and 5th exons of the human 11beta-HSD1 gene. Fragments of the expected size were consistently detected on agarose gels, and sequencing showed complete identity with the corresponding sequence deposited in GenBank. The occurrence of 11beta-HSD1 protein was established by Western immunoblot analysis with a specific polyclonal antibody. Enzyme oxo-reductase activity was investigated by incubating 12 samples of MNL isolated from from 8 subjects with [3H]cortisone and formation of cortisol was established only in 4 subjects (yield range: 0.15-1.3%) after acetylation and TLC, blank subtraction and correction for losses. 18beta-Glycyrrhetinic acid, an inhibitor of 11 beta-HSD1, reduced cortisol production below detection limit. Dehydrogenase activity could not be demonstrated. It is suggested that, although enzyme activity of 11beta-HSD1 in circulating MNL is low, it is apparently ready for enhancement after MNL migration to sites of inflammation. PMID:19235128

  2. Assignment of the {beta}B1 crystallin gene (CRYBB1) to human chromosome 22 and mouse chromosome 5

    SciTech Connect

    Hulsebos, T.J.M.; Westerveld, A.; Gilbert, D.J.; Jenkins, N.A.; Copeland, N.G.

    1995-10-10

    By using primers complementary to the rat {beta}B1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences. CRYBB1 was assigned to the group 5 region in 22q11.2-q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products of CRYBB1 were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other {beta} crystallin genes ({beta}B2(-l), {beta}B3, and {beta}A4) have previously been mapped to the same regions in human and mouse. We demonstrate that the {beta}B1 and {beta}A4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major {beta} crystallins genes that are expressed in the bovine lens. 39 refs., 5 figs., 1 tab.

  3. Transcriptional regulation of the human iNOS gene by IL-1beta in endothelial cells.

    PubMed Central

    Kolyada, A. Y.; Madias, N. E.

    2001-01-01

    BACKGROUND: Vascular endothelium participates in the control of vascular tone and function via the release of nitric oxide (NO) by the endothelial-type NO synthase (eNOS). Inducible NO synthase (iNOS) expression in endothelial cells occurs in many clinical conditions following induction by lipopolysaccharide or cytokines and generates large quantities of NO that result in endothelial cell activation and dysfunction. No information exists on the transcriptional regulation of the human iNOS gene (or that of other species) in endothelial cells. MATERIALS AND METHODS: We examined the transcriptional regulation of the human iNOS gene by interleukin-1beta (IL-1beta) in rat pulmonary microvascular endothelial cells (PVEC) by transient cotransfections of different iNOS-promoter constructs and cDNA of different transcription factors and regulatory proteins. RESULTS: The -1034/+88 bp iNOS promoter was strongly induced by IL-1beta, the regulatory elements for such induction being localized downstream of -205 bp. Cotransfection experiments with NF-kappaB isoforms, IkappaB isoforms, and IKK mutants suggested that the NF-kappaB site at -115/-106 bp is important, but not sufficient, for induction of iNOS promoter and that the role of NF-kappaB is partially independent of its binding site. C/EBP sites within the -205/+88 bp region were shown to be responsible, along with NF-kappaB site, for induction of iNOS promoter by IL-1beta. Overexpression of C/EBPalpha, C/EBPdelta, and liver-enriched activator protein (LAP) activated the promoter, whereas overexpression of liver-enriched inhibitory protein (LIP) strongly suppressed it. C/EBPbeta (LAP and LIP isoforms) was constitutively present in PVEC and was induced (approximately 2-fold) by IL-1beta, whereas C/EBPdelta was not constitutively expressed but was strongly induced by IL-1beta. Both C/EBPbeta and C/EBPdelta participated in DNA-protein complex formation. CONCLUSION: Both NF-kappaB and C/EBP pathways are important for the

  4. Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

    SciTech Connect

    Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.; Pessin, J.E.

    1988-05-03

    Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.

  5. Simultaneous extraction of. beta. -endorphin and leu- and met-enkephalins from human and rat plasma

    SciTech Connect

    Bhathena, S.J.; Smith, P.M.; Kennedy, B.W. ); Voyles, N.R.; Recant, L. )

    1989-01-01

    A simple, rapid and reliable procedure is described to simultaneously concentrated and purify {beta}-endorphin, leu-and met-enkephalins from small volumes of human and rat plasma before radioimmunoassay is performed. It uses C{sub 18} Sep-Pak reverse phase cartridges. The effectiveness of different protease inhibitors in preventing degradation of opiates by plasma and different solvent systems for eluting opiates is also evaluated.

  6. Novel monoclonal antibody against beta 1 integrin enhances cisplatin efficacy in human lung adenocarcinoma cells

    PubMed Central

    Kim, Min-Young; Cho, Woon-Dong; Hong, Kwon Pyo; Choi, Da Bin; Hong, Jeong won; Kim, Soseul; Moon, Yoo Ri; Son, Seung-Myoung; Lee, Ok-Jun; Lee, Ho-Chang; Song, Hyung Geun

    2016-01-01

    Abstract The use of anti-beta 1 integrin monoclonal antibody in lung cancer treatment has proven beneficial. Here, we developed a novel monoclonal antibody (mAb), called P5, by immunizing mice with human peripheral blood mononuclear cells (PBMC). Its anti-tumor effect is now being tested, in a clinical phase III trial, in combinatorial treatments with various chemical drugs. To confirm that P5 indeed binds to beta 1 integrin, cell lysates were immunoprecipitated with commercial anti-beta 1 integrin mAb (TS2/16) and immunoblotted against P5 to reveal a 140 kDa molecular weight band, as expected. Immunoprecipitation with P5 followed by LC/MS protein sequence analysis further verified P5 antigen to be beta 1 integrin. Cisplatin treatment upregulated cell surface expression of beta 1 integrin in A549 cells, while causing inhibition of cell growth. When cells were co-treated with different concentrations of P5 mAb, the cisplatin-mediated inhibitory effect was enhanced in a dose-dependent manner. Our findings show that a combinatorial treatment of P5 mAb and cisplatin in A549 cells resulted in a 30% increase in apoptosis, compared to baseline, and significantly more when compared to either the cisplatin or P5 alone group. The entire peptide sequences in CDR from variable region of Ig heavy and light chain gene for P5 mAb are also disclosed. Together, these results provide evidence of the beneficial effect of P5 mAb in combinatorial treatment of human lung adenocarcinoma. PMID:27533932

  7. Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets

    PubMed Central

    Broca, Christophe; Varin, Elodie; Armanet, Mathieu; Tourrel-Cuzin, Cécile; Bosco, Domenico; Dalle, Stéphane; Wojtusciszyn, Anne

    2014-01-01

    The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes. PMID:24642635

  8. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

    PubMed

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-07-18

    Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

  9. Immunogenicity of recombinant human interferon beta interacting with particles of glass, metal, and polystyrene.

    PubMed

    Van Beers, Miranda M C; Gilli, Francesca; Schellekens, Huub; Randolph, Theodore W; Jiskoot, Wim

    2012-01-01

    Aggregates play a major role in the immunogenicity of recombinant human interferon beta (rhIFNβ), a protein used to treat multiple sclerosis. A possible cause of aggregation is interaction between therapeutic protein and surfaces encountered during processing, storage, and administration. Moreover, proteins may adsorb to particles shed from these surfaces. In this work, we studied the immunogenicity of recombinant human interferon beta-1a (rhIFNβ-1a) interacting with glass microparticles, stainless steel microparticles, and polystyrene nanoparticles. At physiological pH, rhIFNβ-1a readily adsorbed to the particles, while the degree of adsorption was influenced by the ionic strength of the phosphate buffer. Front-face fluorescence showed that the tertiary structure of rhIFNβ-1a slightly changed upon adsorption to glass. The interaction with stainless steel microparticles resulted in increased levels of aggregates in the free protein fraction. Furthermore, protein adsorbed to stainless steel microparticles was more difficult to desorb than protein adsorbed to glass. Incubation with stainless steel considerably enhanced the immunogenicity of rhIFNβ-1a in transgenic mice immune tolerant for human interferon beta. The protein fraction adsorbed on stainless steel particles was responsible for this. In conclusion, rhIFNβ-1a adsorbs to common hydrophilic surface materials, possibly increasing the immunogenicity of the protein.

  10. Localized decrease of {beta}-catenin contributes to the differentiation of human embryonic stem cells

    SciTech Connect

    Lam, Hayley; Patel, Shyam; Wong, Janelle; Chu, Julia; Li, Adrian; Li, Song

    2008-08-08

    Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, {beta}-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of {beta}-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down {beta}-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of {beta}-catenin contributes to the spatial pattern of differentiation in hESC colonies.

  11. Expression of beta 2-microglobulin by human benign and malignant mesenchymal and neurogenic tumours.

    PubMed Central

    Petersen, B. L.; Braendstrup, O.

    1993-01-01

    Human myosarcomas, liposarcomas, meningosarcomas, glioblastomas and malignant schwannomas, their benign counterparts and normal cells from which these tumours derive, were examined for the expression of beta 2-microglobulin (beta 2m). Formalin fixed specimens from these tumours were studied by light microscopy employing the immunoperoxidase method with the use of antibodies directed towards beta 2m. The malignant tumours showed a broad spectrum from unstained to strongly stained tumours, most pronounced among myosarcomas. In addition, most stained tumours displayed a mosaic staining pattern in that unstained areas alternated with stained. There was a tendency towards increased staining for beta 2m in malignant compared to normal cells; this was also observed in the benign tumours although to a lesser degree. The results differ from most earlier studies, mainly of carcinomas which have shown a tendency towards down-regulation of MHC I molecules on the tumour cells. The results are discussed in relation to concepts of immune surveillance of tumours. Images Figure 1 Figure 2 PMID:8398813

  12. Identification of functional nuclear export sequences in human topoisomerase IIalpha and beta.

    PubMed

    Mirski, Shelagh E L; Bielawski, Juliane C; Cole, Susan P C

    2003-07-11

    Nuclear localization of topoisomerase IIalpha and beta is important for normal cell function as well as being a determinant of tumour cell sensitivity to topoisomerase II-targeting chemotherapeutic agents. However, topoisomerase II is cytoplasmic under certain circumstances, indicating that it may undergo active nuclear export. We have examined the ability of Leu-rich potential nuclear export signal (NES) sequences present in human topoisomerase IIalpha and beta to direct the export of a green fluorescent protein-glutathione-S-transferase fusion protein following microinjection into HeLa cell nuclei. Of 12 sequences tested, only one potential NES sequence from the comparable location in each isoform (alphaNES(1018-1028) and betaNES(1034-1044)) was active. Mutation of hydrophobic residues in alphaNES(1018-1028) and betaNES(1034-1044) substantially reduced their nuclear export activity as did leptomycin B treatment of microinjected cells. Our results provide the first evidence of active nuclear export of topoisomerase II and suggest it is mediated by a CRM1-dependent pathway.

  13. Partial rescue of epithelial phenotype in integrin beta4 null mice by a keratin-5 promoter driven human integrin beta4 transgene.

    PubMed

    van der Neut, R; Cachaço, A S; Thorsteinsdóttir, S; Janssen, H; Prins, D; Bulthuis, J; van der Valk, M; Calafat, J; Sonnenberg, A

    1999-11-01

    Integrin beta4 null mice exhibit extensive epidermal detachment, reminiscent of the human skin blistering disease junctional epidermolysis bullosa associated with pyloric atresia. Hemidesmosomes, the stable adhesion structures of squamous epithelia, are not formed in the absence of alpha6beta4. Null mutant mice die shortly after birth, but apart from their striking epithelial phenotype, no obvious developmental defects have been observed. To elucidate the cause of death in these mice, we generated transgenic mice with a heterologous construct consisting of the squamous epithelial-specific keratin-5 promoter and a human integrin beta4 subunit cDNA. The transgene was not expressed in the presence of endogenous beta4, probably as a result of competition for a limited pool of alpha6 subunits. In a beta4 null background, however, the transgene was expressed, and its expression pattern followed that of squamous epithelial-specific keratins. These rescued pups appeared healthy and ultrastructural analysis revealed that the interspecies heterodimer alpha6(mouse)/beta4(human) was sufficient to trigger the assembly of hemidesmosomes. After a variable period of up to 48 hours after birth these animals began to exhibit haemorrhages at the plantar and palmar areas. We observed the formation of small blisters and found that the transgene was not detectably expressed in this region, which is devoid of hair follicles. The rescued neonates became increasingly cyanotic and died soon after the onset of this phenomenon. We performed a developmental study of the expression of beta4 in the complete respiratory tract, but we found no correlation between the spatiotemporal distribution of beta4 and the onset of the respiratory insufficiency. It became clear, however, that there was a gradual detachment of squamous epithelia in the oral and nasal cavities which led to obstruction of the respiratory tract, suggesting that in beta4 null and rescued mice, neonatal death was a direct

  14. Contribution of coupling between human myometrial beta2-adrenoreceptor and the BK(Ca) channel to uterine quiescence.

    PubMed

    Chanrachakul, Boonsri; Broughton Pipkin, Fiona; Khan, Raheela N

    2004-12-01

    The beta(2)-adrenergic receptor (beta(2)-AR) and the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both beta(2)-AR and BK(Ca) channel proteins in pregnant human myometrium decrease by approximately 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the beta(2)-AR and the BK(Ca) channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of beta(2)-AR colocalizing with the BK(Ca) channel. Coimmunoprecipitation studies indicate that BK(Ca) and beta(2)-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BK(Ca) channel blocker, paxilline, significantly antagonized the relaxant effect of the beta(2)-AR agonist ritodrine. These novel findings provide an insight into the coupling between the beta(2)-AR and BK(Ca) channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor.

  15. Dielectronic satellites of the He{sub {beta}} line of the Si XIII ion in a dense laser plasma

    SciTech Connect

    Skobelev, I Yu; Faenov, A Ya; Bartnik, A; Kostecki, J; Fiedorowicz, H; Szczurek, M; Jarocki, R; Behar, E; Doron, R; Mandelbaum, P; Schwob, J L; Dyakin, V M

    1998-08-31

    The first precision measurements of the wavelengths and identifications were made of the satellites of the Heb line of the Si XIII ion in the emission spectrum of a laser plasma. These satellites were the result of radiation decay of the 1s3l{sub 1}2l{sub 2} levels of the Si XII ion. The wavelengths were determined more accurately for the 1s3l{sub 1}3l{sub 2} configurations. The experimental results obtained were compared with calculations carried out by various methods. The structure of the relative intensities of the 1s3l{sub 1}3l{sub 2} satellites indicated that their emission occurred mainly in a thin overdense plasma region with N{sub e}>N{sub e}{sup cr}. (interaction of laser radiation with matter)

  16. The human beta fibrinogen promoter contains a hepatocyte nuclear factor 1-dependent interleukin-6-responsive element.

    PubMed Central

    Dalmon, J; Laurent, M; Courtois, G

    1993-01-01

    Acute-phase reactants are liver proteins whose synthesis is positively or negatively regulated during inflammation. The main mediators of this phenomenon are glucocorticoids and interleukin-6 (IL-6), a pleiotropic cytokine that also controls hematopoiesis. Functional analysis of several acute-phase reactant promoter regions has identified two major DNA motifs used by IL-6-regulated genes. The first one corresponds to a CTGG(G/A)AA sequence, and the other is a binding site for members of the C/EBP family of nuclear proteins. We have previously shown that the human beta fibrinogen (beta Fg) promoter contains an IL-6-responsive region, located between bp -150 and -67 (P. Huber, M. Laurent, and J. Dalmon, J. Biol. Chem. 265:5695-5701, 1990). In this study, using DNase I footprinting, mobility shift assays, and mutagenesis, we demonstrate that at least three subdomains of this region are necessary to observe a full response to IL-6. The most distal contains a CTGGGAA motif, and its mutation inhibits IL-6 stimulation. Another, which is able to interact with several distinct nuclear proteins, among them members of the C/EBP family, is dispensable for IL-6 induction but plays an important role in the constitutive expression of beta Fg. Finally, a proximal hepatocyte nuclear factor 1 binding site, already described as the major determinant of beta Fg tissue-specific expression, is also required for IL-6 stimulation. These results indicate a complex interplay between nuclear proteins within the beta Fg IL-6-responsive region and suggest a tight functional coupling between the tissue-specific and inducible elements. Images PMID:8423785

  17. Calcium channel beta 4 (CACNB4): human ortholog of the mouse epilepsy gene lethargic.

    PubMed

    Escayg, A; Jones, J M; Kearney, J A; Hitchcock, P F; Meisler, M H

    1998-05-15

    The mouse neurological mutant lethargic (lh) is characterized by ataxia, focal myoclonus, and absence epilepsy due to a loss-of-function mutation in the beta4 subunit of the voltage-gated calcium channel. To evaluate the role of this channel subunit in human neurological disease, we determined the chromosomal location and intron/exon structure of the human CACNB4 gene. The 1560-bp open reading frame of the CACNB4 cDNA predicts a 58-kDa protein with an amino acid sequence that is 99% identical to the rat protein. The 13 coding exons of CACNB4 span >55 kb of genomic DNA. Human cerebellar RNA contains one major CACNB4 transcript that is 9 kb in length. Expression of CACNB4 was detected in cerebellum, kidney, testis, retina, lymphoblasts, and circulating lymphocytes. Retinal transcripts were localized by in situ hybridization to ganglion cells and the inner nuclear layer. Analysis of the GeneBridge 4 radiation hybrid mapping panel localized CACNB4 to position 791 cR on human chromosome 2, in a conserved linkage group on human 2q22-q31 and mouse chromosome 2. We localized CACNB4 to the 1.3-Mb YAC clone 952F10 in Whitehead contig WC861, along with the polymorphic markers D2S2236 and D2S2299. The chromosomal linkage of three of the four beta subunit genes to homeobox gene clusters associates the evolutionary origin of the beta gene family with the events that generated the four HOX clusters early in vertebrate evolution.

  18. Mechanism of mismatch recognition revealed by human MutS[beta] bound to unpaired DNA loops

    SciTech Connect

    Gupta, Shikha; Gellert, Martin; Yang, Wei

    2012-04-17

    DNA mismatch repair corrects replication errors, thus reducing mutation rates and microsatellite instability. Genetic defects in this pathway cause Lynch syndrome and various cancers in humans. Binding of a mispaired or unpaired base by bacterial MutS and eukaryotic MutS{alpha} is well characterized. We report here crystal structures of human MutS{beta} in complex with DNA containing insertion-deletion loops (IDL) of two, three, four or six unpaired nucleotides. In contrast to eukaryotic MutS{alpha} and bacterial MutS, which bind the base of a mismatched nucleotide, MutS{beta} binds three phosphates in an IDL. DNA is severely bent at the IDL; unpaired bases are flipped out into the major groove and partially exposed to solvent. A normal downstream base pair can become unpaired; a single unpaired base can thereby be converted to an IDL of two nucleotides and recognized by MutS{beta}. The C-terminal dimerization domains form an integral part of the MutS structure and coordinate asymmetrical ATP hydrolysis by Msh2 and Msh3 with mismatch binding to signal for repair.

  19. A unique chromatin complex occupies young α-satellite arrays of human centromeres

    PubMed Central

    Henikoff, Jorja G.; Thakur, Jitendra; Kasinathan, Sivakanthan; Henikoff, Steven

    2015-01-01

    The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes. PMID:25927077

  20. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

    PubMed Central

    Cone, R D; Weber-Benarous, A; Baorto, D; Mulligan, R C

    1987-01-01

    We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element. Images PMID:3029570

  1. Crystal structure of human renal dipeptidase involved in beta-lactam hydrolysis.

    PubMed

    Nitanai, Yasushi; Satow, Yoshinori; Adachi, Hideki; Tsujimoto, Masafumi

    2002-08-01

    Human renal dipeptidase is a membrane-bound glycoprotein hydrolyzing dipeptides and is involved in hydrolytic metabolism of penem and carbapenem beta-lactam antibiotics. The crystal structures of the saccharide-trimmed enzyme are determined as unliganded and inhibitor-liganded forms. They are informative for designing new antibiotics that are not hydrolyzed by this enzyme. The active site in each of the (alpha/beta)(8) barrel subunits of the homodimeric molecule is composed of binuclear zinc ions bridged by the Glu125 side-chain located at the bottom of the barrel, and it faces toward the microvillar membrane of a kidney tubule. A dipeptidyl moiety of the therapeutically used cilastatin inhibitor is fully accommodated in the active-site pocket, which is small enough for precise recognition of dipeptide substrates. The barrel and active-site architectures utilizing catalytic metal ions exhibit unexpected similarities to those of the murine adenosine deaminase and the catalytic domain of the bacterial urease.

  2. Beta 2-microglobulin and neopterin: predictive markers for human immunodeficiency virus type 1 infection in children?

    PubMed Central

    Chan, M M; Campos, J M; Josephs, S; Rifai, N

    1990-01-01

    The value of beta 2-microglobulin and neopterin concentrations in serum for early diagnosis of infants born to human immunodeficiency virus type 1 (HIV-1)-infected mothers was assessed. Concentrations of both markers were measured in serum samples from pediatric patients (Centers for Disease Control classifications P0, P1, and P2), as well as in age-matched normal subjects. Both beta 2-microglobulin and neopterin were significantly increased in HIV-1-infected symptomatic subjects (P2) compared to controls. Seventy-five percent of asymptomatic patients (P1) also had increased values. On the other hand, a significant overlap in concentrations of both markers in serum was found between controls and P0 patients. Thirty-eight percent of the P0 patients had values comparable to those of the P2 group. Persistently high concentrations of both markers in P0 patients may be indicative of HIV-1 infection. PMID:2229344

  3. Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation.

    PubMed

    Kan, Y W; Dozy, A M

    1978-11-01

    Restriction endonuclease mapping of the human globin genes revealed a genetic variation in a Hpa I recognition site about 5000 nucleotides from the 3' end of the beta-globin structural gene. Instead of a normal 7.6-kilobase (kb) fragment which contains the beta-globin structural gene, 7.0-kb and 13.0-kb variants were detected. Both variants were found in people of African origin and were not detected in Asians or Caucasians. The 13.0-kb variant is frequently associated with the sickle hemoglobin mutation and may be useful for the prediction of the sickle cell gene in prenatal diagnosis. Polymorphism in a restriction enzyme site could be considered as a new class of genetic marker and may offer a new approach to linkage analysis and anthropological studies.

  4. The functional importance of a cap site-proximal region of the human prointerleukin 1[beta] gene is defined

    SciTech Connect

    Hunninghake, G.W.; Geist, L.J.; Monick, M.M.; Stinski, M.F. ); Monks, B.G.; Monroy, M.A.; Fenton, M.J. ); Webb, A.C. ); Dayer, J.M. ); Auron, P.E. )

    1992-08-01

    Prointerleukin 1[beta] (IL-1[beta]) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1[beta] transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1[beta] gene. We identified a protein, termed NFIL-1[beta]A (NF[beta]A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1[beta] genes. The IL-1[alpha] gene, which lacks a TATA motif, does not possess an NF[beta]A-binding sequence within the promoter region, suggesting that NF[beta]A may selectively regulate IL-1[beta] expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varied rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF[beta]A was assessed in transient transfection assays. These data indicate the NF[beta]A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF[beta]A in order to trans-activate the proximal IL-1[beta] promoter in a monocytic cell line. We propose that NF[beta]A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation.

  5. Responsiveness to transforming growth factor-beta (TGF-beta)-mediated growth inhibition is a function of membrane-bound TGF-beta type II receptor in human breast cancer cells.

    PubMed

    Lynch, M A; Petrel, T A; Song, H; Knobloch, T J; Casto, B C; Ramljak, D; Anderson, L M; DeGroff, V; Stoner, G D; Brueggemeier, R W; Weghorst, C M

    2001-01-01

    Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth and proliferation of breast epithelial cells, and loss of sensitivity to its effects has been associated with malignant transformation and tumorigenesis. The biological effects of TGF-beta are mediated by the TGF-beta receptor complex, a multimer composed of TGF-beta receptor type I (TbetaR-I) and TGF-beta receptor type II (TbetaR-II) subunits. Evidence suggests that loss of expression of Tbeta3R-II is implicated in the loss of sensitivity of tumorigenic breast cell lines to TGF-beta-mediated growth inhibition. A panel of human breast cell lines, including the immortalized MCF-10F and tumorigenic MCF-7, ZR75-1, BT474, T47-D, MDA-MB231, BT20, and SKBR-3 cell lines, was characterized for responsiveness to TGF-beta-induced G1 growth arrest. Only the nontumorigenic MCF-10F and the tumorigenic MDA-MB231 cell lines demonstrated a significant inhibitory response to TGF-beta1 and a significant binding of 125I-labeled TGF-beta ligand. While expression of TbetaR-I mRNA was similar across the panel of cell lines, TbetaR-II mRNA expression was decreased significantly in all seven tumorigenic cell lines in comparison with the nontumorigenic MCF- 10F cell line. When total cellular protein was fractionated by centrifugation, TbetaR-I protein was observed in both the cytosolic and membrane fractions at similar levels in all cell lines; however, TbetaR-II protein was present in the cytosolic fraction in all cell lines, but was observed in the membrane fraction of only the TGF-beta-responsive MCF-10F and MDA-MB231 cells. Thus, lack of membrane-bound TbetaR-II protein appears to be an important determinant of resistance to TGF-beta-mediated growth inhibition in this group of breast cell lines. PMID:11444526

  6. Images of war: using satellite images for human rights monitoring in Turkish Kurdistan.

    PubMed

    de Vos, Hugo; Jongerden, Joost; van Etten, Jacob

    2008-09-01

    In areas of war and armed conflict it is difficult to get trustworthy and coherent information. Civil society and human rights groups often face problems of dealing with fragmented witness reports, disinformation of war propaganda, and difficult direct access to these areas. Turkish Kurdistan was used as a case study of armed conflict to evaluate the potential use of satellite images for verification of witness reports collected by human rights groups. The Turkish army was reported to be burning forests, fields and villages as a strategy in the conflict against guerrilla uprising. This paper concludes that satellite images are useful to validate witness reports of forest fires. Even though the use of this technology for human rights groups will depend on some feasibility factors such as prices, access and expertise, the images proved to be key for analysis of spatial aspects of conflict and valuable for reconstructing a more trustworthy picture.

  7. Crystal Structure of Human [Beta]-Hexosaminidase B: Understanding the Molecular Basis of Sandhoff and Tay-Sachs Disease

    SciTech Connect

    Mark, Brian L.; Mahuran, Don J.; Cherney, Maia M.; Zhao, Dalian; Knapp, Spencer; James, Michael N.G.

    2010-12-01

    In humans, two major {beta}-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits {alpha} and {beta} (60% identity), whereas Hex B is a homodimer of {beta}-subunits. Interest in human {beta}-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G{sub M2}-ganglioside (G{sub M2}). Hex A degrades G{sub M2} by removing a terminal N-acetyl-D-galactosamine ({beta}-GalNAc) residue, and this activity requires the G{sub M2}-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4 {angstrom}) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2 {angstrom}) or NAG-thiazoline (2.5 {angstrom}). From these, and the known X-ray structure of the G{sub M2}-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how {alpha} and {beta}-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease ({beta}-subunit mutations) and Tay-Sachs disease ({alpha}-subunit mutations).

  8. Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells.

    PubMed Central

    Chen, J K; Hoshi, H; McKeehan, W L

    1987-01-01

    Myo-intimal proteoglycan metabolism is thought to be important in blood vessel homeostasis, blood clotting, atherogenesis, and atherosclerosis. Human platelet-derived transforming growth factor type beta (TGF-beta) specifically stimulated synthesis of at least two types of chondroitin sulfate proteoglycans in nonproliferating human adult arterial smooth muscle cells in culture. Stimulation of smooth muscle cell proteoglycan synthesis by smooth muscle cell growth promoters (epidermal growth factor, platelet-derived growth factor, and heparin-binding growth factors) was less than 20% of that elicited by TGF-beta. TGF-beta neither significantly stimulated proliferation of quiescent smooth muscle cells nor inhibited proliferating cells. The extent of TGF-beta stimulation of smooth muscle cell proteoglycan synthesis was similar in both nonproliferating and growth-stimulated cells. TGF-beta, which is a reversible inhibitor of endothelial cell proliferation, had no comparable effect on endothelial cell proteoglycan synthesis. These results are consistent with the hypothesis that TGF-beta is a cell-type-specific regulator of proteoglycan synthesis in human blood vessels and may contribute to the myo-intimal accumulation of proteoglycan in atherosclerotic lesions. Images PMID:3474655

  9. Reduction of 3-keto-5 beta-cholanoic acid to lithocholic and isolithocholic acids by human liver cytosol in vitro.

    PubMed

    Amuro, Y; Yamade, W; Maebo, A; Hada, T; Higashino, K

    1985-10-23

    The formation of lithocholic and isolithocholic acids from 3-keto-5 beta-cholanoic acid by human liver cytosol was examined in vitro. Liver cytosol was incubated at various pH levels with 3-keto-5 beta-cholanoic acid in a phosphate buffer containing NADPH or NADH; the products formed were analyzed by gas chromatography. Results showed that human liver cytosol reduced 3-keto-5 beta-cholanoic acid to lithocholic acid at a pH level of 7.0 or above and to isolithocholic acid at a pH level of 6.0 or below when NADPH was used as a coenzyme, and it was reduced to isolithocholic acid only when NADH was used. Furthermore, two peaks for the reducing enzymes could be clearly found by column chromatography of Affi-Gel Blue. These results indicate that human liver cytosol contains two enzymes acting on reduction of 3-keto-5 beta-cholanoic acid to lithocholic and isolithocholic acids, which are dependent on the pH level and the use of NADPH or NADH in vitro. Since the 3 beta-dehydrogenation was inhibited by the addition of pyrazole, an alcohol dehydrogenase inhibitor or ethanol, and the major peak of 3 beta-hydroxysteroid dehydrogenase coincided with the peak of alcohol dehydrogenase on Affi-Gel Blue chromatography, at least some of the cytosolic 3 beta-hydroxysteroid dehydrogenase seemed to be identical to or to have characteristics similar to alcohol dehydrogenase. PMID:2932163

  10. Suppression of beta oscillations in the subthalamic nucleus following cortical stimulation in humans

    PubMed Central

    Doyle Gaynor, L M F; Kühn, A A; Dileone, M; Litvak, V; Eusebio, A; Pogosyan, A; Androulidakis, A G; Tisch, S; Limousin, P; Insola, A; Mazzone, P; Di Lazzaro, V; Brown, P

    2008-01-01

    It is unclear how subthalamic nucleus activity is modulated by the cerebral cortex. Here we investigate the effect of transcranial magnetic stimulation (TMS) of the cortex on oscillatory subthalamic local field potential activity in the 8–35 Hz (alpha/beta) band, as exaggerated synchronization in this band is implicated in the pathophysiology of parkinsonism. We studied nine patients with Parkinson’s disease (PD) to test whether cortical stimulation can modulate synchronized oscillations in the human subthalamic nucleus. With patients at rest, single-pulse TMS was delivered every 5 s over each primary motor area and supplementary motor area at intensities of 85–115% resting motor threshold. Subthalamic local field potentials were recorded from deep brain stimulation electrodes implanted into this nucleus for the treatment of PD. Motor cortical stimulation suppressed beta activity in the subthalamic nucleus from ∼0.2 to 0.6 s after TMS (repeated measures anova; main effect of time, P<0.01; main effect of side, P=0.03), regardless of intensity. TMS over the supplementary motor area also reduced subthalamic beta activity at 95% (P=0.05) and 115% resting motor threshold (P=0.01). The oscillatory activity decreased to 80 ± 26% of baseline (averaged across sites and stimulation intensities). Suppression with subthreshold stimuli confirmed that these changes were centrally driven and not due to peripheral afference. The results may have implications for mechanisms underlying the reported therapeutic benefits of cortical stimulation. PMID:18657185

  11. Alterations in brain dopamine and serotonin metabolism during the development of tolerance to human beta-endorphin in rats.

    PubMed

    Van Loon, G R; De Souza, E B; Kim, C

    1978-12-01

    Repeated intracisternal injections of human beta-endorphin lead to development of tolerance with respect to the catalepsy, analgesia, and hypothermia which are seen following a single injection. The initial injection of beta-endorphin results in increases in the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in neostriatum, as well as increases in the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in hypothalamus and brainstem and a decrease in 5-HIAA in hippocampus. In the present study, we report changes in metabolism of dopamine and serotonin in specific brain areas during the development of tolerance to beta-endorphin. Thus, the development of tolerance to beta-endorphin with respect to catalepsy, analgesia, and hypothermia may be mediated by development of tolerance to the effects of beta-endorphin on brain dopamine and serotonin release.

  12. T cell receptor V beta gene usage in a human alloreactive response. Shared structural features among HLA-B27-specific T cell clones

    PubMed Central

    1990-01-01

    A strategy, based on using V beta family-specific oligonucleotides, was developed for specific amplification and direct sequencing of human TCR V beta genes. With this strategy, it was possible to undertake a structural analysis of TCRs from human T cell clones in specific responses. 12 HLA-B27-specific cytotoxic clones were examined. The results reveal a nonrandom use of V beta gene diversity in this alloreactive response in that: (a) the clones express a restricted number of V beta segments, including a subset of V beta families that are significantly more related to one another than to most other V beta families; (b) five of seven clones having a particular reaction pattern with HLA-B27 subtypes possess Alanine at the D-J junction; and (c) identical J beta segments are found associated in several instances with identical or highly homologous V beta gene segments. In addition, two new V beta 13 members are reported. PMID:1691261

  13. Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.

    PubMed Central

    Lotz, M; Rosen, F; McCabe, G; Quach, J; Blanco, F; Dudler, J; Solan, J; Goding, J; Seegmiller, J E; Terkeltaub, R

    1995-01-01

    Articular cartilage chondrocytes have the unique ability to elaborate large amounts of extracellular pyrophosphate (PPi), and transforming growth factor beta (TGF beta) appears singular among cartilage regulatory factors in stimulating PPi production. TGF beta caused a time and dose-dependent increase in intracellular and extracellular PPi in human articular chondrocyte cultures. TGF beta and interleukin 1 beta (IL-1 beta) antagonistically regulate certain chondrocyte functions. IL-1 beta profoundly inhibited basal and TGF beta-induced PPi elaboration. To address mechanisms involved with the regulation of PPi synthesis by IL-1 beta and TGF beta, we analyzed the activity of the PPi-generating enzyme NTP pyrophosphohydrolase (NTPPPH) and the PPi-hydrolyzing enzyme alkaline phosphatase. Human chondrocyte NTPPPH activity was largely attributable to plasma cell membrane glycoprotein 1, PC-1. Furthermore, TGF beta induced comparable increases in the activity of extracellular PPi, intracellular PPi, and cellular NTPPPH and in the levels of PC-1 protein and mRNA in chondrocytes as well as a decrease in alkaline phosphatase. All of these TGF beta-induced responses were completely blocked by IL-1 beta. Thus, IL-1 beta may be an important regulator of mineralization in chondrocytes by inhibiting TGF beta-induced PPi production and PC-1 expression. Images Fig. 4 Fig. 5 PMID:7479785

  14. Dual promoter activation by the human beta-globin locus control region.

    PubMed Central

    Bresnick, E H; Felsenfeld, G

    1994-01-01

    The human beta-globin locus control region (LCR) is necessary for high-level and position-independent expression of globin genes in erythroid cells. A variety of mechanisms have been proposed for the cis-activation of individual members of the beta-globin gene family by the LCR located 10-50 kilobases upstream. It is not known, however, whether a given LCR can activate all developmentally appropriate globin family members on its chromosome or whether, within a given chromosome, the LCR must be committed to activating only a single gene. We have devised an experiment to distinguish between these possibilities. This experiment takes advantage of the fact that if two genes in a cluster are transcriptionally active and their promoters, therefore, are in a conformation hypersensitive to nucleases, restriction enzymes that cleave the promoters will excise the intervening chromatin fragment. The Apa I sites on human fetal G gamma- and A gamma-globin gene promoters are accessible to cleavage in nuclei from the human erythroleukemia cell line K562, which expresses these genes, but not in HeLa cells. We find that Apa I digestion leads to excision in high yield of the fragment spanning these promoters, showing that a LCR element is capable of sharing its activating function among members of a gene cluster on a single chromosome. Images PMID:8108408

  15. Overexpression of human aspartyl(asparaginyl)beta-hydroxylase in hepatocellular carcinoma and cholangiocarcinoma.

    PubMed Central

    Lavaissiere, L; Jia, S; Nishiyama, M; de la Monte, S; Stern, A M; Wands, J R; Friedman, P A

    1996-01-01

    To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the

  16. Generation of Beta Cells from Human Pluripotent Stem Cells: Potential for Regenerative Medicine

    PubMed Central

    Nostro, Maria Cristina; Keller, Gordon

    2015-01-01

    The loss of beta cells in Type I Diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I Diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic and induced pluripotent stem cells. Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from hPSCs and as a consequence, it is now possible to routinely generate human insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the ‘first transition’. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hPSCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this

  17. beta 1 integrin inhibition dramatically enhances radiotherapy efficacy in human breast cancer xenografts

    SciTech Connect

    Park, Catherine C.; Park, Catherine C.; Zhang, Hui J.; Yao, Evelyn S.; Park, Chong J.; Bissell, Mina J.

    2008-06-02

    {beta}1 integrin signaling has been shown to mediate cellular resistance to apoptosis after exposure to ionizing radiation (IR). Other signaling molecules that increase resistance include Akt, which promotes cell survival downstream of {beta}1 integrin signaling. We showed previously that {beta}1 integrin inhibitory antibodies, AIIB2, enhance apoptosis and decrease growth in human breast cancer cells in 3 dimensional laminin-rich extracellular matrix (3D lrECM) cultures and in vivo. Here we asked whether AIIB2 could synergize with IR to modify Akt-mediated IR resistance. We used 3D lrECM cultures to test the optimal combination of AIIB2 with IR treatment of two breast cancer cell lines, MCF-7 and HMT3522-T4-2, as well as T4-2 myr-Akt breast cancer colonies or HMT3522-S-1, which form normal organotypic structures in 3D lrECM. Colonies were assayed for apoptosis and {beta}1 integrin/Akt signaling pathways were evaluated using western blot. In addition, mice bearing MCF-7 xenografts were used to validate the findings in 3D lrECM. We report that AIIB2 increased apoptosis optimally post-IR by down regulating Akt in breast cancer colonies in 3D lrECM. In vivo, addition of AIIB2 after IR significantly enhanced tumor growth inhibition and apoptosis compared to either treatment alone. Remarkably, the degree of tumor growth inhibition using AIIB2 plus 2 Gy radiation was similar to that of 8 Gy alone. We showed previously that AIIB2 had no discernible toxicity in mice; here, its addition allowed for a significant reduction in the IR dose that was necessary to achieve comparable growth inhibition and apoptosis in breast cancer xenografts in vivo.

  18. Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes.

    PubMed

    George, M D; Vollberg, T M; Floyd, E E; Stein, J P; Jetten, A M

    1990-07-01

    This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis. PMID:1972706

  19. Ionizing radiation predisposes non-malignant human mammaryepithelial cells to undergo TGF beta-induced epithelial to mesenchymaltransition

    SciTech Connect

    Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Costes, Sylvain; Gascard, Philippe; Mott, Joni D.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2007-04-06

    Transforming growth factor {beta}1 (TGF{beta}) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGF{beta}, activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGF{beta}-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGF{beta} (0.4 ng/ml), or double-treated. All double-treated (IR+TGF{beta}) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, {beta}-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel{trademark}. Neither radiation nor TGF{beta} alone elicited EMT, even though IR increased chronic TGF{beta} signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGF{beta}-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGF{beta}, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  20. Differential response of nontumorigenic and tumorigenic human papillomavirus type 16-positive epithelial cells to transforming growth factor beta 1.

    PubMed

    Braun, L; Dürst, M; Mikumo, R; Gruppuso, P

    1990-11-15

    The transforming growth factor (TGF) beta s are multifunctional polypeptide growth factors with diverse biological effects, including inhibition of epithelial cell proliferation both in vitro and in vivo. To investigate the possible role of TGF beta 1 in the regulation of papillomavirus infection and papillomavirus-associated transformation, we compared the response to TGF beta 1 of normal keratinocytes, human papillomavirus, type 16 (HPV 16)-positive-immortalized keratinocytes (nontumorigenic), and HPV 16-positive cervical carcinoma cells (tumorigenic) with respect to DNA synthesis and protooncogene expression. All HPV 16-immortalized cell lines were nearly as inhibited by TGF beta 1 as normal keratinocytes, whereas two cervical carcinoma cell lines (Caski and Siha) were refractory to growth inhibition by TGF beta 1. Cell surface receptors for TGF beta 1 were present on both normal and carcinoma cell lines. In all cases, growth inhibition by TGF beta 1 was accompanied by suppression of Steady-state levels of c-myc mRNA. In contrast, TGF beta 1 induced the expression of c-jun mRNA transcripts in normal, immortalized, and tumorigenic cells. We also studied the effect of TGF beta 1 on HPV 16 mRNA expression. Steady-state levels of HPV 16 mRNA transcripts were suppressed by TGF beta 1 in the nontumorigenic HPK cells but were unaffected in the tumorigenic lines. These findings suggest that TGF beta 1 may be an in vivo modulator of HPV infection and that loss of responsiveness to this growth inhibitory signal may be involved in HPV-associated malignant transformation. PMID:2171761

  1. Beta-lipotropin is the major component of the plasma opioid response to surgical stress in humans

    SciTech Connect

    Porro, C.A.; Facchinetti, F.; Bertellini, E.; Petraglia, F.; Stacca, R.; Barbieri, G.C.; Genazzani, A.R.

    1987-12-07

    There is growing experimental evidence that beta-endorphin immunoreactivity is raised by surgical stress in patients undergoing general anesthesia. As the assay methods employed to date did not allow to fully discriminate between beta-endorphin and its immediate precursor, beta-lipotropin, the authors have investigated in the present study plasma levels of these two peptides by separating them by chromatography on plasma extracts prior to radioimmunoassay. Beta-lipotropin, but not beta-endorphin, plasma levels were found to be significantly elevated during surgery in the general anesthesia group, while no change was found in either peptide concentration in the spinal one. Cortisol plasma levels also increased significantly 90 minutes after the beginning of surgery. Although the sampling time they adopted may have prevented them from detecting an early peak of beta-endorphin during the first 30 minutes of surgery, the major component of the pituitary opioid response to surgical stress appears to be related to beta-lipotropin. This is in agreement with results of experimental work on various kinds of stress in animals and humans and seems to rule out a role for plasma beta-endorphin in post-operative analgesia. 38 references, 1 figure, 1 table.

  2. Mutation of tyrosine-141 inhibits insulin-promoted tyrosine phosphorylation and increased responsiveness of the human beta 2-adrenergic receptor.

    PubMed Central

    Valiquette, M; Parent, S; Loisel, T P; Bouvier, M

    1995-01-01

    The ability of insulin to promote phosphorylation of the human beta 2-adrenergic receptor (beta 2AR) was assessed in Chinese hamster fibroblasts transfected with beta 2AR cDNA. Phosphotyrosine residues were detected in purified beta 2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulin-promoted phosphorylation of the beta 2AR was accompanied by an increase in the beta-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the beta 2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the beta 2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor. Images PMID:8521811

  3. Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-{beta} signaling

    SciTech Connect

    Kim, Reuben H.; Lieberman, Mark B.; Lee, Rachel; Shin, Ki-Hyuk; Mehrazarin, Shebli; Oh, Ju-Eun; Park, No-Hee; Kang, Mo K.

    2010-10-01

    We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-{beta} signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-{beta} signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-{beta}1 levels, phosphorylation of Smad2/3, and increased expression of p15{sup INK4B} and p57{sup KIP2}. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-{beta}1 induced dephosphorylation of Smad2/3, and diminished the level of p15{sup INK4B} and p57{sup KIP2}. Moreover, Bmi-1 expression led to the inhibition of TGF-{beta}-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15{sup INK4B}, and p57{sup KIP2}. In addition, an exposure of senescent NHOK to TGF-{beta} receptor I kinase inhibitor or anti-TGF-{beta} antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-{beta} signaling pathway in NHOK.

  4. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

    PubMed

    Wang, Peng; Alvarez-Perez, Juan-Carlos; Felsenfeld, Dan P; Liu, Hongtao; Sivendran, Sharmila; Bender, Aaron; Kumar, Anil; Sanchez, Roberto; Scott, Donald K; Garcia-Ocaña, Adolfo; Stewart, Andrew F

    2015-04-01

    Types 1 and 2 diabetes affect some 380 million people worldwide. Both ultimately result from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with a peak percentage (∼2%) engaged in the cell cycle in the first year of life. In embryonic life and after early childhood, beta cell replication is barely detectable. Whereas beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts. Hence, there remains an urgent need for antidiabetic therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, using a high-throughput small-molecule screen (HTS), we find that analogs of the small molecule harmine function as a new class of human beta cell mitogenic compounds. We also define dual-specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine and the nuclear factors of activated T cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation and differentiation. Using three different mouse and human islet in vivo-based models, we show that harmine is able to induce beta cell proliferation, increase islet mass and improve glycemic control. These observations suggest that harmine analogs may have unique therapeutic promise for human diabetes therapy. Enhancing the potency and beta cell specificity of these compounds are important future challenges. PMID:25751815

  5. Accelerated phagocytosis of amyloid-beta by mouse and human microglia overexpressing the macrophage colony-stimulating factor receptor.

    PubMed

    Mitrasinovic, Olivera M; Murphy, Greer M

    2002-08-16

    Microglia surrounding A beta plaques in Alzheimer's disease and in the APPV717F transgenic mouse model of Alzheimer's disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated A beta. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of A beta in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-microm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of A beta was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of A beta. M-CSFR antibody blocking experiments demonstrated that increased A beta uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of A beta after immunization.

  6. Regenerative capacity of human satellite cells: the mitotic clock in cell transplantation.

    PubMed

    Di Donna, S; Renault, V; Forestier, C; Piron-Hamelin, G; Thiesson, D; Cooper, R N; Ponsot, E; Decary, S; Amouri, R; Hentati, F; Butler-Browne, G S; Mouly, V

    2000-01-01

    In this communication, we will review the problems caused by cell-mediated gene therapy, taking skeletal muscle as a physiological model. In particular we have utilised vectors transferring telomerase under the control of retroviral promoters into human satellite cells. The set of results presented here has several implications regarding gene therapy trials. Nevertheless, more experiments will be required to fully validate this cellular model and to use telomerase to safely extend the lifespan of putative gene therapy vectors.

  7. The effect of a short-term ritodrine treatment on the concentration of beta-adrenergic receptors in human myometrium.

    PubMed

    Ekblad, U; Grenman, S; Kaila, T

    1987-01-01

    The concentration of beta-adrenoceptors in human myometrium has been studied of women giving birth by elective cesarean section at term. Eight of the woman were treated with intravenous ritodrine two hours prior to the operation. In the control group seven women received physiological saline with the same infusion rate as ritodrine in the study group. The concentration of beta-receptors in the myometrium of the lower uterine segment was determined with radioligand binding assay. Ritodrine treatment decreased the available beta-receptors significantly.

  8. Evidence for beta3-adrenoceptor subtypes in relaxation of the human urinary bladder detrusor: analysis by molecular biological and pharmacological methods.

    PubMed

    Takeda, M; Obara, K; Mizusawa, T; Tomita, Y; Arai, K; Tsutsui, T; Hatano, A; Takahashi, K; Nomura, S

    1999-03-01

    The purpose of the present study was to confirm the presence of beta3-adrenoceptor subtype in the relaxation of human urinary bladder detrusor tissue by reverse transcription-polymerase chain reaction (PCR); direct sequencing of the PCR product, in situ hybridization; and isometric contraction. Using reverse transcription-PCR, the mRNAs of three receptor subtypes (beta1, beta2, and beta3) were expressed in the human urinary bladder detrusor tissue. Direct sequencing of the PCR product of the above beta3-adrenoceptor revealed no mutation in the amplified regions. In situ hybridization with digoxygenin-labeled oligonucleotide probe revealed the presence of the mRNA of beta3-adrenoceptor subtype in the smooth muscle of the urinary bladder. The relaxant effects of isoproterenol (a nonselective beta-adrenoceptor agonist); ZD7114, BRL37344, and CGP12177A (putative selective beta3-adrenoceptor agonists); and SR59230A (a putative selective beta3-adrenoceptor antagonist) were tested using an isometric contraction technique. Isoproterenol in either the presence or absence of both atenolol (a beta1-adrenoceptor-selective antagonist) and butoxamine (a beta2-adrenoceptor-selective antagonist) revealed a relaxant effect on the carbachol-induced contraction of the human urinary bladder detrusor. Both BRL37344 and CGP12177A also revealed relaxant effects on the human urinary bladder detrusor, but ZD7114 did not elicit any relaxation. These results suggest that beta3-adrenoceptor may have some role in urine storage in the human urinary bladder. PMID:10027879

  9. The Use of a Satellite Human Interaction System in Conjunction with a Satellite Media Distribution System. Satellite Technology Demonstration, Technical Report No. 0217.

    ERIC Educational Resources Information Center

    Dale, Joyce B.

    Satellite Technology Demonstration (STD) was designed to provide data on the use of a satellite to deliver educational programs to 56 rural-isolated schools in eight Rocky Mountain States. Three series were broadcast: (1) a junior high school career development, (2) career development for public school administrators and teachers, and (3) topical…

  10. Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes.

    PubMed Central

    Peterson, K R; Clegg, C H; Huxley, C; Josephson, B M; Haugen, H S; Furukawa, T; Stamatoyannopoulos, G

    1993-01-01

    Transgenic mice were generated using a purified 248-kb yeast artificial chromosome (YAC) bearing an intact 82-kb human beta-globin locus and 148 kb of flanking sequence. Seventeen of 148 F0 pups were transgenic. RNase protection analysis of RNA isolated from the blood of 13 gamma- and beta-globin-positive founders showed that only the human beta-globin gene was expressed in the adult founders. Studies of F1 and F2 fetuses demonstrated that the genes of the beta-locus YAC displayed the proper developmental switches in beta-like globin gene expression. Expression of epsilon- and gamma-globin, but not beta-globin, was observed in the yolk sac, there was only minor gamma and mostly beta expression in the 14-day liver, and only beta mRNA in the blood of the adult animals. Structural data showed that the locus was intact. These results indicate that it is now possible to dissect regulatory mechanisms within the context of an entire locus in vivo by using the ability to perform mutagenesis efficiently in yeast via homologous recombination, followed by purification of the altered YAC and its introduction into mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8356061

  11. Rising serum values of beta-subunit human chorionic gonadotrophin (hCG) in patients with progressive vulvar carcinomas.

    PubMed Central

    de Bruijn, H. W.; ten Hoor, K. A.; Krans, M.; van der Zee, A. G.

    1997-01-01

    Elevated serum levels of the beta-subunit of human chorionic gonadotrophin (hCG) were measured in 50% of patients with locoregional recurrences or progressive vulvar carcinoma (n = 14). At diagnosis of vulvar cancer, however, the incidence of elevated serum levels was low (5%) in 104 patients. The rising serum levels during progression of disease indicate that the synthesis of the beta-subunit hCG can be increased in vulvar carcinoma. PMID:9099973

  12. Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.

    PubMed Central

    Jörnvall, H; Hempel, J; Vallee, B L; Bosron, W F; Li, T K

    1984-01-01

    The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme. PMID:6374651

  13. Beta modulation reflects name retrieval in the human anterior temporal lobe: an intracranial recording study.

    PubMed

    Abel, Taylor J; Rhone, Ariane E; Nourski, Kirill V; Ando, Timothy K; Oya, Hiroyuki; Kovach, Christopher K; Kawasaki, Hiroto; Howard, Matthew A; Tranel, Daniel

    2016-06-01

    Naming people, places, and things is a fundamental human ability that is often impaired in patients with language-dominant anterior temporal lobe (ATL) dysfunction or ATL resection as part of epilepsy treatment. Convergent lines of evidence point to the importance of the ATL in name retrieval. The physiologic mechanisms that mediate name retrieval in the ATL, however, are not well understood. The purpose of this study was to characterize the electrophysiologic responses of the human ATL during overt cued naming of famous people and objects. Eight neurosurgical patients with suspected temporal lobe epilepsy who underwent implantation of intracranial electrodes for seizure focus localization were the subjects of this study. Specialized coverage of the ATL was achieved in each subject. The subjects named pictures of U.S. presidents and images of common hand-held tools. Event-related band power was measured for each ATL recording site. Both the left and right ATL demonstrated robust and focal increases in beta-band (14-30 Hz) power during person and tool naming. The onset of this response typically occurred at 400 ms but sometimes as early as 200 ms. Visual naming of famous people and tools is associated with robust and localized modulation of the beta band in both the left and right ATL. Measurement of visual naming responses may provide the groundwork for future mapping modalities to localize eloquent cortex in the ATL. PMID:27075536

  14. The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds.

    PubMed

    Johnson, James D

    2016-10-01

    The production of fully functional insulin-secreting cells to treat diabetes is a major goal of regenerative medicine. In this article, I review progress towards this goal over the last 15 years from the perspective of a beta cell biologist. I describe the current state-of-the-art, and speculate on the general approaches that will be required to identify and achieve our ultimate goal of producing functional beta cells. The need for deeper phenotyping of heterogeneous cultures of stem cell derived islet-like cells in parallel with a better understanding of the heterogeneity of the target cell type(s) is emphasised. This deep phenotyping should include high-throughput single-cell analysis, as well as comprehensive 'omics technologies to provide unbiased characterisation of cell products and human beta cells. There are justified calls for more detailed and well-powered studies of primary human pancreatic beta cell physiology, and I propose online databases of standardised human beta cell responses to physiological stimuli, including both functional and metabolomic/proteomic/transcriptomic profiles. With a concerted, community-wide effort, including both basic and applied scientists, beta cell replacement will become a clinical reality for patients with diabetes. PMID:27473069

  15. The quest to make fully functional human pancreatic beta cells from embryonic stem cells: climbing a mountain in the clouds.

    PubMed

    Johnson, James D

    2016-10-01

    The production of fully functional insulin-secreting cells to treat diabetes is a major goal of regenerative medicine. In this article, I review progress towards this goal over the last 15 years from the perspective of a beta cell biologist. I describe the current state-of-the-art, and speculate on the general approaches that will be required to identify and achieve our ultimate goal of producing functional beta cells. The need for deeper phenotyping of heterogeneous cultures of stem cell derived islet-like cells in parallel with a better understanding of the heterogeneity of the target cell type(s) is emphasised. This deep phenotyping should include high-throughput single-cell analysis, as well as comprehensive 'omics technologies to provide unbiased characterisation of cell products and human beta cells. There are justified calls for more detailed and well-powered studies of primary human pancreatic beta cell physiology, and I propose online databases of standardised human beta cell responses to physiological stimuli, including both functional and metabolomic/proteomic/transcriptomic profiles. With a concerted, community-wide effort, including both basic and applied scientists, beta cell replacement will become a clinical reality for patients with diabetes.

  16. CENPT bridges adjacent CENPA nucleosomes on young human α-satellite dimers

    PubMed Central

    Thakur, Jitendra; Henikoff, Steven

    2016-01-01

    Nucleosomes containing the CenH3 (CENPA or CENP-A) histone variant replace H3 nucleosomes at centromeres to provide a foundation for kinetochore assembly. CENPA nucleosomes are part of the constitutive centromere associated network (CCAN) that forms the inner kinetochore on which outer kinetochore proteins assemble. Two components of the CCAN, CENPC and the histone-fold protein CENPT, provide independent connections from the ∼171-bp centromeric α-satellite repeat units to the outer kinetochore. However, the spatial relationship between CENPA nucleosomes and these two branches remains unclear. To address this issue, we use a base-pair resolution genomic readout of protein–protein interactions, comparative chromatin immunoprecipitation (ChIP) with sequencing, together with sequential ChIP, to infer the in vivo molecular architecture of the human CCAN. In contrast to the currently accepted model in which CENPT associates with H3 nucleosomes, we find that CENPT is centered over the CENPB box between two well-positioned CENPA nucleosomes on the most abundant centromeric young α-satellite dimers and interacts with the CENPB/CENPC complex. Upon cross-linking, the entire CENPA/CENPB/CENPC/CENPT complex is nuclease-protected over an α-satellite dimer that comprises the fundamental unit of centromeric chromatin. We conclude that CENPA/CENPC and CENPT pathways for kinetochore assembly are physically integrated over young α-satellite dimers. PMID:27384170

  17. Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans.

    PubMed

    Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat H; Ricordi, Camillo; Roper, Stephen D; Berggren, Per-Olof; Caicedo, Alejandro

    2011-06-19

    Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.

  18. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

    SciTech Connect

    Li, Xiaoou; Liu, Lian; Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan; Wen, Fuqiang

    2014-11-28

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.

  19. Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.

    PubMed

    Takekoshi, K J; Oh, Y H; Westerman, K W; London, I M; Leboulch, P

    1995-03-28

    Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients.

  20. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

    PubMed Central

    Sadelain, M; Wang, C H; Antoniou, M; Grosveld, F; Mulligan, R C

    1995-01-01

    Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of

  1. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  2. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  3. Increased thermogenic responsiveness to intravenous beta-adrenergic stimulation in habitually exercising humans is not related to skeletal muscle beta2-adrenergic receptor density.

    PubMed

    Stob, Nicole R; Seals, Douglas R; Jørgen, Jensen; van Baak, Marleen A; Steig, Amy J; Lindstrom, Rachel C; Bikman, Benjamin T; Bell, Christopher

    2007-09-01

    Habitually exercising adults demonstrate greater thermogenic responsiveness to beta-adrenergic receptor (beta-AR) stimulation compared with their sedentary peers, but the molecular mechanisms involved are unknown. To determine the possible role of increased beta-AR density, we studied 32 healthy adults: 17 habitual aerobic exercisers (age 45 +/- 5 years, 11 males) and 15 sedentary (49 +/- 5 years, 7 males). Maximal oxygen uptake (43.7 +/- 2.5 versus 31.6 +/- 2.9 ml kg(-1) min(-1), P = 0.002, mean +/- S.E.M.) and vastus lateralis muscle maximal citrate synthase activity (1.70 +/- 0.36 versus 0.58 +/- 0.11 micromol min(-1) g(-1), P = 0.008) were higher in the habitually exercising subjects. Resting energy expenditure (EE) adjusted for fat-free mass (FFM) was similar in the habitually exercising (5903 +/- 280 kJ day(-1)) and sedentary adults (6054 +/- 289 kJ day(-1), P = 0.43). The percentage increase in EE (DeltaEE%; indirect calorimetry, ventilated hood) above resting EE in response to beta-AR stimulation (intravenous isoproterenol at 6, 12 and 24 ng (kg FFM)(-1) min(-1)) was greater (7.1 +/- 1.2, 13.7 +/- 1.0, 20.7 +/- 1.3 versus 5.9 +/- 0.9, 9.9 +/- 1.4, 15.9 +/- 1.70%, respectively, P = 0.04), and the dose of isoproterenol required to increase EE by 10% above resting EE was lower (8.2 +/- 1.5 versus 17.1 +/- 4.1 ng (kg FFM)(-1) min(-1), P = 0.03) in the habitually exercising adults. In contrast, vastus lateralis muscle beta(2)-AR density was similar in the habitually exercising and sedentary subjects (7.46 +/- 0.29 versus 7.44 +/- 0.60 fmol (mg dry weight muscle)(-1), P = 0.98), and was not related to DeltaEE% (r = 0.02, P = 0.94) or to the isoproterenol dose required to increase EE by 10% above resting EE (r = -0.06, P = 0.76). These findings indicate that increased beta(2)-AR density is not a mechanism contributing to the greater thermogenic responsiveness to beta-AR stimulation in adult humans who regularly perform aerobic exercise.

  4. Physiologically based pharmacokinetics of radioiodinated human beta-endorphin in rats. An application of the capillary membrane-limited model

    SciTech Connect

    Sato, H.; Sugiyama, Y.; Sawada, Y.; Iga, T.; Hanano, M.

    1987-07-01

    In order to simulate the distribution and elimination of radioiodinated human beta-endorphin (/sup 125/I-beta-EP) after iv bolus injection in rats, we proposed a physiologically based pharmacokinetic model incorporating diffusional transport of /sup 125/I-beta-EP across the capillary membrane. This model assumes that the distribution of /sup 125/I-beta-EP is restricted only within the blood and the tissue interstitial fluid, and that a diffusional barrier across the capillary membrane exists in each tissue except the liver. The tissue-to-blood partition coefficients were estimated from the ratios of the concentration in tissues to that in arterial plasma at the terminal (pseudoequilibrium) phase. The total body plasma clearance (9.0 ml/min/kg) was appropriately assigned to the liver and kidney. The transcapillary diffusion clearances of /sup 125/I-beta-EP were also estimated and shown to correlate linearly with that of inulin in several tissues. Numerically solving the mass-balance differential equations as to plasma and each tissue simultaneously, simulated concentration curves of /sup 125/I-beta-EP corresponded well with the observed data. It was suggested by the simulation that the initial rapid disappearance of /sup 125/I-beta-EP from plasma after iv injection could be attributed in part to the transcapillary diffusion of the peptide.

  5. Transfer of cow's milk beta-lactoglobulin to human serum after a milk load: a pilot study.

    PubMed Central

    Lovegrove, J A; Osman, D L; Morgan, J B; Hampton, S M

    1993-01-01

    A sensitive and specific enzyme linked immunosorbent assay (ELISA) was developed for the quantification of cow's milk beta-lactoglobulin in human serum. The assay had a sensitivity of 80 pg/ml (2 standard deviations from zero) and did not cross react significantly with any other tested milk proteins. The absorption of cow's milk beta-lactoglobulin and beta-lactoglobulin immunoglobulin G antibody levels after a milk load was investigated in eight healthy non-allergic individuals. There was a significant variation in the circulating beta-lactoglobulin antigen levels during the six hour period after feeding (p < 0.01) for the combined data. A biphasic absorption pattern was seen in all subjects except one subject who had undetectable levels. No significant variation with time was observed for the beta-lactoglobulin IgG antibody levels and no correlation was found between the beta-lactoglobulin antigen levels and the beta-lactoglobulin IgG antibody levels. This pilot study offered information on the quantity of immunogenic cows' milk protein transferred to the blood after a milk load in non-allergic healthy adults and confirmed a biphasic profile of antigen transfer. The sensitivity of the assay used has shown that transfer of food antigen does occur in the majority of healthy nonallergic subjects. PMID:8432474

  6. Primary adult human astrocytes as an ex vivo vehicle for beta-glucuronidase delivery in the brain.

    PubMed

    Serguera, C; Sarkis, C; Ridet, J L; Colin, P; Moullier, P; Mallet, J

    2001-06-01

    Astrocytes are a good candidate cell type for brain transplantation: They are endogenous to the CNS, they have efficient secretory machinery, and they play a major role in neuronal support. We assessed the potential of genetically modified primary adult human astrocytes as vehicles for the delivery of secreted molecules in the mammalian CNS. We report that such cells can be efficiently transduced by a recombinant adenoviral vector carrying the human beta-glucuronidase cDNA (Ad/CMV*beta-glu) and that the transduced astrocytes produce large amounts of the enzyme. Released beta-glucuronidase could be captured, in vitro, by primary neurons and astrocytes and by a neuroblastoma cell line and beta-glucuronidase-deficient fibroblasts. Following grafting into the mouse striatum, adult human astrocytes survived and expressed the transgene for at least 8 weeks. Moreover, the dosage of beta-glucuronidase activity within the grafted brains revealed high enzymatic levels at a long distance from the graft. These experiments document the grafting of engineered primary adult human astrocytes, allowing the release of a secreted therapeutic factor throughout the brain.

  7. Human beta-globin gene polymorphisms characterized in DNA extracted from ancient bones 12,000 years old.

    PubMed Central

    Béraud-Colomb, E; Roubin, R; Martin, J; Maroc, N; Gardeisen, A; Trabuchet, G; Goosséns, M

    1995-01-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the beta-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for beta-globin frameworks by sequencing through two variable positions and for a polymorphic (AT) chi (T) gamma microsatellite 500 bp upstream of the beta-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. Images Figure 2 Figure 3 PMID:8533755

  8. Beta 2-adrenergic receptor regulation of human neutrophil function is sexually dimorphic.

    PubMed

    de Coupade, Catherine; Gear, Robert W; Dazin, Paul F; Sroussi, Herve Y; Green, Paul G; Levine, Jon D

    2004-12-01

    While the mechanisms underlying the marked sexual dimorphism in inflammatory diseases are not well understood, the sexually dimorphic sympathoadrenal axis profoundly affects the inflammatory response. We tested whether adrenergic receptor-mediated activation of human neutrophil function is sexually dimorphic, since neutrophils provide the first line of defense in the inflammatory response. There was a marked sexual dimorphism in beta(2)-adrenergic receptor binding, using the specific beta(2)-adrenergic receptor ligand, [(3)H]-dihydroalprenolol, with almost three times more binding sites on neutrophils from females (20,878 +/- 2470) compared to males (7331 +/- 3179). There was also a marked sexual dimorphism in the effects of isoprenaline, a beta-adrenergic receptor agonist, which increased nondirected locomotion (chemokinesis) in neutrophils obtained from females, while having no effect on neutrophils from males. Isoprenaline stimulated the release of a chemotactic factor from neutrophils obtained from females, but not from males. This chemotactic factor acts on the G protein-coupled CXC chemokine receptor 2 (CXCR2) chemokine receptor, since an anti-CXCR2 antibody and the selective nonpeptide CXCR2 antagonist SB225002, inhibited chemotaxis produced by this factor. While interleukin- (IL-) 8 is a principal CXCR2 ligand, isoprenaline did not produce an increase in IL-8 release from neutrophils. IL-8-induced chemotaxis was inhibited in a sexually dimorphic manner by isoprenaline, which also stimulated release of a mediator from neutrophils that induced chemotaxis, that was inhibited by anti-CXCR2 antibodies. These findings indicate an important role for adrenergic receptors in the modulation of neutrophil trafficking, which could contribute to sex-differences in the inflammatory response. PMID:15477226

  9. beta. -Endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

    SciTech Connect

    Diamant, M.; Henricks, P.A.J.; Nijkamp, F.P.; de Wied, D. )

    1989-01-01

    In the present study, the immunomodulatory effect of {beta}-endorphin ({beta}-E) and shorter pro-opiomelancortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity and their non-opioid derivatives were tested. With the exception of {alpha}-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10{sup {minus}17}M and higher than 10{sup {minus}8}M were without effect. {beta}-E and dT{beta}E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations. {gamma}-E and dT{gamma}E proved to be less potent inhibitors, reaching maximal effect at higher concentrations. DE{gamma}E exerted an even less pronounced but still significant suppressive effect at the concentration of 10{sup {minus}10}M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone.

  10. Haplotyping the human T-cell receptor. beta. -chain gene complex by use of restriction fragment length polymorphisms

    SciTech Connect

    Charmley, P.; Chao, A.; Gatti, R.A. ); Concannon, P. ); Hood, L. )

    1990-06-01

    The authors have studied the genetic segregation of human T-cell receptor {beta}-chain (TCR{beta}) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR{beta} gene complex. Analysis of allele distributions between TCR{beta} genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR{beta} gene complex. The results should provide new insight into recent reports of disease associations with the TCR{beta} gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.

  11. Effects of beta 2-adrenoceptor agonists on anti-IgE-induced contraction and smooth muscle reactivity in human airways.

    PubMed Central

    Gorenne, I; Labat, C; Norel, X; De Montpreville, V; Guillet, M C; Cavero, I; Brink, C

    1995-01-01

    1. The beta 2-adrenoceptor agonists, salbutamol, salmeterol and RP 58802 relaxed basal tone of human isolated bronchial smooth muscle. Salmeterol- and RP 58802-induced relaxations persisted for more than 4 h when the medium was constantly renewed after treatment. 2. Salbutamol, salmeterol and RP 58802 reversed histamine-induced contractions in human airways (pD2 values: 6.15 +/- 0.21, 6.00 +/- 0.19 and 6.56 +/- 0.12, respectively). 3. Anti-IgE-induced contractions were significantly inhibited immediately after pretreatment of preparations with beta 2-adrenoceptor agonists (10 microM). However, when tissues were treated with beta 2-agonists and then washed for a period of 4 h, salmeterol was the only agonist which significantly inhibited the anti-IgE response. 4. Histamine response curves were shifted to the right immediately after pretreatment of tissues with the beta 2-adrenoceptor agonists (10 microM; 20 min), but maximal contractions were not affected. After a 4 h washing period, the histamine curves were not significantly different from controls. Concentration-effect curves to acetylcholine (ACh) or leukotriene C4 (LTC4) were not significantly modified after beta 2-agonist pretreatment. 5. These results suggest that beta 2-adrenoceptor agonists may prevent anti-IgE-induced contraction by inhibition of mediator release rather than alterations of those mechanisms involved in airway smooth muscle contraction. PMID:7780648

  12. The Wnt/beta-catenin pathway is activated during advanced arterial aging in humans.

    PubMed

    Marchand, Alexandre; Atassi, Fabrice; Gaaya, Amira; Leprince, Pascal; Le Feuvre, Claude; Soubrier, Florent; Lompré, Anne-Marie; Nadaud, Sophie

    2011-04-01

    Aging is the main risk factor for cardiovascular diseases, but the associated molecular mechanisms are poorly understood. The Wnt signaling pathway was shown to be induced during aging in muscle and in the skin, but the regulation and role of Wnt signaling in the aged vessel have not yet been addressed. While screening for age-related changes in gene expression in the intima/media of human mammary arteries, we observed that the expression of frizzled 4 (Fzd4), a Wnt receptor, and of several targets of the Wnt/β-catenin/TCF signaling pathway [Wnt-inducible secreted protein 1 (WISP1), versican, osteopontin (SPP1), insulin-like growth factor binding protein 2 (IGFBP-2), and p21] were modified with age, suggesting an activation of the Wnt/β-catenin pathway. In contrast, we did not observe any regulation of forkhead transcription factor (FoxO) target genes. Beta-catenin-activating phosphorylation at position Ser675 was increased in aging mammary arteries, confirming the activation of this pathway. We confirmed in vitro that Wnt3a or Wnt1 treatment of human vascular smooth muscle cells (VSMCs) induced β-catenin phosphorylation at Ser675 and WISP1, SPP1, and IGFBP-2 expression. In vitro, Wnt treatment induced proliferation and cyclin D1 expression in VSMC from young (6 weeks old) rats but not in cells from older rats (8 months old), even though low-density lipoprotein receptor-related protein 6 and β-catenin phosphorylation, and β-catenin nuclear translocation demonstrated β-catenin activation in both cell types. Beta-catenin silencing demonstrated that Wnt induction of cyclin D1 expression is β-catenin dependent. Altogether, our data show that the Wnt/β-catenin/TCF pathway is activated in aging human mammary artery cells, but fails to induce the proliferation of aging vascular cells. PMID:21108734

  13. Crystallization and preliminary X-ray analysis of recombinant human acid beta-glucocerebrosidase, a treatment for Gaucher's disease

    NASA Technical Reports Server (NTRS)

    Roeber, Dana; Achari, Aniruddha; Manavalan, Partha; Edmunds, Tim; Scott, David L.

    2003-01-01

    Acid beta-glucocerebrosidase (N-acylsphingosyl-1-O-beta-D-glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme (Genzyme Corporation, Cambridge, MA, USA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is used in the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. Here, the crystallization and the initial diffraction analysis of Cerezyme are reported. The crystals are C-centered orthorhombic, with unit-cell parameters a = 285.0, b = 110.2, c = 91.7 A. A 99.9% complete data set has been collected to 2.75 A with an R(sym) of 8.8%.

  14. Crystallization and Preliminary X-ray analysis of Human Recombinant Acid beta-glucocerebrosidase, a treatment for Gaucher's Disease

    NASA Technical Reports Server (NTRS)

    Roeber, Dana F.; Achari, Aniruddha; Manavalan, Partha; Edmunds, Tim; Scott, David L.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Acid beta-glucocerebrosidase (N-acylsphingosyl - O - beta-D - glucoside:glucohydrolase) is a lysosomal glycoprotein that catalyzes the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Inadequate levels of this enzyme underly the pathophysiology of Gaucher's disease. Cerezyme(R) (Genzyme Corporation, Cambridge, MA) is a partially deglycosylated form of recombinant human acid beta-glucocerebrosidase that is commercially available for the treatment of Gaucher patients. Although acid beta-glucocerebrosidase belongs to a large family of glycosidases, relatively little is known regarding its structural biology. We report the crystallization and the initial diffraction analysis of Cerezyme(R). The crystals are C-centered orthorhombic, with unit-cell parameters of a = 285.0 A, b = 110.2 A, and c = 91.7 A. A 99.9 A complete data set has been collected to 2.75 A with an R(sub sym) of 8.8 %.

  15. Simulating alpha/beta selectivity at the human thyroid hormone receptor: consensus scoring using multidimensional QSAR.

    PubMed

    Vedani, Angelo; Zumstein, Martin; Lill, Markus A; Ernst, Beat

    2007-01-01

    We present a consensus-scoring study on the human thyroid hormone receptor alpha and beta using two receptor-modeling concepts (software Quasar and Raptor) that are based on multidimensional QSAR and allow for the explicit simulation of induced fit. The binding mode of 82 agonists and indirect antagonists, spanning an activity range of seven orders of magnitude in K(i), was identified through flexible docking to the respective X-ray crystal structures (Yeti software) and represented by a 4D data set with up to four conformations per compound. The receptor surrogates for the thyroid alpha receptor converged at a cross-validated r(2) of 0.846/0.919 (64 training compounds; for Quasar and Raptor, respectively) and yielded a predictive r(2) of 0.812/0.814 (18 test compounds); the models for the thyroid beta receptor resulted in a cross-validated r(2) of 0.823/0.909 and a predictive r(2) of 0.665/0.796, respectively. Consensus was achieved as, on average, the calculated activities of the training set differ only by a factor of 2.2 in K(i) and those of the test set by a factor of 2.8 when predicted by Quasar and Raptor, respectively.

  16. Secondary structure determination of human. beta. -endorphin by /sup 1/H NMR spectroscopy

    SciTech Connect

    Lichtarge, O.; Jardetzky, O.; Li, C.H.

    1987-09-08

    The /sup 1/H NMR spectra of human ..beta..-endorphin indicate that the peptide exists in random-coil form in aqueous solution but becomes helical in mixed solvent. Thermal denaturation NMR experiments show that in water there is no transition between 24 and 75/sup 0/C, while a slow noncooperative thermal unfolding is observed in a 60% methanol-40% water mixed solvent in the same temperature range. These findings are consistent with circular dichroism studies by other workers concluding that ..beta..-endorphin is a random coil in water but that it forms 50% ..cap alpha..-helix or more in mixed solvents. The peptide in the mixed water-methanol solvent was further studied by correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments. These allow a complete set of assignments to be made and establish two distinct stretches over which the solvent induces formation of ..cap alpha..-helices: the first occurs between Tyr-1 and Thr-12 and the second between Leu-14 and extending to Lys-28. There is evidence that the latter is capped by a turn occurring between Lys-28 and Glu-31. These helices form at the enkephalin receptor binding site, which is at the amino terminus, and at the morphine receptor binding site, located at the carboxyl terminus. The findings suggest that these two receptors may specifically recognize ..cap alpha..-helices.

  17. Dose assessment of bremsstrahlung induced by beta-emitting radioisotopes of uranium-238 series and lead in human tissues.

    PubMed

    Manjunatha, H C

    2014-01-01

    In the natural uranium-238 decay series, pure beta isotopes such as (234)Th, (234)Pa, (214)Pb, (214)Bi, (210)Pb and (210)Bi are released. The few lead isotopes such as (211)Pb, (212)Pb, (213)Pb and (215)Pb are good beta emitters. In certain nuclear reactions of reactor these isotopes are released. These beta isotopes have maximum beta energies, which induce the bremsstrahlung radiation. The bremsstrahlung component of these beta isotopes has been traditionally ignored in dosimetry calculations. The shapes of bremsstrahlung spectra are a basic ingredient in the understanding and quantification of beta-ray dosimetry. The bremsstrahlung spectra produced by these high-energy isotopes such as (234)Th, (234)Pa, (214)Pb, (214)Bi, (210)Pb, (210)Bi, (211)Pb, (212)Pb, (213)Pb and (215)Pb in bone, muscle and teeth are studied, and the computed spectral distributions are presented. The spectral shapes are primarily responsible for variations in the shapes of depth-dose distributions. They are intended to provide a quick and convenient reference for spectral shapes and to give an indication of the wide variation in these shapes. The evaluated beta bremsstrahlung dose as a function distance for the studied nuclides is also presented. The efficiency, intensity and dose rate of bremsstrahlung induced by beta isotopes of natural uranium-238 decay series and beta-emitting lead isotopes in human tissues such as brain, breast, heart, kidney, liver, muscle, pancreas and bone have also been studied in the present investigation. The values of bremsstrahlung dosimetric parameters are low for pancreas, but they are high for bone. For all studied tissues these parameters are high for (234)Pa, but low for (210)Pb.

  18. Selective killing of human monocytes and cytokine release provoked by sphingomyelinase (beta-toxin) of Staphylococcus aureus.

    PubMed Central

    Walev, I; Weller, U; Strauch, S; Foster, T; Bhakdi, S

    1996-01-01

    The best-known activity of Staphylococcus aureus sphingomyelinase C, alias beta-toxin, is as a hemolysin that provokes hot-cold lysis of erythrocytes which contain substantial amounts of sphingomyelin in the plasma membrane. Sheep erythrocytes are most susceptible, and we found that one hemolytic unit, representing the toxin concentration that elicits 50% hemolysis of 2.5 X 10(8) erythrocytes per ml, corresponds to 0.05 enzyme units or to approximately 0.25 microg of sphingomyelinase per ml. The cytotoxic action of beta-toxin on nucleated cells has not been described in any detail before, and the present investigation was undertaken to fill this information gap. We now identify beta-toxin as a remarkably potent monocytocidal agent. At a concentration of 0.001 U/ml, corresponding to approximately 5 ng/ml, beta-toxin killed over 50% of human monocytes (10(6) cells per ml) within 60 min. By contrast, 1 to 5 microg of beta-toxin per ml had no cytocidal effects on human granulocytes, fibroblasts, lymphocytes, or erythrocytes. A selective monocytocidal action was also observed with sphingomyelinase C from Bacillus cereus and a Streptomyces sp., whereas phospholipase A2 and phospholipase D at 100 U/ml were without effect. Monocytes succumbing to the action of beta-toxin processed and released interleukin-1beta, soluble interleukin-6 receptor, and soluble CD14 into the supernatant. Thus, monocyte killing by beta-toxin is associated with cytokine-related events that are important for the initiation and progression of infectious disease. These findings uncover a potentially important role for sphingomyelinase as a determinant of microbial pathogenicity. PMID:8757823

  19. High divergence in primate-specific duplicated regions: Human and chimpanzee Chorionic Gonadotropin Beta genes

    PubMed Central

    2008-01-01

    Background Low nucleotide divergence between human and chimpanzee does not sufficiently explain the species-specific morphological, physiological and behavioral traits. As gene duplication is a major prerequisite for the emergence of new genes and novel biological processes, comparative studies of human and chimpanzee duplicated genes may assist in understanding the mechanisms behind primate evolution. We addressed the divergence between human and chimpanzee duplicated genomic regions by using Luteinizing Hormone Beta (LHB)/Chorionic Gonadotropin Beta (CGB) gene cluster as a model. The placental CGB genes that are essential for implantation have evolved from an ancestral pituitary LHB gene by duplications in the primate lineage. Results We shotgun sequenced and compared the human (45,165 bp) and chimpanzee (39,876 bp) LHB/CGB regions and hereby present evidence for structural variation resulting in discordant number of CGB genes (6 in human, 5 in chimpanzee). The scenario of species-specific parallel duplications was supported (i) as the most parsimonious solution requiring the least rearrangement events to explain the interspecies structural differences; (ii) by the phylogenetic trees constructed with fragments of intergenic regions; (iii) by the sequence similarity calculations. Across the orthologous regions of LHB/CGB cluster, substitutions and indels contributed approximately equally to the interspecies divergence and the distribution of nucleotide identity was correlated with the regional repeat content. Intraspecies gene conversion may have shaped the LHB/CGB gene cluster. The substitution divergence (1.8–2.59%) exceeded two-three fold the estimates for single-copy loci and the fraction of transversional mutations was increased compared to the unique sequences (43% versus ~30%). Despite the high sequence identity among LHB/CGB genes, there are signs of functional differentiation among the gene copies. Estimates for dn/ds rate ratio suggested a purifying

  20. Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.

    PubMed Central

    Guénette, S Y; Chen, J; Jondro, P D; Tanzi, R E

    1996-01-01

    We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855266

  1. Role of acetylation and charge in antimicrobial peptides based on human beta-defensin-3.

    PubMed

    Papanastasiou, Emilios Andrew; Hua, Quyen; Sandouk, Aline; Son, U Hyon; Christenson, Andrew James; Van Hoek, Monique Louise; Bishop, Barney Michael

    2009-07-01

    Cationic antimicrobial peptides are an evolutionarily ancient and essential element of innate immunity in higher organisms. The precise mechanism by which these peptides exert their antimicrobial activity on bacteria is not well understood. Decapeptides based on the C-terminus of human beta-defensin-3 were designed and evaluated to study the role of charge in defining the antimicrobial activity and selectivity of these peptides against Escherichia coli. Acetylated derivatives of these peptides were prepared in order to further evaluate how positively charged primary amines contribute to potency in these small antimicrobial peptides. These peptides enabled us to explore the relationship between net charge, charge distribution and antimicrobial activity. While the results indicate that net charge is a major factor in antimicrobial activity in these peptides, the actual relationship between charge and potency appears to be more complex.

  2. Regulation of the human. beta. -actin promoter by upstream and intron domains

    SciTech Connect

    Ng, Sunyu )); Gunning, P.; Kedes, L. ); Liu, Shuhui National Tsing Hua Univ., Hsinchu ); Leavitt, J. )

    1989-01-25

    The authors have identified three regulatory domains of the complex human {beta}-actin gene promoter. They span a region of about 3,000 bases, from not more than {minus}2,011 bases upstream of the mRNA cap site to within the 5{prime} intron (832 bases long). A distal upstream domain contains at least one enhancer-like element. A proximal upstream domain, with a CArG (for CC(A+T rich){sub 6}GG) motif found in all known mammalian actin genes, seems to confer serum, but not growth factor, inducibility. The third domain is within the evolutionarily conserved 3{prime} region of the first intron and contains a 13 base-pair sequence, identical to the upstream sequence with the CArG motif. This domain also contains sequences that are both serum and fibroblast growth inducible.

  3. Production of human beta interferon in insect cells infected with a Baculovirus expression vector

    SciTech Connect

    Smith, G.E.; Summers, M.D.; Fraser, M.J.

    1983-12-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) was used as an expression vector for human beta interferon. By using specially constructed plasmids, the protein-coding sequences for interferon were linked to the AcNPV promoter for the gene encoding for polyhedrin, the major occlusion protein. The interferon gene was inserted at various locations relative to the AcNPV polyhedrin transcriptional and translational signals, and the interferon-polyhedrin hybrid genes were transferred to infectious AcNPV expression vectors. Biologically active interferon was produced, and greater than 95% was secreted from infected insect cells. A maximum of ca. 5 x 10/sup 6/ U of interferon activity was produced by 10/sup 6/ infected cells. These results demonstrate that AcNPV should be suitable for use as a eucaryotic expression vector for the production of products from cloned genes.

  4. Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets.

    PubMed Central

    Oka, Y; Orth, D N

    1983-01-01

    Human epidermal growth factor (hEGF) has previously been isolated from urine and probably is identical to human beta-urogastrone (hUG). Immunoreactive hEGF/UG has been found in the plasma of normal subjects. In this study, using immunoaffinity chromatography to extract hEGF/UG from plasma, we found that immunoreactive hEGF/UG in blood was associated with blood platelets. It was present in platelet-rich, but not platelet-poor plasma and serum, and was found predominantly in the platelet fraction of whole blood. Sephadex G-50 Fine gel-exclusion chromatography of an extract of outdated blood bank platelets revealed two hEGF/UG components, one of which eluted in the void volume, and the other of which coeluted with purified standard hEGF/UG. The former hEGF/UG component was a high-molecular weight form that was cleaved into hEGF/UG by incubation with either mouse EGF/UG-associated arginine esterase or trypsin. It appeared to be identical to the high-molecular weight hEGF/UG previously reported in human urine, except for its apparently equal activities in radioimmunoassay and radioreceptor assay. The latter hEGF/UG component was immunologically, biologically, and physiochemically indistinguishable from highly purified hEGF/UG from human urine and was immunologically different from purified human platelet-derived growth factor. Platelet-associated hEGF/UG may account for the mitogenic activity of serum in cell lines in which platelet-derived growth factor is not active. Since hEGF/UG appears to be liberated from platelets during coagulation, platelet-associated EGF/UG may be involved in normal vascular and tissue repair and in the pathogenesis of atherosclerotic lesions. The discovery that the EGF/UG in plasma is associated with blood platelets raises important new possibilities for its role in human health and disease. PMID:6603475

  5. Compounds exhibiting selective efficacy for different beta subunits of human recombinant gamma-aminobutyric acid A receptors.

    PubMed

    Smith, Alison J; Oxley, Beth; Malpas, Sallie; Pillai, Gopalan V; Simpson, Peter B

    2004-11-01

    Inhibitory GABA(A) receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk(-) cells stably expressing human recombinant GABA(A) subunits (alpha1beta1-3gamma2s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl(-) buffer using a voltage/ion probe reader. The influence of different betasubunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at alpha1beta1gamma2, alpha1beta2gamma2, and alpha1beta3gamma2 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC(50) values of 2.4 +/- 0.2, 2.5 +/- 0.2, and 1.3 +/- 0.1 microM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC(50) values of 8.7 +/- 0.9, 9.2 +/- 0.5, and 11.7 +/- 1.2, and 43.7 +/- 6.4, 24.8 +/- 1.6, and 26.1 +/- 2.4 microM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, pentobarbital, and steroids, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one, were unaffected by the beta isoform present in the receptor complex. However, several compounds displayed beta2/3 subunit selectivity, notably loreclezole, R(-)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at beta2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at alpha1beta1gamma2. Diflunisal was the most efficacious compound, eliciting greater

  6. Beta-Cryptoxanthin Suppresses the Growth of Immortalized Human Bronchial Epithelial Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent findings of an inverse association between beta-cryptoxanthin and lung cancer risk in several observational epidemiologic studies suggests that beta-cryptoxanthin could potentially act as a chemopreventive agent against lung cancer. However, the biological activity of beta-cryptoxanthin and m...

  7. Genomic organization of the human {beta}-catenin gene (CTNNB1)

    SciTech Connect

    Nollet, F.; Berx, G.; Molemans, F.; Roy, F. van

    1996-03-05

    The cytoplasmic {beta}-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human {beta}-catenin gene (CTNNB1) by analysis cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 pb and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of {beta}-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5{prime} end and 766 at the 3{prime} end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3{prime} UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5{prime}-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NFkB, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5{prime}-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line. 53 refs., 5 figs., 2 tabs.

  8. Myomir dysregulation and reactive oxygen species in aged human satellite cells.

    PubMed

    Di Filippo, Ester Sara; Mancinelli, Rosa; Pietrangelo, Tiziana; La Rovere, Rita Maria Laura; Quattrocelli, Mattia; Sampaolesi, Maurilio; Fulle, Stefania

    2016-04-29

    Satellite cells that reside on the myofibre surface are crucial for the muscle homeostasis and regeneration. Aging goes along with a less effective regeneration of skeletal muscle tissue mainly due to the decreased myogenic capability of satellite cells. This phenomenon impedes proper maintenance and contributes to the age-associated decline in muscle mass, known as sarcopenia. The myogenic potential impairment does not depend on a reduced myogenic cell number, but mainly on their difficulty to complete a differentiation program. The unbalanced production of reactive oxygen species in elderly people could be responsible for skeletal muscle impairments. microRNAs are conserved post-transcriptional regulators implicated in numerous biological processes including adult myogenesis. Here, we measure the ROS level and analyze myomiR (miR-1, miR-133b and miR-206) expression in human myogenic precursors obtained from Vastus lateralis of elderly and young subjects to provide the molecular signature responsible for the differentiation impairment of elderly activated satellite cells.

  9. Human factors analysis of workstation design: Earth Radiation Budget Satellite Mission Operations Room

    NASA Technical Reports Server (NTRS)

    Stewart, L. J.; Murphy, E. D.; Mitchell, C. M.

    1982-01-01

    A human factors analysis addressed three related yet distinct issues within the area of workstation design for the Earth Radiation Budget Satellite (ERBS) mission operation room (MOR). The first issue, physical layout of the MOR, received the most intensive effort. It involved the positioning of clusters of equipment within the physical dimensions of the ERBS MOR. The second issue for analysis was comprised of several environmental concerns, such as lighting, furniture, and heating and ventilation systems. The third issue was component arrangement, involving the physical arrangement of individual components within clusters of consoles, e.g., a communications panel.

  10. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

    SciTech Connect

    Zhao, Yong; Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema; Zhang, Yongkang; Jain, Sumit; Skidgel, Randal A.; Prabhakar, Bellur S.; Mazzone, Theodore; Holterman, Mark J.

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

  11. The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

    PubMed Central

    Kato, Y; Muto, T; Tomura, T; Tsumura, H; Watarai, H; Mikayama, T; Ishizaka, K; Kuroki, R

    1996-01-01

    Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different. Images Fig. 1 Fig. 3 PMID:8610159

  12. Beta-interferon treatment reduces human herpesvirus-6 viral load in multiple sclerosis relapses but not in remission.

    PubMed

    Alvarez-Lafuente, Roberto; De Las Heras, Virginia; Bartolomé, Manuel; Picazo, Juan José; Arroyo, Rafael

    2004-01-01

    To determine whether the DNA prevalence of human herpesvirus-6 (HHV-6), the viral load and the prevalence of both HHV-6 variants in relapsing-remitting multiple sclerosis (RRMS) patients in exacerbation are altered by beta-interferon (IFN-beta) treatment, in comparison with RRMS patients in remission, we analyzed HHV-6 (A and B) genomes in 189 serum samples by quantitative real-time polymerase chain reaction: 105 of the RRMS patients were receiving IFN-beta treatment (48 in exacerbation) and 84 were untreated (36 in relapse). The results were as follows. (1) Prevalence decrease because of IFN-beta treatment was not significant: 25% of RRMS patients in relapse vs. 15.9% in remission (p = 0.45). (2) Viral load was twice as much in untreated patients in relapse than in treated ones. (3) We only found variant A. Since IFN-beta treatment is able to significantly reduce HHV-6 viral load in RRMS patients in relapse, but not in remission, we suggest a role for HHV-6 in the pathogenesis of multiple sclerosis exacerbations and an antiviral role for IFN-beta treatment in RRMS.

  13. Assimilation of Real-Time Satellite And Human Sensor Networks for Modeling Natural Disasters

    NASA Astrophysics Data System (ADS)

    Aulov, O.; Halem, M.; Lary, D. J.

    2011-12-01

    We describe the development of underlying technologies needed to address the merging of a web of real time satellite sensor Web (SSW) and Human Sensor Web (HSW) needed to augment the US response to extreme events. As an initial prototyping step and use case scenario, we consider the development of two major system tools that can be transitioned from research to the responding operational agency for mitigating coastal oil spills. These tools consist of the capture of Situation Aware (SA) Social Media (SM) Data, and assimilation of the processed information into forecasting models to provide incident decision managers with interactive virtual spatial temporal animations superimposed with probabilistic data estimates. The system methodologies are equally applicable to the wider class of extreme events such as plume dispersions from volcanoes or massive fires, major floods, hurricane impacts, radioactive isotope dispersions from nuclear accidents, etc. A successful feasibility demonstration of this technology has been shown in the case of the Deepwater Horizon Oil Spill where Human Sensor Networks have been combined with a geophysical model to perform parameter assessments. Flickr images of beached oil were mined from the spill area, geolocated and timestamped and converted into geophysical data. This data was incorporated into General NOAA Operational Modeling Environment (GNOME), a Lagrangian forecast model that uses near real-time surface winds, ocean currents, and satellite shape profiles of oil to generate a forecast of plume movement. As a result, improved estimates of diffusive coefficients and rates of oil spill were determined. Current approaches for providing satellite derived oil distributions are collected from a satellite sensor web of operational and research sensors from many countries, and a manual analysis is performed by NESDIS. A real time SA HSW processing system based on geolocated SM data from sources such as Twitter, Flickr, YouTube etc., greatly

  14. Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341.

    PubMed Central

    Moffett, S; Mouillac, B; Bonin, H; Bouvier, M

    1993-01-01

    Exposure of beta 2-adrenergic receptors to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist-promoted phosphorylation of the beta 2-adrenergic receptor (beta 2AR) by protein kinase A and the beta-adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2-adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide-sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor-stimulated adenylyl cyclase response was observed but resulted from the agonist-induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist-promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8381352

  15. Serum levels of interleukin-6, interleukin-1beta and human chorionic gonadotropin in pre-eclamptic and normal pregnancy.

    PubMed

    Casart, Ysabel C; Tarrazzi, Katiuska; Camejo, María I

    2007-05-01

    Studies in placentas from the first trimester and in vitro models indicate that interleukin (IL)-1beta and IL-6 induce the release of human chorionic gonadotropin (hCG). During pre-eclampsia there is an increase of pro-inflammatory cytokines; however, its relationship with hCG levels during the third trimester of pregnancy has not been determined. The aim of the present study was to evaluate the relationship between blood levels of IL-6, IL-1beta and hCG in normal pregnancy and pre-eclampsia. Blood samples during the third trimester of pregnancy from women with severe pre-eclampsia (n = 20) or normal pregnancy (n = 20) were assayed for hCG by immunoassay, IL-6 and IL-1beta by enzyme-linked immunosorbent assay. Serum level of IL-6 was significantly higher in pre-eclamptic than in normal women (16.5 +/- 2.1 vs. 4.9 +/- 1.1 pg/ml); however, IL-1beta was similar in both groups. Although hCG was higher in pre-eclampsia than normal pregnancy, the difference was not statistically significant. Furthermore, IL-1beta in normal pregnancy was correlated negatively with hCG (r = -0.69, p < 0.001). In conclusion, serum levels of IL-6 were increased in pre-eclampsia but were not correlated with hCG or IL-1beta; however, in normal pregnancy there was a negative correlation between IL-1beta and hCG. The interaction between IL-1beta and hCG at the third trimester needs to be investigated.

  16. Induction of TNF-alpha production from human peripheral blood monocytes with beta-1,3-glucan oligomer prepared from laminarin with beta-1,3-glucanase from Bacillus clausii NM-1.

    PubMed

    Miyanishi, Nobumitsu; Iwamoto, Yoshiko; Watanabe, Etsuo; Odaz, Tatsuya

    2003-01-01

    We prepared a beta-1,3-glucan oligomer (DP> or = 4) from laminarin (DP: 25-30) derived from Laminaria digitata with beta-1,3-glucanase, and examined its effect on human peripheral blood monocytes. Conditioned medium prepared by incubating monocytes (MC-CM) with the beta-1,3-glucan oligomer showed strong inhibitory activity against the proliferation of human leukemic U937 cells. Since the beta-1,3-glucan oligomer had no direct cytotoxic effect on U937 cells up to 1000 microg/ml, the cytotoxicity of the MC-CM may be due to cytotoxic cytokines produced from monocytes stimulated by the beta-1,3-glucan oligomer. On the other hand, the MC-CM prepared with original laminarin had little effect on the growth of U937 cells. The cytotoxicity of the MC-CM prepared with the beta-1,3-glucan oligomer was significantly reduced by an anti-TNF-alpha antibody, but the anti-TNF-beta antibody had no effect. Our results suggest that the enzymatically depolymerized beta-1,3-glucan oligomer induces TNF-alpha production from human monocytes. PMID:16233391

  17. Increased expression of heme oxygenase-1 in human retinal pigment epithelial cells by transforming growth factor-beta.

    PubMed

    Kutty, R K; Nagineni, C N; Kutty, G; Hooks, J J; Chader, G J; Wiggert, B

    1994-05-01

    Antibodies specific for heme oxygenase-1 (HO-1) were produced in rabbits, using the multiple antigen peptide (MAP) technique, and were employed to investigate the ability of transforming growth factor-beta 1 (TGF-beta 1) to induce the HO-1 protein in cultured human retinal pigment epithelial (RPE) cells. Western blot analyses showed that the cytokine induced HO-1 in these cells in a time- and dose-dependent manner. TGF-beta 1 also increased the mRNA for HO-1 in treated cells prior to the increase in HO-1 protein. The induction was effectively blocked by a neutralizing antibody preparation against TGF-beta 1. When tested under similar conditions, other growth factors such as basic fibroblast growth factor-I, platelet-derived growth factor, insulin-like growth factor, transforming growth factor-alpha, and epidermal growth factor did not show appreciable induction of HO-1. Lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma were also not inducers, although TGF-beta 2 effectively induced HO-1. Heavy metal ions and thiol reagents were also highly potent inducers of HO-1 in human RPE cells. The induction of HO-1 by TGF-beta 1 was also observed in bovine choroid fibroblasts, but not in HELA, HEL or bovine corneal fibroblasts. Our results demonstrate for the first time that HO-1 can be induced by an important cytokine, TGF-beta 1, causing an increase in the expression of both HO-1 message and protein in specific neuroepithelial and fibroblast cells.

  18. Expression of human {beta}-defensin-2 gene induced by CpG-DNA in human B cells

    SciTech Connect

    Han, Su Ho; Kim, Young-Eun; Park, Jeong-A; Park, Jae-Bong; Kim, Yong-Sun; Lee, Younghee; Choi, Ihn-Geun; Kwon, Hyung-Joo

    2009-11-20

    Defensins have a broad range of antimicrobial activity against bacteria, fungi, and viruses. The expression of human {beta}-defensin-2 (hBD-2) is prevalently observed in epithelial cells and is induced by bacterial infection. Here, we have shown that the expression of the hBD-2 gene and release of hBD-2 protein into the medium is up-regulated in response to CpG-DNA in human B cell line RPMI 8226. The induction of hBD-2 was dependent on CG sequence and phosphorothioate backbone-modification. This was also confirmed in primary human lymphocytes. To shed light on the molecular mechanism involved in hBD-2 induction by CpG-DNA, we examined the contribution of the NF-{kappa}B signaling pathway in RPMI 8226 cells. Suppression of MyD88 function and inhibition of NF-{kappa}B nuclear localization blocked hBD-2 induction. The NF-{kappa}B pathway inhibitors also abolished hBD-2 induction. These results may contribute to a better understanding on the therapeutic effects of CpG-DNA against infectious diseases.

  19. Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin.

    PubMed

    Bigliardi-Qi, M; Sumanovski, L T; Büchner, S; Rufli, T; Bigliardi, P L

    2004-01-01

    We have previously shown that human epidermal keratinocytes express a functionally active micro-opiate receptor, which adds a new dimension to the recently developed research in neuroimmunodermatology and neurogenic inflammation in skin diseases. Human keratinocytes specifically bind and also produce beta-endorphin, the endogenous micro-opiate receptor ligand. Using confocal imaging microscopy, we could now demonstrate that micro-opiate receptors are not only expressed in keratinocytes, but also on unmyelinated peripheral nerve fibers in the dermis and epidermis. Some of the peripheral nerve fibers also express the ligand beta-endorphin. The keratinocytes positive for beta-endorphin staining are clustered around the terminal ends of the unmyelinated nerve fibers. Therefore the opiate receptor system seems to be crucial in the direct communication between nerves and skin. The keratinocytes can influence the unmyelinated nerve fibers in the epidermis directly via secreting beta-endorphin. On the other hand, nerve fibers can also secrete beta-endorphin and influence the migration, differentiation and probably also the cytokine production pattern of keratinocytes.

  20. TRIBUTYLTIN ALTERS SECRETION OF INTERLEUKIN 1 BETA FROM HUMAN IMMUNE CELLS

    PubMed Central

    Brown, Shyretha; Whalen, Margaret

    2014-01-01

    Tributyltin (TBT) has been used as a biocide in industrial applications such as wood preservation, antifouling paint, and antifungal agents. Due to its many uses, it contaminates the environment and has been found in human blood samples. Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that promotes cell growth, tissue repair, and immune response regulation. Produced predominately by both monocytes and macrophages, IL-1β appears to increase the invasiveness of certain tumors. This study shows that TBT modifies the secretion of IL-1β from increasingly reconstituted preparations of human immune cells. IL-1β secretion was examined after 24h, 48h, or 6 day exposures to TBT in highly enriched human NK cells, monocyte-depleted (MD) peripheral blood mononuclear cells (MD-PBMCs), PBMCs, granulocytes, and a preparation combining both PBMCs and granulocytes (PBMCs+granulocytes). TBT altered IL-1β secretion from all of the cells preparations. The 200 nM concentration of TBT normally blocked the secretion of IL-1β, while lower concentrations (usually 5-50 nM) elevated secretion of IL-1β. Examination of the signaling pathway(s) responsible for the elevated secretion of IL-1β were carried out in MD-PBMCs. Pathways examined were IL-1β processing (Caspase-1), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa B (NFκB). Results indicated that MAPK pathways (p44/42 and p38) appear to be the targets of TBT that lead to increased IL-1β secretion from immune cells. These results from human immune cells show IL-1β dysregulation by TBT is occurring ex vivo. Thus, potential for in vivo effects on pro-inflammatory cytokine levels may possibly be a consequence of TBT exposures. PMID:25382723

  1. The FBXW7 {beta}-form is suppressed in human glioma cells

    SciTech Connect

    Gu, Zhaodi; Inomata, Kenichi; Ishizawa, Kota; Horii, Akira . E-mail: horii@mail.tains.tohoku.ac.jp

    2007-03-23

    FBXW7 (F-box and WD40 domain protein 7) is an F-box protein with 7 tandem WDs (tryptophan-aspartic acid) that functions as a phosphoepitope-specific substrate recognition component of SCF (Skp1-Cul1-F-box protein) ubiquitin ligases and catalyzes the ubiquitination of proteins promoting cell proliferation, such as CCNE1, MYC, AURKA, NOTCH1, and JUN, which are frequently activated in a wide range of human cancers. FBXW7 is a candidate tumor suppressor, and mutations have been reported in some human tumors. In this study, we analyzed 84 human tumor cell lines in search for genetic alterations of FBXW7, as well as mRNA and protein expressional changes, and compared them with expression levels of the CCNE1, MYC, and AURKA proteins. We found a novel nonsense mutation in a colon cancer cell line SCC and confirmed the missense mutations in SKOV3, an ovarian cancer cell line, and LoVo, a colon cancer cell line. Moreover, suppressed expression of FBXW7 accompanied by activation of the target proteins were observed in ovarian, colon, endometrial, gastric, and prostate cancers. It is notable that highly suppressed mRNA expression of the FBXW7 {beta}-form was found in all the human glioma cell lines analyzed; enhanced expressions of CCNE1, MYC, and AURKA were observed in these cells. Our present results imply that FBXW7 plays a pivotal role in many tissues by controlling the amount of cell cycle promoter proteins and that dysfunction of this protein is one of the essential steps in carcinogenesis in multiple organs.

  2. Linking Regional Satellite Observations with Coupled Human-Ecological Systems in Global Drylands

    NASA Astrophysics Data System (ADS)

    Hutchinson, C.; Reynolds, J. F.

    2009-12-01

    The African Sahel has attracted consistent attention since a series of droughts in the 1970s and 1980s caused widespread famine and land degradation (desertification). These events spawned international conventions and sustained development efforts to increase food security and reverse poverty for the local populations, and to arrest environmental degradation. Since 1985, several studies using satellite data have described a general “greening” in response to increased rainfall trends. However, some areas show more greening while others less greening than can be explained by precipitation alone (Glob. Env. Change 15- 2005). The debated question is how to explain the residual changes: management, policy, human adaptation, or something else? Placing results in an human-ecological framework could help answer this question. Providing a meaningful assessment will allow national and international agencies to evaluate the effectiveness of alternative approaches to poverty alleviation and environmental restoration in drylands at regional and global scales.

  3. Effect of ions on antibacterial activity of human beta defensin 2.

    PubMed

    Tomita, T; Hitomi, S; Nagase, T; Matsui, H; Matsuse, T; Kimura, S; Ouchi, Y

    2000-01-01

    Human beta defensin 2 (HBD-2), the most recently discovered human defensin, has been considered to work as a host defense substance against microbial infection. Using Escherichia coli ATCC 25922, we investigated how some cations and anions influenced the antimicrobial activity of HBD-2. This activity, measured in 10 mM phosphate buffer at a concentration of 20 microg/ml, reduced significantly in the presence of 100 and 150 mM sodium or potassium chloride. The reduction was not significantly different when the total amounts of sodium and potassium ions were equal. The kind and the valence of anions (chlorine and sulfate ions) did not affect the bactericidal activity as long as the concentrations of sodium ions were equal. Divalent ions (calcium and magnesium ions) added to 10 mM of Tris buffer significantly inactivated HBD-2 at much lower concentrations (more than or equal to 0.01 mM and 0.05 mM, respectively) than the monovalent ions did. These findings suggest that HBD-2 kills the bacteria through at least two phases, which are affected independently by either monovalent or divalent ions and unaffected by anions.

  4. Effects of long-acting beta 2-adrenoceptor agonists on mast cells of rat, guinea pig, and human.

    PubMed

    Lau, H Y; Wong, P L; Lai, C K; Ho, J K

    1994-10-01

    The effects of two recently developed long-acting beta 2-adrenoceptor agonists, formoterol and salmeterol, on mast cells from different sources were compared with those of the prototype short-acting analogue, salbutamol. With the exception of high concentrations of salmeterol (> 10(-5) M), none of the tested beta 2-adrenoceptor agonists inhibited the anti-IgE-induced histamine release from rat peritoneal mast cells. In contrast, all three compounds dose dependently inhibited the immunologically induced histamine release from isolated lung mast cells of guinea pig and human at concentrations < or = 10(-5) M.

  5. Bile acid N-acetylglucosaminidation. In vivo and in vitro evidence for a selective conjugation reaction of 7 beta-hydroxylated bile acids in humans.

    PubMed Central

    Marschall, H U; Matern, H; Wietholtz, H; Egestad, B; Matern, S; Sjövall, J

    1992-01-01

    The aim of this study was to define whether N-acetylglucosaminidation is a selective conjugation pathway of structurally related bile acids in humans. The following bile acids released enzymatically from N-acetylglucosaminides were identified: 3 alpha,7 beta-dihydroxy-5 beta-cholanoic (ursodeoxycholic), 3 beta, 7 beta-dihydroxy-5 beta-cholanoic (isoursodeoxycholic), 3 beta,7 beta-dihydroxy-5 alpha-cholanoic (alloisoursodeoxycholic), 3 beta,7 beta-dihydroxy-5-cholenoic, 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic, and 3 alpha,6 alpha,7 beta-trihydroxy-5 beta-cholanoic acids. The selectivity of conjugation was studied by administration of 0.5 g ursodeoxycholic (UDCA) or hyodeoxycholic (HDCA) acids, labeled with 13C, to patients with extrahepatic cholestasis, and of 0.5 g of 13C-labeled chenodeoxycholic acid (CDCA) to patients with extra- or intrahepatic cholestasis. After administration of [24-13C]-CDCA, labeled glucosides, and the glucuronide of CDCA were excreted in similar amounts. Labeled N-acetylglucosaminides of UDCA and isoUDCA were also formed. When [24-13C]-UDCA was given, 13C-label was detected in the N-acetylglucosaminide, the glucosides, and the glucuronide of UDCA, and in the N-acetylglucosaminide of isoUDCA. In the patient studied, 32% of the total UDCA excreted in urine was conjugated with N-acetylglucosamine. In contrast, 96% of the excreted amount of [24-13C]HDCA was glucuronidated, and 13C-labeled glucosides but no N-acetylglucosaminide were detected. The selectivity of N-acetylglucosaminidation towards bile acids containing a 7 beta-hydroxyl group was confirmed in vitro using human liver and kidney microsomes and uridine diphosphate glucose (UDP)-N-acetylglucosamine. These studies show that N-acetylglucosaminidation is a selective conjugation pathway for 7 beta-hydroxylated bile acids. PMID:1602004

  6. Human-Specific SNP in Obesity Genes, Adrenergic Receptor Beta2 (ADRB2), Beta3 (ADRB3), and PPAR γ2 (PPARG), during Primate Evolution

    PubMed Central

    Takenaka, Akiko; Nakamura, Shin; Mitsunaga, Fusako; Inoue-Murayama, Miho; Udono, Toshifumi; Suryobroto, Bambang

    2012-01-01

    Adrenergic-receptor beta2 (ADRB2) and beta3 (ADRB3) are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM) in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG) is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP). All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques) had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. Conclusions These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods. PMID:22937051

  7. Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL.

    PubMed

    Plaisance, Valérie; Brajkovic, Saška; Tenenbaum, Mathie; Favre, Dimitri; Ezanno, Hélène; Bonnefond, Amélie; Bonner, Caroline; Gmyr, Valéry; Kerr-Conte, Julie; Gauthier, Benoit R; Widmann, Christian; Waeber, Gérard; Pattou, François; Froguel, Philippe; Abderrahmani, Amar

    2016-01-01

    Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment. PMID:27636901

  8. Endoplasmic Reticulum Stress Links Oxidative Stress to Impaired Pancreatic Beta-Cell Function Caused by Human Oxidized LDL

    PubMed Central

    Favre, Dimitri; Ezanno, Hélène; Bonnefond, Amélie; Bonner, Caroline; Gmyr, Valéry; Kerr-Conte, Julie; Gauthier, Benoit R.; Widmann, Christian; Waeber, Gérard; Pattou, François; Froguel, Philippe; Abderrahmani, Amar

    2016-01-01

    Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1α, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment. PMID:27636901

  9. Overexpression of IRS2 in isolated pancreatic islets causes proliferation and protects human {beta}-cells from hyperglycemia-induced apoptosis

    SciTech Connect

    Mohanty, S.; Spinas, G.A.; Maedler, K.; Zuellig, R.A.; Lehmann, R.; Donath, M.Y.; Trueb, T.; Niessen, M. . E-mail: markus.niessen@usz.ch

    2005-02-01

    Studies in vivo indicate that IRS2 plays an important role in maintaining functional {beta}-cell mass. To investigate if IRS2 autonomously affects {beta}-cells, we have studied proliferation, apoptosis, and {beta}-cell function in isolated rat and human islets after overexpression of IRS2 or IRS1. We found that {beta}-cell proliferation was significantly increased in rat islets overexpressing IRS2 while IRS1 was less effective. Moreover, proliferation of a {beta}-cell line, INS-1, was decreased after repression of Irs2 expression using RNA oligonucleotides. Overexpression of IRS2 in human islets significantly decreased apoptosis of {beta}-cells, induced by 33.3 mM D-glucose. However, IRS2 did not protect cultured rat islets against apoptosis in the presence of 0.5 mM palmitic acid. Overexpression of IRS2 in isolated rat islets significantly increased basal and D-glucose-stimulated insulin secretion as determined in perifusion experiments. Therefore, IRS2 is sufficient to induce proliferation in rat islets and to protect human {beta}-cells from D-glucose-induced apoptosis. In addition, IRS2 can improve {beta}-cell function. Our results indicate that IRS2 acts autonomously in {beta}-cells in maintenance and expansion of functional {beta}-cell mass in vivo.

  10. Modeling of conformational transitions of fibrillogenic peptide, homologous to beta-domain of human alpha-lactalbumin

    NASA Astrophysics Data System (ADS)

    Kadochnikov, V. V.; Egorov, V. V.; Shvetsov, A. V.; Kuklin, A. I.; Isaev-Ivanov, V. V.; Lebedev, D. V.

    2016-01-01

    The behavior of the peptide corresponding to beta domain of human alpha-lactalbumin (GYDTQAIVENNESTEYG, WT) has been simulated by the molecular dynamics method. It is shown that, within the model considered, the monomer of this peptide does not tend to form a stable secondary structure; however, simulation of the behavior of several peptide molecules revealed the occurrence of beta structures due to the formation of intermolecular hydrogen bonds. Since the aforementioned interactions involve the terminal portions of peptides, the influence of the tetrapeptide corresponding to the N-terminal portion of WT, TDYG (R), on the secondary structure has been analyzed. The model calculations show that the interaction of this peptide with WT monomer facilitates formation of beta-structures. It is suggested that peptide R may affect the quaternary structure of WT.

  11. Carotenoid absorption in humans consuming tomato sauces obtained from tangerine or high-beta-carotene varieties of tomatoes.

    PubMed

    Unlu, Nuray Z; Bohn, Torsten; Francis, David; Clinton, Steven K; Schwartz, Steven J

    2007-02-21

    Tomato sauces were produced from unique tomato varieties to study carotenoid absorption in humans. Tangerine tomatoes, high in cis-lycopene, especially prolycopene (7Z,9Z,7'Z,9'Z), and high-beta-carotene tomatoes as an alternative dietary source of beta-carotene were grown and processed. Sauces were served after 2 week washout periods and overnight fasting for breakfast to healthy subjects (n = 12, 6M/6F) in a randomized crossover design. The serving size was 150 g (containing 15 g of corn oil), tangerine sauce containing 13 mg of lycopene (97.0% as cis-isomers) and high-beta-carotene sauce containing 17 mg of total beta-carotene (1.6% as the 9-cis-isomer) and 4 mg of lycopene. Blood samples were collected 0, 2, 3, 4, 5, 6, 8, and 9.5 h following test meal consumption and carotenoids determined in the plasma triacylglycerol-rich lipoprotein fraction by HPLC-electrochemical detection. Baseline-corrected areas under the concentration vs time curves (AUC) were used as a measure of absorption. AUC0-9.5h values for total lycopene in the tangerine sauce group were 870 +/- 187 (nmol.h)/L (mean +/- SEM) with >99% as cis-isomers (59% as the tetra-cis-isomer). The AUC0-9.5h values for total beta-carotene and lycopene after consumption of the high-beta-carotene sauce were 304 +/- 54 (4% as 9-cis-carotene) and 118 +/- 24 (nmol.h)/L, respectively. Lycopene dose-adjusted triacylglycerol-rich lipoprotein AUC responses in the tangerine sauce group were relatively high when compared to those in the literature and the high-beta-carotene group. The results support the hypothesis that lycopene cis-isomers are highly bioavailable and suggest that special tomato varieties can be utilized to increase both the intake and bioavailability of health-beneficial carotenoids.

  12. A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia

    SciTech Connect

    Rouyer-Fessard, P.; Garel, M.C.; Domenget, C.; Guetarni, D.; Bachir, D.; Colonna, P.; Beuzard, Y. )

    1989-11-15

    The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with ({sup 3}H)N-ethylmaleimide. This pool of soluble alpha chains was 0.067 {plus minus} 0.017% of hemoglobin in blood of normal adult, 0.11 {plus minus} 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using ({sup 3}H)N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

  13. Nuclear matrix association of the human beta-globin locus utilizing a novel approach to quantitative real-time PCR.

    PubMed

    Ostermeier, G Charles; Liu, Zhandong; Martins, Rui Pires; Bharadwaj, Rikki R; Ellis, James; Draghici, Sorin; Krawetz, Stephen A

    2003-06-15

    The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.

  14. Distribution of precursor amyloid-. beta. -protein messenger RNA in human cerebral cortex: relationship to neurofibrillary tangles and neuritic plaques

    SciTech Connect

    Lewis, D.A.; Higgins, G.A.; Young, W.G.; Goldgaber, D.; Gajdusek, D.C.; Wilson, M.C.; Morrison, J.H.

    1988-03-01

    Neurofibrillary tangles (NFT) and neuritic plaques (NP), two neuropathological markers of Alzheimer disease, may both contain peptide fragments derived from the human amyloid ..beta.. protein. However, the nature of the relationship between NFT and NP and the source of the amyloid ..beta.. proteins found in each have remained unclear. The authors used in situ hybridization techniques to map the anatomical distribution of precursor amyloid-..beta..-protein mRNA in the neocortex of brains from three subjects with no known neurologic disease and from five patients with Alzheimer disease. In brains from control subjects, positively hybridizing neurons were present in cortical regions and layers that contain a high density of neuropathological markers in Alzheimer disease, as well as in those loci that contain NP but few NFT. Quantitative analyses of in situ hybridization patterns within layers III and V of the superior frontal cortex revealed that the presence of high numbers of NFT in Alzheimer-diseased brains was associated with a decrease in the number of positively hybridizing neurons compared to controls and Alzheimer-diseased brains with few NFT. These findings suggest that the expression of precursor amyloid-..beta..-protein mRNA may be a necessary but is clearly not a sufficient prerequisite for NFT formation. In addition, these results may indicate that the amyloid ..beta.. protein, present in NP in a given region or layer of cortex, is not derived from the resident neuronal cell bodies that express the mRNA for the precursor protein.

  15. Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer's disease.

    PubMed Central

    Goedert, M

    1987-01-01

    Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:3322812

  16. Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

    PubMed

    Kumar, Sanjeev; Nagl, Sylvia; Kalsi, J K; Ravirajan, C T; Athwal, Dee; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2003-12-01

    We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

  17. Prodigiosin induces the proapoptotic gene NAG-1 via glycogen synthase kinase-3beta activity in human breast cancer cells.

    PubMed

    Soto-Cerrato, Vanessa; Viñals, Francesc; Lambert, James R; Kelly, Julie A; Pérez-Tomás, Ricardo

    2007-01-01

    Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) is a bacterial metabolite that has anticancer and antimetastatic properties. However, the molecular mechanisms responsible for these abilities are not fully understood. Gene expression profiling of the human breast cancer cell line MCF-7 treated with prodigiosin was analyzed by cDNA array technology. The majority of the significantly modified genes were related to apoptosis, cell cycle, cellular adhesion, or transcription regulation. The dramatic increase of the nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1) made this gene an interesting candidate regarding the possible mechanism by which prodigiosin induces cytotoxicity in MCF-7 cells. Our results show that prodigiosin triggers accumulation of the DNA-damage response tumor-suppressor protein p53 but that NAG-1 induction was independent of p53 accumulation. Moreover, prodigiosin caused AKT dephosphorylation and glycogen synthase kinase-3beta (GSK-3beta) activation, which correlated with NAG-1 expression. Prodigiosin-induced apoptosis was recovered by inhibiting GSK-3beta, which might be due, at least in part, to the blockade of the GSK-3beta-dependent up-regulation of death receptors 4 and 5 expression. These findings suggest that prodigiosin-mediated GSK-3beta activation is a key event in regulating the molecular pathways that trigger the apoptosis induced by this anticancer agent.

  18. Purification and characterization of a cytosolic broad specificity beta-glucosidase from human liver.

    PubMed

    Daniels, L B; Coyle, P J; Chiao, Y B; Glew, R H; Labow, R S

    1981-12-25

    A cytoplasmic beta-glucosidase has been isolated and purified 9,000-fold to homogeneity from the liver of a case of type 1 Gaucher's disease to a specific activity of 400,000 nmol/h/mg of protein. Although markedly elevated above control levels in this case of adult Gaucher's disease, the activity of this cytosolic liver enzyme was found to be markedly deficient in two cases of neurologic Gaucher's disease. The purification scheme employs QAE-Sephadex, DE52 cellulose, CM-Sephadex, hydroxylapatite, and Cibacron blue-Sepharose chromatography, and preparative isoelectric focusing. The beta-glucosidase preparations isolated from the liver of the case of adult Gaucher's disease and control liver have similar physical properties. Both enzymes have a molecular weight of approximately 53,000, sw,20 of 4.3, pI of 4.5-4.6, a pH optimum between 5 and 6, and a high affinity for 4-methylumbelliferyl-beta-D-glucopyranoside (Km = 0.06-0.07 mM). The enzymes from both sources also have a broad specificity and will hydrolyze the 4-methylumbelliferyl derivatives of beta-D-galactose, beta-D-fucose, beta-D-xylose, and alpha-L-arabinose in addition to several aryl-galactosides and steroid-glucosides. The cytoplasmic beta-glucosidase will not hydrolyze glucocerebroside and shows no cross-reactivity with antibodies prepared against lysosomal glucocerebrosidase. Both cytoplasmic beta-glucosidase and glucocerebrosidase will hydrolyze 17 beta-estradiol-17'-beta-D-glucose, and the activity of both enzymes on this substrate is increased more than 15-fold in the presence of the Gaucher spleen heat-stable factor. The role of this cytoplasmic beta-glucosidase in the etiology of Gaucher's disease and its possible relationship to lysosomal glucocerebrosidase are discussed.

  19. Human N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin): genomic structure of the alpha and beta subunits.

    PubMed Central

    Hahn, D; Illisson, R; Metspalu, A; Sterchi, E E

    2000-01-01

    N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), a zinc-metalloendopeptidase of the astacin family, consists of two similar subunits. As well as in small-intestinal epithelial cells, the enzyme is found in lamina propria leucocytes, human cancer cells and colorectal cancer tissue, making it a potential candidate for a role in tumour formation and cancer progression. To elucidate the mechanisms that control PPH gene expression and to gain more insights into the evolutionary relationship of the two subunits, we analysed the complete exon-intron organization and searched for putative regulatory elements in 3 kb of the upstream region of both genes. The human gene for the alpha subunit is approx. 35 kb in size and contains 14 exons. The gene for the beta subunit is organized in 15 exons and spans approx. 27 kb. A comparison of both genes indicates strong structural similarities. The exons are almost identical in size, except exon 13 in PPHalpha, which codes for an additional I domain not present in PPHbeta. The locations of the respective exon-intron junctions and the intron phases are almost identical; five of them contain conserved split codons. The main variation is in the intron lengths. It can be concluded that PPHalpha and PPHbeta are derived from a common ancestor. Sequence analysis of the 5' flanking DNA with a computer search for promoter elements and different promoter constructs transfected into Caco-2 cells revealed a number of potential regulatory motifs and suggests that each of the two genes is regulated independently. PMID:10657243

  20. Application of satellite imagery to monitoring human rights abuse of vulnerable communities, with minimal risk to relief staff

    NASA Astrophysics Data System (ADS)

    Lavers, C.; Bishop, C.; Hawkins, O.; Grealey, E.; Cox, C.; Thomas, D.; Trimel, S.

    2009-07-01

    Space imagery offers remote surveillance of ethnic people groups at risk of human rights abuse. We highlight work in alleged violations in Burma and Sudan, using satellite imagery for verification with Amnesty International. We consider how imaging may effectively support small to medium-sized Non Governmental Organisations and charities, e.g. HART, working in dangerous zones on the ground. Satellite based sensing applications are now at a sufficiently mature stage for moderate Governmental funding levels to help prevent human rights abuse, rather than the greater cost of rebuilding communities and healing sectarian divisions after abuse has taken place.

  1. Enhanced polymerization of recombinant human deoxyhemoglobin. beta. 6 Glu yields Ile

    SciTech Connect

    Baudin-Chich, V.; Pagnier, J.; Marden, M.; Bohn, B.; Lacaze, N.; Kister, J.; Poyart, C. ); Schaad, O.; Edelstein, S.J. )

    1990-03-01

    Polymerization of the deoxy form of sickle cell hemoglobin involves both hydrophobic and electrostatic intermolecular contacts. These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands. X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the {beta}6 valines of each molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer. To estimate the relative importance of this key hydrophobic contact in polymer formation the authors have generated a polymerizing Hb with isoleucine at the {beta}6 position ({beta}E6I) by site-directed mutagenesis. The mutated {beta} chains were produced in Escherichia coli and reassembled into functional tetramers with native {alpha} chains. Compared to native Hb S, the {beta}E6I mutant polymerizes faster and with a shortened delay time in 1.8 M phosphate buffer, indicating an increased stability of the nuclei preceding fiber growth. Computer modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induced increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue. The agreement between the predicted and experimental differences in solubility suggests that the transfer of the {beta}6 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.

  2. MicroRNA-320a suppresses human colon cancer cell proliferation by directly targeting {beta}-catenin

    SciTech Connect

    Sun, Jian-Yong; Huang, Yi; Li, Ji-Peng; Zhang, Xiang; Wang, Lei; Meng, Yan-Ling; Yan, Bo; Bian, Yong-Qian; Zhao, Jing; Wang, Wei-Zhong; and others

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer miR-320a is downregulated in human colorectal carcinoma. Black-Right-Pointing-Pointer Overexpression of miR-320a inhibits colon cancer cell proliferation. Black-Right-Pointing-Pointer {beta}-Catenin is a direct target of miR-320a in colon cancer cells. Black-Right-Pointing-Pointer miR-320a expression inversely correlates with mRNA expression of {beta}-catenin's target genes in human colon carcinoma. -- Abstract: Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-320a) in human colorectal carcinoma. However, its expression pattern and underlying mechanisms in the development and progression of colorectal carcinoma has not been elucidated clearly. Here, we performed real-time PCR to examine the expression levels of miR-320a in colon cancer cell lines and tumor tissues. And then, we investigated its biological functions in colon cancer cells by a gain of functional strategy. Further more, by the combinational approaches of bioinformatics and experimental validation, we confirmed target associations of miR-320a in colorectal carcinoma. Our results showed that miR-320a was frequently downregulated in cancer cell lines and colon cancer tissues. And we demonstrated that miR-320a restoration inhibited colon cancer cell proliferation and {beta}-catenin, a functionally oncogenic molecule was a direct target gene of miR-320a. Finally, the data of real-time PCR showed the reciprocal relationship between miR-320a and {beta}-catenin's downstream genes in colon cancer tissues. These findings indicate that miR-320a suppresses the growth of colon cancer cells by directly targeting {beta}-catenin, suggesting its application in prognosis prediction and cancer treatment.

  3. The fatty acid beta-oxidation pathway is important for decidualization of endometrial stromal cells in both humans and mice.

    PubMed

    Tsai, Jui-He; Chi, Maggie M-Y; Schulte, Maureen B; Moley, Kelle H

    2014-02-01

    Embryo implantation and development requires the endometrial stromal cells (ESCs) to undergo decidualization. This differentiation process requires glucose utilization, and blockade of the pentose phosphate pathway inhibits decidualization of ESCs both in vitro and in vivo. Glucose and fatty acids are energy substrates for many cell types, and fatty acid beta-oxidation is critical for embryo implantation. Here, we investigated whether beta-oxidation is required for decidualization of ESCs. As assessed by marker gene expression, decidualization of human primary ESCs was blocked by reducing activity of carnitine calmitoyltransferase I, the rate-limiting enzyme in beta-oxidation, either by short hairpin RNA-mediated silencing or by treatment with the inhibitor etomoxir. Ranolazine (RAN), a partial beta-oxidation inhibitor, blocked early decidualization of a human ESC line. However, decidualization resumed after several days, most likely due to a compensatory up-regulation of GLUT1 expression and an increase in glucose metabolism. Simultaneous inhibition of the beta-oxidation pathway with RAN and the pentose phosphate pathway with glucosamine (GlcN) impaired in vitro decidualization of human ESCs more strongly than inhibition of either pathway alone. These findings were confirmed in murine ESCs in vitro, and exposure to RAN plus GlcN inhibited decidualization in vivo in a deciduoma model. Finally, intrauterine implantation of time-release RAN and GlcN pellets reduced pup number. Importantly, pup number returned to normal after the end of the pellet-active period. This work indicates that both fatty acids and glucose metabolism pathways are important for ESC decidualization, and suggests novel pathways to target for the design of future nonhormonal contraceptives.

  4. The fatty acid beta-oxidation pathway is important for decidualization of endometrial stromal cells in both humans and mice.

    PubMed

    Tsai, Jui-He; Chi, Maggie M-Y; Schulte, Maureen B; Moley, Kelle H

    2014-02-01

    Embryo implantation and development requires the endometrial stromal cells (ESCs) to undergo decidualization. This differentiation process requires glucose utilization, and blockade of the pentose phosphate pathway inhibits decidualization of ESCs both in vitro and in vivo. Glucose and fatty acids are energy substrates for many cell types, and fatty acid beta-oxidation is critical for embryo implantation. Here, we investigated whether beta-oxidation is required for decidualization of ESCs. As assessed by marker gene expression, decidualization of human primary ESCs was blocked by reducing activity of carnitine calmitoyltransferase I, the rate-limiting enzyme in beta-oxidation, either by short hairpin RNA-mediated silencing or by treatment with the inhibitor etomoxir. Ranolazine (RAN), a partial beta-oxidation inhibitor, blocked early decidualization of a human ESC line. However, decidualization resumed after several days, most likely due to a compensatory up-regulation of GLUT1 expression and an increase in glucose metabolism. Simultaneous inhibition of the beta-oxidation pathway with RAN and the pentose phosphate pathway with glucosamine (GlcN) impaired in vitro decidualization of human ESCs more strongly than inhibition of either pathway alone. These findings were confirmed in murine ESCs in vitro, and exposure to RAN plus GlcN inhibited decidualization in vivo in a deciduoma model. Finally, intrauterine implantation of time-release RAN and GlcN pellets reduced pup number. Importantly, pup number returned to normal after the end of the pellet-active period. This work indicates that both fatty acids and glucose metabolism pathways are important for ESC decidualization, and suggests novel pathways to target for the design of future nonhormonal contraceptives. PMID:24403548

  5. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    NASA Technical Reports Server (NTRS)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  6. Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice.

    PubMed

    Chen, Xin; Yang, Yange; Shi, Zhaopeng; Gao, Ming-Qing; Zhang, Yong

    2016-01-01

    This study was performed to investigate the effects of genetically modified (GM) milk containing human beta-defensin-3 (HBD3) on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT). The emphasis was placed on the effects on gastrointestinal (GI) tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health. PMID:27438026

  7. Human dopamine {beta}-hydroxylase locus and the chromosome 9q34 region in alcoholism

    SciTech Connect

    Parsian. A.; Suarez, B.K.; Hampe, C.

    1994-09-01

    Human dopamine {beta}-hydroxylase (DBH) is responsible for conversion of dopamine to norepinephrine in catecholamine neurons. Potential inhibitors of this enzyme do exist, but they are generally not effective in vivo in reducing tissue concentrations of catecholamines. The gene for DBH has been localized to 9q34 by linkage analysis and in situ hybridization. Recently there have been reports indicating a suggestive evidence of linkage between DNA markers in 9q34 region and alcoholism. In order to test for this suggestive linkage, we have genotyped a sample of 134 subjects with alcoholism, 30 alcoholic families (n=302) and 92 normal controls. The alcoholic subjects are probands of multiple incidence families. The normal controls are an epidemiologically ascertained samples of middle-aged, unrelated individuals. The two groups were matched for sex and ethnic background. The markers used in this study were dinucleotide repeats in the DBH gene, and two highly informative (CA) markers (D9S64, D9S66) flanking the DBH gene. A preliminary affected-sib-pair analysis was carried out under two diagnostic schemes. Regardless of whether `probable` alcoholics are classified as unaffected (t=0.63) or affected (t=1.50), these data do not reveal a significant excess in DBH marker sharing among affected-sib-pairs. However, the comparison of the DBH marker allele frequencies between the unrelated alcoholic panel and the unrelated normal control panel was significant at the p=0.04 level.

  8. Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice

    PubMed Central

    Yang, Yange; Shi, Zhaopeng; Gao, Ming-Qing; Zhang, Yong

    2016-01-01

    This study was performed to investigate the effects of genetically modified (GM) milk containing human beta-defensin-3 (HBD3) on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT). The emphasis was placed on the effects on gastrointestinal (GI) tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health. PMID:27438026

  9. Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor.

    PubMed

    Selwood, Trevor; Elrod, Kyle C; Schechter, Norman M

    2003-12-01

    Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding. PMID:14719803

  10. Beta-adrenergic regulation of cyclic AMP synthesis in cultured human syncytiotrophoblast.

    PubMed

    Grullon, K; Jacobs, M M; Li, S X; Illsley, N P

    1995-10-01

    Isolated elements of the beta-adrenergic/adenyl cyclase signal transduction system have been studied previously using purified membranes. We used cultured syncytiotrophoblast cells to identify components of this signalling system and the interactions which regulate syncytial adenyl cyclase. Generation of cyclic AMP (cAMP) was stimulated in these cells by both forskolin and isoproterenol but not by dopamine, adenosine, carbachol or prostaglandin E1. Synthesis was also stimulated by treatment with cholera toxin, indicating the involvement of the G-protein, Gs. Somatostatin inhibited isoproterenol- or forskolin-stimulated cAMP generation, an effect which could be blocked by pretreatment of the cells with pertussis toxin, demonstrating the mediation of somatostatin action by Gi. Furthermore, secretion of human chorionic gonadotrophin (hCG) was increased significantly by isoproterenol while somatostatin blocked the isoproterenol-stimulated release of hCG. These results clearly demonstrate that adenyl cyclase in syncytiotrophoblast is controlled by a stimulatory pathway operating through Gs and inhibitory pathway acting through Gi.

  11. Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice.

    PubMed

    Chen, Xin; Yang, Yange; Shi, Zhaopeng; Gao, Ming-Qing; Zhang, Yong

    2016-01-01

    This study was performed to investigate the effects of genetically modified (GM) milk containing human beta-defensin-3 (HBD3) on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT). The emphasis was placed on the effects on gastrointestinal (GI) tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health.

  12. Physical mapping of the human t-cell receptor beta gene complex, using yeast artificial chromosomes

    SciTech Connect

    Hashim, Y.; So, A.K.; Kearney, L.

    1995-04-01

    Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell {beta} chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280, 110, 90, and 125 kbp and was in accordance with published data. In addition, comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600-620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Fluorescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences. 32 refs., 5 figs., 1 tab.

  13. Human T-cell receptor v{beta} gene polymorphism and multiple sclerosis

    SciTech Connect

    Wei, S.; Charmley, P.; Birchfield, R.I.; Concannon, P.

    1995-04-01

    Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the {beta} chain of the T-cell receptor for antigen (RCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of {approximately}110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility. The TCRBV coding region-specific markers generated in these studies, as well as the approach of testing for associations with specific functionally relevant polymorphic sites within individual BV genes, should be useful in the evaluation of the many reported disease associations involving the human TCRB region. 22 refs., 1 fig., 3 tabs.

  14. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  15. Assessment of human health impact from exposure to multiple air pollutants in China based on satellite observations

    NASA Astrophysics Data System (ADS)

    Yu, Tao; Wang, Wen; Ciren, Pubu; Zhu, Yan

    2016-10-01

    Assessment of human health impact caused by air pollution is crucial for evaluating environmental hazards. In this paper, concentrations of six air pollutants (PM10, PM2.5, NO2, SO2, O3, and CO) were first derived from satellite observations, and then the overall human health risks in China caused by multiple air pollutants were assessed using an aggregated health risks index. Unlike traditional approach for human health risks assessment, which relied on the in-situ air pollution measurements, the spatial distribution of aggregated human health risks in China were obtained using satellite observations in this research. It was indicated that the remote sensing data have advantages over in-situ data in accessing human health impact caused by air pollution.

  16. A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite

    PubMed Central

    1989-01-01

    We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170- bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere. PMID:2808515

  17. Comparative beta-adrenoceptor blocking effects of propranolol, bisoprolol, atenolol, acebutolol and diacetolol on the human isolated bronchus.

    PubMed Central

    Naline, E; Sarria, B; Ertzbischoff, O; Ozanne, P; Advenier, C

    1990-01-01

    Five beta-adrenoceptor blockers, propranolol, acebutolol, diacebutolol, atenolol and bisoprolol, were tested for their antagonistic effect against isoprenaline on human isolated bronchi. The results showed (1) that only propranolol exerted a competitive antagonistic effect against isoprenaline (pA2 = 9.40 +/- 0.22, n = 7) whereas the other drugs did not, and (2) that, in the presence of beta-adrenoceptor blockers in the plasma concentrations reported after a single usual therapeutic dose, the doses of isoprenaline giving the same bronchodilator effect must be multiplied by 32.6, 5.51, 4.63, 2.82 and 1.95 respectively with propranolol, diacetolol, acebutolol, atenolol, and bisoprolol. It was concluded that (1) atenolol and bisoprolol were the least potent drugs at bronchial level in therapeutic plasma concentrations and (2) that tests performed on the human isolated bronchus might be a useful screening procedure for new drugs with potential activity on the airways. PMID:1975197

  18. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

    NASA Technical Reports Server (NTRS)

    Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

    2003-01-01

    Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

  19. Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

    PubMed

    Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

    2015-10-01

    Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

  20. Regulation of MUC5AC mucin secretion and airway surface liquid metabolism by IL-1beta in human bronchial epithelia.

    PubMed

    Gray, Thomas; Coakley, Ray; Hirsh, Andrew; Thornton, David; Kirkham, S; Koo, Ja-Seok; Burch, Lauranell; Boucher, Richard; Nettesheim, Paul

    2004-02-01

    Mucociliary transport in the airways significantly depends on the liquid and mucin components of the airway surface liquid (ASL). The regulation of ASL water and mucin content during pathological conditions is not well understood. We hypothesized that airway epithelial mucin production and liquid transport are regulated in response to inflammatory stimuli and tested this hypothesis by investigating the effects of the pleiotropic, early-response cytokine, IL-1beta, on cultured primary human bronchial epithelial and second-passage, normal human tracheo-bronchial epithelial (NHTBE) cell cultures. Fully differentiated NHTBE cultures secreted two major airway mucins, MUC5AC and MUC5B. IL-1beta, in a dose- and time-dependent manner, increased the secretion of MUC5AC, but not MUC5B. MUC5AC mRNA levels were only transiently increased at 1 and 4 h after the start of IL-1beta treatment and returned to control levels thereafter, even though MUC5AC mucin production remained elevated for at least 72 h. Synchronous with elevated MUC5AC secretion, ASL volume increased, its percentage of solid was reduced, and the pH/[HCO(3)(-)] of the ASL was elevated. ASL volume changes reflected altered ion transport, including an upregulation of Cl(-) secretory currents (via CFTR and Ca(2+)-activated Cl(-) conductance) and an inhibition of epithelial sodium channel (ENaC)-mediated absorptive Na(+) currents. IL-1beta increased CFTR mRNA levels without affecting those for ENaC subunits. The synchronous regulation of ASL mucin and liquid metabolism triggered by IL-1beta may be an important defense mechanism of the airway epithelium to enhance mucociliary clearance during airway inflammation. PMID:14527933

  1. A human serotonin 1D receptor variant (5HT1D beta) encoded by an intronless gene on chromosome 6.

    PubMed Central

    Demchyshyn, L; Sunahara, R K; Miller, K; Teitler, M; Hoffman, B J; Kennedy, J L; Seeman, P; Van Tol, H H; Niznik, H B

    1992-01-01

    An intronless gene encoding a serotonin receptor (5HT1D beta) has been cloned and functionally expressed in mammalian fibroblast cultures. Based on the deduced amino acid sequence, the gene encodes a 390-amino acid protein displaying considerable homology, within putative transmembrane domains (approximately 75% identity) to the canine and human 5HT1D receptors. Membranes prepared from CHO cells stably expressing the receptor bound [3H]serotonin with high affinity (Kd 4 nM) and displayed a pharmacological profile consistent, but not identical, with that of the characterized serotonin 5HT1D receptor. Most notably, metergoline and serotonergic piperazine derivatives, as a group, display 3- to 8-fold lower affinity for the 5HT1D beta receptor than for the 5HT1D receptor, whereas both receptors display similar affinities for tryptamine derivatives, including the antimigraine drug sumatriptan. Northern blot analysis revealed an mRNA of approximately 5.5 kilobases expressed in human and monkey frontal cortex, medulla, striatum, hippocampus and amygdala but not in cerebellum, olfactory tubercle, and pituitary. The 5HT1D beta gene maps to human chromosome 6. The existence of multiple neuronal 5HT1D-like receptors may help account for some of the complexities associated with [3H]serotonin binding patterns in native membranes. Images PMID:1351684

  2. Impaired beta-cell functions induced by chronic exposure of cultured human pancreatic islets to high glucose.

    PubMed

    Marshak, S; Leibowitz, G; Bertuzzi, F; Socci, C; Kaiser, N; Gross, D J; Cerasi, E; Melloul, D

    1999-06-01

    In type 2 diabetes, chronic hyperglycemia has been suggested to be detrimental to beta-cell function, causing reduced glucose-stimulated insulin secretion and disproportionately elevated proinsulin. In the present study, we investigated the effect on several beta-cell functions of prolonged in vitro exposure of human pancreatic islet cultures to high glucose concentrations. Islets exposed to high glucose levels (33 mmol/l) for 4 and 9 days showed dramatic decreases in glucose-induced insulin release and in islet insulin content, with increased proportion of proinsulin-like peptides relative to insulin. The depletion in insulin stores correlated with the reduction in insulin mRNA levels and human insulin promoter transcriptional activity. We also demonstrated that high glucose dramatically lowered the binding activity of pancreatic duodenal homeobox 1 (the glucose-sensitive transcription factor), whereas the transcription factor rat insulin promoter element 3b1 activator was less influenced and insulin enhancer factor 1 remained unaffected. Most of these beta-cell impairments were partially reversible when islets first incubated for 6 days in high glucose were transferred to normal glucose (5.5 mmol/l) concentrations for 3 days. We conclude that cultured human islets are sensitive to the deleterious effect of high glucose concentrations at multiple functional levels, and that such mechanisms may play an important role in the decreased insulin production and secretion of type 2 diabetic patients. PMID:10342809

  3. Dissociation between renin and arterial pressure responses to beta-adrenergic blockade in human essential hypertension.

    PubMed

    Bravo, E L; Tarazi, R C; Dustan, H P; Lewis, J W

    1975-06-01

    Studies were carried out in 69 patients with essential hypertension to examine the relationship between changes in plasma renin activity (PRA) and arterial pressure (BP) in response to a beta-adrenergic blocking agent, propranolol. PRA had no consistent relationship with BP during treatment, either in patients receiving propranolol alone (r = 0.12) or in those receiving a combination of diuretics and propranolol (r = 0.18). Furthermore, long-term beta-adrenergic blockade failed to inhibit increases of PRA induced by diuretics or rapid sodium depletion. These results indicate that (1) beta-adrenergic blockade can reduce BP by mechanisms other than PRA suppression; and (2) the beta-adrenergic nervous system is important, but not essential, for renin release. PMID:236841

  4. Immunoreactive beta-endorphin increases after an aspartame chocolate drink in healthy human subjects.

    PubMed

    Melchior, J C; Rigaud, D; Colas-Linhart, N; Petiet, A; Girard, A; Apfelbaum, M

    1991-11-01

    It has been claimed that sucrose intake induces a rise in beta-endorphins. In an attempt to discriminate between the sensorial and metabolic effects of sucrose intake in this process, the effects of two chocolate drinks were compared: one sweetened with 50 g of sucrose, the other with 80 mg of aspartame. Plasma beta-endorphin concentrations were more elevated after the aspartame drink than after sucrose or fasting, while insulin increased after drinking as much with aspartame as with sucrose. We suggest that the increase in beta-endorphin after aspartame edulcorated chocolate is related with insulin secretion in the absence of marked changes in blood glucose or with a direct effect of aspartame itself on beta-endorphin liberation.

  5. Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

    PubMed Central

    Andersson, Lotta E.; Valtat, Bérengère; Bagge, Annika; Sharoyko, Vladimir V.; Nicholls, David G.; Ravassard, Philippe; Scharfmann, Raphael; Spégel, Peter; Mulder, Hindrik

    2015-01-01

    Aims/Hypothesis Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets. Methods Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets. Results Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells. Conclusions/Interpretation Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings

  6. Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation

    SciTech Connect

    Grasso, P.; Santa-Coloma, T.A.; Reichert, L.E. Jr. )

    1991-06-01

    We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.

  7. Phase Dependency of the Human Primary Motor Cortex and Cholinergic Inhibition Cancelation During Beta tACS

    PubMed Central

    Guerra, Andrea; Pogosyan, Alek; Nowak, Magdalena; Tan, Huiling; Ferreri, Florinda; Di Lazzaro, Vincenzo; Brown, Peter

    2016-01-01

    The human motor cortex has a tendency to resonant activity at about 20 Hz so stimulation should more readily entrain neuronal populations at this frequency. We investigated whether and how different interneuronal circuits contribute to such resonance by using transcranial magnetic stimulation (TMS) during transcranial alternating current stimulation (tACS) at motor (20 Hz) and a nonmotor resonance frequency (7 Hz). We tested different TMS interneuronal protocols and triggered TMS pulses at different tACS phases. The effect of cholinergic short-latency afferent inhibition (SAI) was abolished by 20 Hz tACS, linking cortical beta activity to sensorimotor integration. However, this effect occurred regardless of the tACS phase. In contrast, 20 Hz tACS selectively modulated MEP size according to the phase of tACS during single pulse, GABAAergic short-interval intracortical inhibition (SICI) and glutamatergic intracortical facilitation (ICF). For SICI this phase effect was more marked during 20 Hz stimulation. Phase modulation of SICI also depended on whether or not spontaneous beta activity occurred at ~20 Hz, supporting an interaction effect between tACS and underlying circuit resonances. The present study provides in vivo evidence linking cortical beta activity to sensorimotor integration, and for beta oscillations in motor cortex being promoted by resonance in GABAAergic interneuronal circuits. PMID:27522077

  8. Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.

    PubMed Central

    Vecchiarelli, A; Retini, C; Pietrella, D; Monari, C; Tascini, C; Beccari, T; Kozel, T R

    1995-01-01

    The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised. PMID:7622213

  9. Genistein potentiates the effect of 17-beta estradiol on human hepatocellular carcinoma cell line

    PubMed Central

    Kavoosi, Fraidoon; Dastjerdi, Mehdi Nikbakht; Valiani, Ali; Esfandiari, Ebrahim; Sanaei, Masumeh; Hakemi, Mazdak Ganjalikhani

    2016-01-01

    Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. This cancer may be due to a multistep process with an accumulation of epigenetic alterations in tumor suppressor genes (TSGs), leading to hypermethylation of the genes. Hypermethylation of TSGs is associated with silencing and inactivation of them. It is well-known that DNA hypomethylation is the initial epigenetic abnormality recognized in human tumors. Estrogen receptor alpha (ERα) is one of the TSGs which modulates gene transcription and its hypermethylation is because of overactivity of DNA methyltransferases. Fortunately, epigenetic changes especially hypermethylation can be reversed by pharmacological compounds such as genistein (GE) and 17-beta estradiol (E2) which involve in preventing the development of certain cancers by maintaining a protective DNA methylation. The aim of the present study was to analyze the effects of GE on ERα and DNMT1 genes expression and also apoptotic and antiproliferative effects of GE and E2 on HCC. Materials and Methods: Cells were treated with various concentrations of GE and E2 and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used. Furthermore, cells were treated with single dose of GE and E2 (25 μM) and flow cytometry assay was performed. The expression level of the genes was determined by quantitative real-time reverse transcription polymerase chain reaction. Results: GE increased ERα and decreased DNMT1 genes expression, GE and E2 inhibited cell viability and induced apoptosis significantly. Conclusion: GE can epigenetically increase ERα expression by inhibition of DNMT1 expression which in turn increases apoptotic effect of E2. Furthermore, a combination of GE and E2 can induce apoptosis more significantly. PMID:27656602

  10. Genistein potentiates the effect of 17-beta estradiol on human hepatocellular carcinoma cell line

    PubMed Central

    Kavoosi, Fraidoon; Dastjerdi, Mehdi Nikbakht; Valiani, Ali; Esfandiari, Ebrahim; Sanaei, Masumeh; Hakemi, Mazdak Ganjalikhani

    2016-01-01

    Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. This cancer may be due to a multistep process with an accumulation of epigenetic alterations in tumor suppressor genes (TSGs), leading to hypermethylation of the genes. Hypermethylation of TSGs is associated with silencing and inactivation of them. It is well-known that DNA hypomethylation is the initial epigenetic abnormality recognized in human tumors. Estrogen receptor alpha (ERα) is one of the TSGs which modulates gene transcription and its hypermethylation is because of overactivity of DNA methyltransferases. Fortunately, epigenetic changes especially hypermethylation can be reversed by pharmacological compounds such as genistein (GE) and 17-beta estradiol (E2) which involve in preventing the development of certain cancers by maintaining a protective DNA methylation. The aim of the present study was to analyze the effects of GE on ERα and DNMT1 genes expression and also apoptotic and antiproliferative effects of GE and E2 on HCC. Materials and Methods: Cells were treated with various concentrations of GE and E2 and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used. Furthermore, cells were treated with single dose of GE and E2 (25 μM) and flow cytometry assay was performed. The expression level of the genes was determined by quantitative real-time reverse transcription polymerase chain reaction. Results: GE increased ERα and decreased DNMT1 genes expression, GE and E2 inhibited cell viability and induced apoptosis significantly. Conclusion: GE can epigenetically increase ERα expression by inhibition of DNMT1 expression which in turn increases apoptotic effect of E2. Furthermore, a combination of GE and E2 can induce apoptosis more significantly.

  11. Gonadotropin-releasing hormone/human chorionic gonadotropin beta based recombinant antibodies and vaccines.

    PubMed

    Talwar, G P; Vyas, Hemant K; Purswani, Shilpi; Gupta, Jagdish C

    2009-12-01

    Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are unique targets for the control of fertility. Immunological approaches to neutralizing these hormones have additional utility in cancer treatment. Vaccines have been developed against both GnRH and hCG and these have undergone Phase I/II clinical trials documenting their safety, reversibility and efficacy. The heterospecies dimer hCG vaccine prevented pregnancy in women of proven fertility without impairment of ovulation or derangement of menstrual regularity and bleeding profiles. The protective threshold of antibody titers to achieve efficacy was determined in these first-ever trials. Recently, a recombinant vaccine against the beta subunit of hCG linked to the B subunit of heat labile enterotoxin has been made and expressed as a glycosylated conjugate in Pichia pastoris. Experiments indicate its ability to generate antibodies above the protective threshold in all immunized Balb/c mice. Ectopic expression of hCG/hCGbeta is observed in many advanced stage cancers of various origins. A chimeric high affinity and specific recombinant antibody against hCGbeta linked to curcumin kills hCGbeta expressing T lymphoblastic leukemia cells without any deleterious effect. Several synthetic and recombinant vaccines have been developed against GnRH. These reduce serum testosterone to castration levels causing atrophy of the prostate. Three Phase I/II clinical trials conducted in India and Austria have shown that these vaccines elicit non-surgical reduction of testosterone, a fall in prostate specific antigen and clinical improvement of prostate carcinoma patients. A multimer recombinant vaccine against GnRH has high efficacy for sterilization of pigs and other animals. PMID:19854518

  12. Evidence for increased beta-adrenoreceptor responsiveness induced by 14 days of simulated microgravity in humans

    NASA Technical Reports Server (NTRS)

    Convertino, V. A.; Polet, J. L.; Engelke, K. A.; Hoffler, G. W.; Lane, L. D.; Blomqvist, C. G.; Eckberg, D. L. (Principal Investigator)

    1997-01-01

    We studied hemodynamic responses to alpha- and beta-receptor agonists in eight healthy men before and after 14 days of 6 degrees head-down tilt (HDT) to test the hypothesis that increased adrenoreceptor responsiveness is induced by prolonged exposure to simulated microgravity. Steady-state infusions of isoproterenol (Iso) at rates of 0.005, 0.01, and 0.02 microgram.kg-1.min-1 were used to assess beta 1- and beta 2-adrenoreceptor responsiveness. Infusions of phenylephrine (PE) at rates of 0.25, 0.50, and 1.00 microgram.kg-1.min-1 were used to assess responsiveness of alpha 1-vascular adrenoreceptors. Slopes calculated from linear regressions between Iso and PE doses and changes in beat-to-beat heart rate, blood pressure, and leg vascular resistance (occlusion plethysmography) for each subject were used as an index of alpha- and beta-adrenoreceptor responsiveness. HDT increased the slopes of heart rate (1,056 +/- 107 to 1,553 +/- 83 beats micrograms-1.kg-1.min-1; P = 0.014) and vasodilation (-469 +/- 111 to -1,446 +/- 309 peripheral resistance units.microgram-1.kg-1.min-1; P = 0.0224) to Iso infusion. There was no alteration in blood pressure or vascular resistance responses to PE infusion after HDT. Our results provide evidence that simulated microgravity causes selective increases in beta 1- and beta 2-adrenoreceptor responsiveness without affecting alpha 1-vascular adrenoreceptor responses.

  13. High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia.

    PubMed Central

    Fabry, M E; Costantini, F; Pachnis, A; Suzuka, S M; Bank, N; Aynedjian, H S; Factor, S M; Nagel, R L

    1992-01-01

    A line of transgenic mice with two cointegrated transgenes, the human beta S- and alpha 2-globin genes, linked to the beta-globin locus control region was produced and bred with mice carrying a deletion of the mouse beta major-globin gene. In transgenic mice homozygous for the beta major deletion (alpha H beta S[beta MDD]; where alpha H is human alpha-globin and MD is mouse deletion), 72.5 +/- 2.4% (mean +/- SD) of the beta-chains are beta S and the ratio of alpha H- to beta S-globin was 0.73. Introduction of a heterozygous mouse alpha-globin deletion into mice homozygous for the beta major deletion (alpha H beta S[alpha MD beta MDD]) resulted in 65.1 +/- 8.5% beta S and a human alpha/beta ratio of 0.89 +/- 0.2. Sickling occurs in 95% of erythrocytes from alpha H beta S[beta MDD] mice after slow deoxygenation. Transmission electron microscopy revealed polymer fiber formation but not fascicles of fiber. Increased organ weight was noted in lung, spleen, and kidney of transgenic mice vs. controls that may be due to hypertrophy or increased blood volume in the lungs and/or increased tissue water content. The hemoglobin content of lung, spleen, and kidney was also elevated in transgenic animals due to trapped hemoglobin and/or increased blood volume. When transgenic and control mice were examined by magnetic resonance imaging at 9.4 tesla, some transgenic animals had enlarged kidneys with prolonged relaxation time, consistent with increased organ weight and water content. The glomerular filtration rate was elevated in transgenic animals, which is characteristic of young sickle cell patients. Furthermore, exposure to hypoxia resulted in significantly decreased hematocrit, increased erythrocyte density, and induced a urine-concentrating defect. We conclude that the transgenic mouse line reported here has chronic organ damage and further hematological and organ dysfunction can be induced by hypoxia. Images PMID:1465455

  14. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    SciTech Connect

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  15. Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite

    SciTech Connect

    Sykora, Peter; Snow, Elizabeth T.

    2008-05-01

    Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase {beta} (Pol {beta}) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol {beta} and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 {mu}M. However, at lower doses Pol {beta} mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol {beta} was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis.

  16. Human peritoneal mesothelial cells synthesize interleukin-8. Synergistic induction by interleukin-1 beta and tumor necrosis factor-alpha.

    PubMed Central

    Topley, N.; Brown, Z.; Jörres, A.; Westwick, J.; Davies, M.; Coles, G. A.; Williams, J. D.

    1993-01-01

    The present study demonstrates the synthesis and secretion of the neutrophil-activating peptide/interleukin-8 (IL-8) by cultured human peritoneal mesothelial cells (HPMC) and examines the regulation of its production by other cytokines. Unstimulated HPMC under growth-arrested conditions released IL-8 in a constitutive and time-dependent manner. Stimulation of HPMC with IL-1 beta or TNF-alpha resulted in a time- and dose-dependent IL-8 generation; after 24 hours the levels induced by IL-1 beta and TNF-alpha (both at 1000 pg/ml) were (mean +/- SEM, n = 5) 101 +/- 26.6 (z = 2.023; P < 0.01) and 35 +/- 8.09 (z = 2.023; P < 0.01) respectively. This release was inhibited following coincubation with the relevant anti-cytokine antibody or preincubation with either cycloheximide or actinomycin D. Treatment of HPMC with IL-1 beta or TNF-alpha resulted in increased levels of IL-8-specific mRNA. Stimulation of HPMC with combinations of IL-1 beta and TNF-alpha resulted in a synergistic increase in IL-8 release. This effect was significant at combined doses of IL-1 beta (50 pg/ml) and TNF-alpha (500 pg/ml) and above, when the release of IL-8 was 88 +/- 27% above the additive IL-8 release values (z = 2.201; P < 0.01). Western blot analysis using specific anti-IL-8 antibody demonstrated the presence of two major immunoreactive bands between 9 and 10 kd, in HPMC culture supernatants. These data demonstrate that HPMC synthesize IL-8 and that its release can be regulated as a result of induction of mRNA expression and de novo protein synthesis by other cytokines. Images Figure 6 Figure 7 PMID:8506955

  17. Kinetic mechanism and characterization of human beta-galactosidase precursor secreted by permanently transfected Chinese hamster ovary cells.

    PubMed

    Zhang, S; McCarter, J D; Okamura-Oho, Y; Yaghi, F; Hinek, A; Withers, S G; Callahan, J W

    1994-11-15

    Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.

  18. Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.

    PubMed

    Fueger, Patrick T; Schisler, Jonathan C; Lu, Danhong; Babu, Daniella A; Mirmira, Raghavendra G; Newgard, Christopher B; Hohmeier, Hans E

    2008-05-01

    Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.

  19. Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

    PubMed Central

    Malinowski, J. J.; Grasberger, B. L.; Trakshel, G.; Huston, E. E.; Helaszek, C. T.; Smallwood, A. M.; Ator, M. A.; Banks, T. M.; Brake, P. G.; Ciccarelli, R. B.

    1995-01-01

    Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease. PMID:8535252

  20. Beta 2 integrin-dependent protein tyrosine phosphorylation and activation of the FGR protein tyrosine kinase in human neutrophils

    PubMed Central

    1994-01-01

    Stimulation of adherent human neutrophils (PMN) with tumor necrosis factor (TNF) triggers protein tyrosine phosphorylation (Fuortes, M., W. W. Jin, and C. Nathan. 1993. J. Cell Biol. 120:777-784). We investigated the dependence of this response on beta 2 integrins by using PMN isolated from a leukocyte adhesion deficiency (LAD) patient, which do not express beta 2 integrins, and by plating PMN on surface bound anti-beta 2 (CD18) antibodies. Protein tyrosine phosphorylation increased in PMN plated on fibrinogen and this phosphorylation was enhanced by TNF. Triggering of protein tyrosine phosphorylation did not occur in LAD PMN plated on fibrinogen either in the absence or the presence of TNF. Surface bound anti-CD18, but not isotype-matched anti- Class I major histocompatibility complex (MHC) antigens, antibodies triggered tyrosine phosphorylation in normal, but not in LAD PMN. As the major tyrosine phosphorylated proteins we found in our assay conditions migrated with an apparent molecular mass of 56-60 kD, we investigated whether beta 2 integrins are implicated in activation of members of the src family of intracellular protein-tyrosine kinases. We found that the fgr protein-tyrosine kinase (p58fgr) activity, and its extent of phosphorylation in tyrosine, in PMN adherent to fibrinogen, was enhanced by TNF. Activation of p58fgr in response to TNF was evident within 10 min of treatment and increased with times up to 30 min. Also other activators of beta 2 integrins such as phorbol-12- myristate 13-acetate (PMA), and formyl methionyl-leucyl-phenylalanine (FMLP), induced activation of p58fgr kinase activity. Activation of p58fgr kinase activity, and phosphorylation in tyrosine, did not occur in PMN of a LAD patient in response to TNF. Soluble anti-CD18, but not anti-Class I MHC antigens, antibodies inhibited activation of p58fgr kinase activity in PMN adherent to fibrinogen in response to TNF, PMA, and FMLP. These findings demonstrate that, in PMN, beta 2 integrins

  1. Interleukin-1 alpha, interleukin-1 beta and interleukin-8 gene expression in human aural cholesteatomas.

    PubMed

    Kim, C S; Lee, C H; Chung, J W; Kim, C D

    1996-03-01

    Bone destruction is a common characteristic feature of chronic otitis media, especially aural cholesteatoma. A number of immunohistochemical studies have suggested that interleukin-1 (IL-1) may be responsible for cholesteatomatous bone destruction. We designed this study to present the mRNA expression patterns of IL-1 alpha, IL-1 beta, and IL-8, which can induce and activate the leukocyte, the major reservoir of potent proteolytic enzymes. Total RNAs were extracted from aural cholesteatomas, external auditory canal skin (EACS), postauricular skin (PAS), and granulation tissues and transcribed into cDNAs. cDNAs were amplified by using PCR technique with primers for IL-1 alpha, IL-1 beta, IL-8, and beta-actin. Amplified products were hybridized with each internal probe and the relative density was measured. In granulation tissues, the relative density of IL-1 alpha was greater than that of other tissues. The ratio of IL-1 beta and IL-8 of aural cholesteatoma was significantly higher than that of EACS and PAS. We suggest that both of IL-1 alpha and IL-1 beta may play a role in the pathological changes, and that IL-8, which is mainly produced from cholesteatomatous epithelium, may have an important role in the pathological changes of cholesteatomas.

  2. Photolabeling of the alpha-neuraminidase/beta-galactosidase complex from human placenta with a photoreactive neuraminidase inhibitor

    SciTech Connect

    Warner, T.G.; Louie, A.; Potier, M. )

    1990-11-30

    Photolabeling of the alpha-neuraminidase/beta-galactosidase complex in human placenta was carried out using the radioactive photoprobe, 9-S-(4-azido-3,5-3H-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9- tetradeoxy-9- thio-D-glycero-D-galacto-non-2-enonic acid. Two intensely labeled bands at 61 and 46 kD were detected with autoradiography. Labeling of the 46 kD protein was blocked with the inclusion of the surfactant Triton X-100 in the photolysis mixture, indicating a nonspecific, hydrophobic interaction. The 61 kD protein was protected from labeling only when the neuraminidase inhibitor 2,3 dehydro N-acetyl neuraminic acid (1 mM) was present during photolysis. These results suggest that the neuraminidase activity resides among the proteins in the 61 kD molecular weight range comigrating with the lysosomal beta-galactosidase, under denaturing conditions.

  3. TGF{beta}-mediated formation of pRb-E2F complexes in human myeloid leukemia cells

    SciTech Connect

    Hu Xiaotang

    2008-05-02

    TGF{beta} is well known for its inhibitory effect on cell cycle G1 checkpoint kinases. However, its role in the control of pRb-E2F complexes is not well established. TGF{beta} inhibits phosphorylation of pRb at several serine and threonine residues and regulates the association of E2F transcription factors with pRb family proteins. Recent studies found that predominantly E2F-4, p130, and histone deacetylase (HDAC) are found to bind to corresponding E2F-responsive promoters in G0/G1 phase. As cells progress through mid-G1, p130-E2F4 complex are replaced by p107-E2F4 followed by activators E2F1, 2, and 3. pRb was not detectable in the promoters containing the E2F-responsive site in cycling cells but was associated with E2F4-p130 complexes or E2F4-p107 complexes during G0/G1 phase. In human myeloid leukemia cell line, MV4-11, TGF{beta} upregulated pRb-E2F-4 and p130-E2F-4, and downregulated p107-E2F-4 complexes. However, pRB-E2F1 and pRb-E2F3 complexes were found in proliferating cells but not in TGF{beta} arrested G1 cells. In addition, electrophoretic gel mobility shift assay (EMSA) could not detect pRb-E2F DNA-binding activities either in S or G1 phase but exhibited the existence of p107-E2F4 in proliferating cells and p130-E2F4 complexes in TGF{beta}-arrested G1 cells, respectively. Our data suggest that p107 and p130, but not pRb, and the repressor E2F, but not activator E2Fs, play a critical role in regulating E2F-responsive gene expression in TGF{beta}-mediated cell cycle control in human myeloid leukemia cells.

  4. A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization

    PubMed Central

    Xu, Shuhua; Soroka, Carol J.; Sun, An-Qiang; Backos, Donald S.; Mennone, Albert; Suchy, Frederick J.; Boyer, James L.

    2016-01-01

    The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization. PMID:27351185

  5. Analysis of Slovak Spotted breed for bovine beta casein A1 variant as risk factor for human health.

    PubMed

    Miluchová, Martina; Gábor, Michal; Trakovická, Anna

    2013-01-01

    The goal of work was identification A1 variant of bovine beta casein which involves ischemic heart disease and diabetes mellitus in human. The digestion of A1beta casein can result in the production of bioactive beta casomorphin-7 (BCM-7); this is not the case with A2. This bioactive peptide has been linked to physiological traits that may elicit effects on components of the vascular and immune systems. The material involved 111 Slovak Spotted breed. Bovine genomic DNA was extracted from whole blood by using commercial kit, and used in order to estimate beta-casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study were detected all three genotypes, homozygote genotype A1A1 (14 animals), heterozygote genotype A1A2 (37 animals) and homozygote genotype A2A2 (60 animals). In the total population of cattle homozygotes A2A2-0.5405 were the most frequent, while homozygotes A1A1-0.1261 were the least frequent ones. This suggests a superiority of allele A2 (0.7072) which does not produce BCM-7, and thus is safe for human consumption. The expected homozygosity for gene CSN2 is in the population stated a slight increase in homozygosity (0.5858). This caused a slight decrease in the level of possible variability realization (41.80%), which corresponds to the effective number of alleles (1.7071).

  6. Analysis of Slovak Spotted breed for bovine beta casein A1 variant as risk factor for human health.

    PubMed

    Miluchová, Martina; Gábor, Michal; Trakovická, Anna

    2013-01-01

    The goal of work was identification A1 variant of bovine beta casein which involves ischemic heart disease and diabetes mellitus in human. The digestion of A1beta casein can result in the production of bioactive beta casomorphin-7 (BCM-7); this is not the case with A2. This bioactive peptide has been linked to physiological traits that may elicit effects on components of the vascular and immune systems. The material involved 111 Slovak Spotted breed. Bovine genomic DNA was extracted from whole blood by using commercial kit, and used in order to estimate beta-casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study were detected all three genotypes, homozygote genotype A1A1 (14 animals), heterozygote genotype A1A2 (37 animals) and homozygote genotype A2A2 (60 animals). In the total population of cattle homozygotes A2A2-0.5405 were the most frequent, while homozygotes A1A1-0.1261 were the least frequent ones. This suggests a superiority of allele A2 (0.7072) which does not produce BCM-7, and thus is safe for human consumption. The expected homozygosity for gene CSN2 is in the population stated a slight increase in homozygosity (0.5858). This caused a slight decrease in the level of possible variability realization (41.80%), which corresponds to the effective number of alleles (1.7071). PMID:24432335

  7. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    PubMed Central

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh; Batchuluun, Battsetseg; Nagy, Kristina; Neely, Eric; Gull, Rida; Nagy, Andras; Wheeler, Michael B.

    2016-01-01

    The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25–30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25–30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function. PMID:27755557

  8. Solar ultraviolet irradiation reduces collagen in photoaged human skin by blocking transforming growth factor-beta type II receptor/Smad signaling.

    PubMed

    Quan, Taihao; He, Tianyuan; Kang, Sewon; Voorhees, John J; Fisher, Gary J

    2004-09-01

    Ultraviolet (UV) irradiation from the sun reduces production of type I procollagen (COLI), the major structural protein in human skin. This reduction is a key feature of the pathophysiology of premature skin aging (photoaging). Photoaging is the most common form of skin damage and is associated with skin carcinoma. TGF-beta/Smad pathway is the major regulator of type I procollagen synthesis in human skin. We have previously reported that UV irradiation impairs transforming growth factor-beta (TGF-beta)/Smad signaling in mink lung epithelial cells. We have investigated the mechanism of UV irradiation impairment of the TGF-beta/Smad pathway and the impact of this impairment on type I procollagen production in human skin fibroblasts, the major collagen-producing cells in skin. We report here that UV irradiation impairs TGF-beta/Smad pathway in human skin by down-regulation of TGF-beta type II receptor (TbetaRII). This loss of TbetaRII occurs within 8 hours after UV irradiation and precedes down-regulation of type I procollagen expression in human skin in vivo. In human skin fibroblasts, UV-induced TbetaRII down-regulation is mediated by transcriptional repression and results in 90% reduction of specific, cell-surface binding of TGF-beta. This loss of TbetaRII prevents downstream activation of Smad2/3 by TGF-beta, thereby reducing expression of type I procollagen. Preventing loss of TbetaRII by overexpression protects against UV inhibition of type I procollagen gene expression in human skin fibroblasts. UV-induced down-regulation of TbetaRII, with attendant reduction of type I procollagen production, is a critical molecular mechanism in the pathophysiology of photoaging.

  9. Effects of human alpha, beta and gamma interferons on varicella zoster virus in vitro.

    PubMed

    Balachandra, K; Thawaranantha, D; Ayuthaya, P I; Bhumisawasdi, J; Shiraki, K; Yamanishi, K

    1994-06-01

    The antiviral effects of interferon (IFN) on varicella zoster virus (VZV) and herpes simplex virus (HSV) in vitro were examined. The values for the 50% inhibitory dose (ID50) of IFN-alpha, beta and gamma determined by plaque reduction assay, were 0.813, 0.650 and 13.750 IU/ml, respectively, against VZV and 18.00, 10.38 and 115.0 IU/ml, respectively, against HSV. Thus IFN-alpha and beta were more effective than IFN-gamma against both VZV and HSV and VZV was more sensitive than HSV to the IFNs. Five mutants of VZV which were resistant to acyclovir (ACV), phosphonoacetic acid (PAA) or bromodeoxyuridine (BUDR) were also sensitive to IFN beta, their average ID50 being 1.31 IU/ml. Analysis of virus-specific proteins by the immunofluorescent technique with various antisera showed that IFN had a significant effect before early protein synthesis.

  10. Antisense targeting of TGF-{beta}1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

    SciTech Connect

    Shen, Zhong-Jian . E-mail: zshen2@wisc.edu; Kook Kim, Sang; Youn Jun, Do; Park, Wan; Ho Kim, Young; Malter, James S.; Jo Moon, Byung . E-mail: bjmoon@mail.knu.ac.kr

    2007-04-15

    Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-{beta}1 on bone formation remains elusive. In particular, the role of autocrine TGF-{beta}1 on human osteoblasts is largely unknown. Here we show the effect of TGF-{beta}1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-{beta}1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-{beta}1. Notably, TGF-{beta}1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-{beta}1. Moreover, TGF-{beta}1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-{beta}1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.

  11. Beta dose calculation in human arteries for various brachytherapy seed types

    NASA Astrophysics Data System (ADS)

    Lee, Sung-Woo

    This dissertation explores beta dose profile of microspheres packed in arteries, various source geometries of 142Pr that can be used for therapeutic purpose, and dose backscatter factors for selected beta sources. A novel treatment method by injecting microspheres into feeding arteries of arteriovenous malformation (AVM) is under pre-clinical investigation. To optimize radiation dose to the clinically important area, i.e. arterial wall, preliminary dosimetric studies were needed. Monte Carlo calculations were performed for several geometries simulating arteries filled with microspheres packed by random packing methods. Arterial radii used in the simulation varied from 50 mum to 3 mm; microsphere radii varied from 10 mum to 0.7 mm. Dose varied significantly as a function of microsphere size, for constant arterial sizes. For the same sizes of arteries, significant dose increase was observed because of inter-artery exposure for large arteries (>0.1 cm rad.) filled with large microspheres (>0.03 cm rad.). Dose increase between small arteries (<0.03 cm rad.) was less significant. The dose profiles of prototype 142Pr beta brachytherapy sources were calculated using MCNP 4C Monte Carlo code as well as dose point kernel (DPK) for selected cases. Dose profiles were similar to beta sources currently used indicating that 142Pr can substitute for current sources for certain cases and the DPK was closely matched with MCNP result. Backscattering of electrons is a prominent secondary effect in beta dosimetry. The backscattering is closely correlated with factors such as geometry of source and scattering material, and composition of scattering material. The backscattering factors were calculated for selected beta sources that are currently used as well as potentially useful sources for therapeutic purpose. The factors were calculated as a function of distance from the interface between water and scatterers. These factors were fit by a simple function for future incorporation into

  12. Evidence for Increased Beta-Adrenoreceptor Responsiveness Induced by 14 Days of Simulated Microgravity in Humans

    NASA Technical Reports Server (NTRS)

    Convertino, Victor A.; Polet, Jill L.; Engelke, Keith A.; Hoffler, G. W.; Lane, Lynda D.

    1996-01-01

    We studied hemodynamic responses to alpha and beta receptor agonists in 8 healthy men ( 38+- 2 yrs) before and after 14 days of 6 degree head-down tilt (HDT) to test the hypothesis that increased adrenergic responsiveness is induced by prolonged exposure to microgravity. Immediately following a 30-min baseline period, a steady-state infusion of isoproterenol (ISO) was used to assess beta 1- and beta 2-adrenergic responsiveness. ISO was infused at three graded constant rates of 0.005, 0.01 and 0.02 ug/kg/min. After heart rate and blood pressure had been allowed to return to baseline levels following ISO infusion graded infusion of phenylephrine (PE) was used to assess responsiveness of alpha I-vascular receptors. PE was infused at three graded constant rates of 0.25, 0.50 and 1.00 ug/kg/min. Each infusion interval for both drugs was 9 min. During the infusions, constant monitoring of beat-to-beat blood pressure and heart rate was performed and leg blood flow was measured with occlusion plethysmography at each infusion level. The slopes calculated from linear regressions between ISO and PE doses and changes in heart rate, blood pressure, and leg vascular resistance for each subject were used to represent alpha- and beta- adrenoreceptor responsiveness. Fourteen days HDT increased the slopes of heart rate (1056 +- 107 to 1553 +- 83 beats/ug/kg/min; P= 0.014) and vasodilation (-469ft +- 111 to -l446 +- 309 PRU/ug/kg/min; P =0.0224) to ISO infusion. There was no alteration in blood pressure or vascular resistance responses to PE infusion after HDT. Our results provide evidence that microgravity causes selective increases in beta 1- and beta 2-adrenergic responsiveness without affecting alpha 1-vascular responses.

  13. Effect of freezing and ultrasonication on the density of human lymphocyte beta 2-adrenoceptors.

    PubMed

    Santala, M; Castrén, O; Saarikoski, S; Penttilä, I

    1989-06-01

    In this study 21 pregnant volunteers were divided into three equal groups; blood samples were taken from each, and after isolation of the lymphocytes the cell suspensions were divided into two equal samples. The density of beta 2-adrenoceptor in intact lymphocytes was compared with the receptor density for lymphocytes stored 7 days or 19 days at -70 degrees C and with cells disrupted by ultrasonication. Storage by lymphocytes at -70 degrees C for 7 days to 19 days decreased the beta 2-adrenoceptor density by 20% and 37%, respectively; ultrasonication decreased the density by 65%.

  14. cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.

    PubMed Central

    Myerowitz, R; Proia, R L

    1984-01-01

    We have isolated a cDNA clone containing sequences complementary to mRNA encoding the alpha-chain of the lysosomal enzyme beta-hexosaminidase. RNA from a human lung fibroblast strain, IMR90, was enriched for beta-hexosaminidase messenger by polysome immunoselection with antiserum against beta-hexosaminidase A. This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure. After transformation of Escherichia coli, 385 ampicillin-resistant colonies were obtained, 44 of which contained inserts in the plasmid DNA. Differential hybridization, with cDNA probes prepared from polysomal RNA enriched or depleted for beta-hexosaminidase messenger, was used to screen the recombinant plasmids for sequences encoding beta-hexosaminidase. One clone, p beta H alpha-1, containing a cDNA insert of approximately equal to 240 base pairs, was identified in this manner. The plasmid hybrid-selected a messenger from placental RNA that programed a translation system to synthesize the alpha-chain of beta-hexosaminidase. p beta H alpha-1 hybridized to an mRNA of approximately equal to 1.9 kilobases in preparations enriched separately in messenger for the alpha-chain or for both alpha- and beta-chains (by polysome immunoselection with antiserum against isolated alpha-chain or against beta-hexosaminidase A, respectively). It did not hybridize to an RNA preparation enriched for messenger of beta-chain by immunoselection with antiserum against beta-hexosaminidase B. The 1.9-kilobase mRNA was observed in poly(A)+ RNA preparations from control fibroblasts and from fibroblasts of a Tay-Sachs patient that synthesize an altered alpha-chain; however, it was not seen in similar preparations from fibroblasts of four Ashkenazi Tay-Sachs patients. Images PMID:6236461

  15. MicroRNA Expression in Alpha and Beta Cells of Human Pancreatic Islets

    PubMed Central

    Vargas, Nancy; Rosero, Samuel; Piroso, Julieta; Ichii, Hirohito; Umland, Oliver; Zhijie, Jiang; Tsinoremas, Nicholas; Ricordi, Camillo; Inverardi, Luca; Domínguez-Bendala, Juan; Pastori, Ricardo L.

    2013-01-01

    microRNAs (miRNAs) play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98%) subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs) and 134 were expressed more in β-cells (β-miRNAs). Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D) community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα) is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels. In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different aspects of islet

  16. Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells

    PubMed Central

    Lee, Sang-Im; Yi, Jin-Kyu; Bae, Won-Jung; Lee, Soojung; Cha, Hee-Jae; Kim, Eun-Cheol

    2016-01-01

    Background Recent reports suggest that thymosin beta-4 (Tβ4) is a key regulator for wound healing and anti-inflammation. However, the role of Tβ4 in osteoclast differentiation remains unclear. Purpose The purpose of this study was to evaluate Tβ4 expression in H2O2-stimulated human periodontal ligament cells (PDLCs), the effects of Tβ4 activation on inflammatory response in PDLCs and osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs), and identify the underlying mechanism. Methods Reverse transcription-polymerase chain reactions and Western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages (BMMs) using conditioned medium (CM) from H2O2-treated PDLCs. Results Tβ4 was down-regulated in H2O2-exposed PDLCs in dose- and time-dependent manners. Tβ4 activation with a Tβ4 peptide attenuated the H2O2-induced production of NO and PGE2 and up-regulated iNOS, COX-2, and osteoclastogenic cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-17) as well as reversed the effect on RANKL and OPG in PDLCs. Tβ4 peptide inhibited the effects of H2O2 on the activation of ERK and JNK MAPK, and NF-κB in PDLCs. Furthermore, Tβ4 peptide inhibited osteoclast differentiation, osteoclast-specific gene expression, and p38, ERK, and JNK phosphorylation and NF-κB activation in RANKL-stimulated BMMs. In addition, H2O2 up-regulated Wnt5a and its cell surface receptors, Frizzled and Ror2 in PDLCs. Wnt5a inhibition by Wnt5a siRNA enhanced the effects of Tβ4 on H2O2-mediated induction of pro-inflammatory cytokines and osteoclastogenic cytokines as well as helping osteoclastic differentiation whereas Wnt5a activation by Wnt5a peptide reversed it. Conclusion In conclusion, this study demonstrated, for the first time, that Tβ4 was down-regulated in ROS-stimulated PDLCs as well as Tβ4 activation exhibited anti-inflammatory effects and anti-osteoclastogenesis in vitro

  17. The effect of pasteurization on transforming growth factor alpha and transforming growth factor beta 2 concentrations in human milk.

    PubMed

    McPherson, R J; Wagner, C L

    2001-01-01

    Transforming growth factor alpha (TGF-alpha) and beta 2 (TGF-beta2) are present in human milk and are involved in growth differentiation and repair of neonatal intestinal epithelia. Heat treatment at 56 degrees C has been shown effective for providing safe banked donor milk, with good retention of other biologically active factors. The purpose of our study was to determine the effect of heat sterilization on TGF-alpha and TGF-beta2 concentrations in human milk. Twenty milk samples were collected from 20 lactating mothers in polypropylene containers and frozen at -20 degrees C for transport or storage. Before heat treatment by holder pasteurization, the frozen milk was thawed and divided into 1-mL aliquots. All samples were heated in an accurately regulated water bath until a holding temperature was achieved, then held for 30 minutes using constant agitation. Holding temperature ranged from 56.5 degrees C to 56.9 degrees C. The milk was then stored at 4 degrees C overnight for analysis the following day. The concentration of TGF-alpha was measured by radioimmunoassay. Mean concentration +/- SD of TGF-alpha in raw milk samples was 119+/-50 pg/mL, range 57 to 234. The mean concentration +/- SD of TGF-alpha in heat treated samples was 113+/-50 pg/mL, range 51 to 227. TGF-alpha concentration was minimally affected by pasteurization, with an overall loss of 6.1%. Of 19 samples, 4 had increased and 15 had decreased concentrations after pasteurization (mean percent SEM: 94%+/-7% of raw milk, range 72%+/-107%). The concentration of acid-activated TGF-beta2 was measured by enzyme-linked immunosorbent assay. Mean concentration +/- SD of TGF-beta2 in raw milk samples was 5624+/-5038 pg/mL, range 195 to 15480. The mean concentration +/- SD of TGF-beta2 in heat-treated samples was 5073+/-4646 pg/mL, range 181 to 15140. TGF-beta2 survived with relatively little loss (0.6%): of 18 samples, 11 had increased and 7 had decreased concentrations after pasteurization (mean percent

  18. Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro.

    PubMed

    Chal, Jérome; Al Tanoury, Ziad; Hestin, Marie; Gobert, Bénédicte; Aivio, Suvi; Hick, Aurore; Cherrier, Thomas; Nesmith, Alexander P; Parker, Kevin K; Pourquié, Olivier

    2016-10-01

    Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches. PMID:27583644

  19. Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro.

    PubMed

    Chal, Jérome; Al Tanoury, Ziad; Hestin, Marie; Gobert, Bénédicte; Aivio, Suvi; Hick, Aurore; Cherrier, Thomas; Nesmith, Alexander P; Parker, Kevin K; Pourquié, Olivier

    2016-10-01

    Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.

  20. Genomic sequence and transcriptional profile of the boundary between pericentromeric satellites and genes on human chromosome arm 10q.

    PubMed

    Guy, J; Spalluto, C; McMurray, A; Hearn, T; Crosier, M; Viggiano, L; Miolla, V; Archidiacono, N; Rocchi, M; Scott, C; Lee, P A; Sulston, J; Rogers, J; Bentley, D; Jackson, M S

    2000-08-12

    The organization of centromeric heterochromatin has been established in a number of eucaryotes but remains poorly defined in human. Here we present 1025 kb of contiguous human genomic sequence which links pericentromeric satellites to the RET proto-oncogene in 10q11.2 and is presumed to span the transition from centric heterochromatin to euchromatin on this chromosome arm. Two distinct domains can be defined within the sequence. The proximal approximately 240 kb consists of arrays of satellites and other tandem repeats separated by tracts of complex sequence which have evolved by pericentromeric-directed duplication. Analysis of 32 human paralogues of these sequences indicates that most terminate at or within repeat arrays, implicating these repeats in the interchromosomal duplication process. Corroborative PCR-based analyses establish a genome-wide correlation between the distribution of these paralogues and the distribution of satellite families present in 10q11. In contrast, the distal approximately 780 kb contains few tandem repeats and is largely chromosome specific. However, a minimum of three independent intrachromosomal duplication events have resulted in >370 kb of this sequence sharing >90% identity with sequences on 10p. Using computer-based analyses and RT-PCR we confirm the presence of three genes within the sequence, ZNF11/33B, KIAA0187 and RET, in addition to five transcripts of unknown structure. All of these transcribed sequences map distal to the satellite arrays. The boundary between satellite-rich interchromosomally duplicated DNA and chromosome-specific DNA therefore appears to define a transition from pericentromeric heterochromatin to euchromatin on the long arm of this chromosome.

  1. Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

    SciTech Connect

    Warburton, P.E.; Gosden, J.; Lawson, D.

    1996-04-15

    Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.

  2. Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

    PubMed Central

    Moon, Sung-Kyun; Lee, Haa-Yung; Pan, Huiqi; Takeshita, Tamotsu; Park, Raekil; Cha, Kiweon; Andalibi, Ali; Lim, David J

    2006-01-01

    Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway. PMID:16433908

  3. Modulation of human corticomuscular beta-range coherence with low-level static forces.

    PubMed

    Witte, Matthias; Patino, Luis; Andrykiewicz, Agnieszka; Hepp-Reymond, Marie-Claude; Kristeva, Rumyana

    2007-12-01

    Although corticomuscular synchronization in the beta range (15-30 Hz) was shown to occur during weak steady-state contractions, an examination of low-level forces around 10% of the maximum voluntary contraction (MVC) is still missing. We addressed this question by investigating coherence between electroencephalogram (EEG) and electromyogram (EMG) as well as cortical spectral power during a visuomotor task. Eight healthy right-handed subjects compensated isometrically static forces at a level of 4% and 16% of MVC with their right index finger. While 4% MVC was accompanied by low coherence values in the middle to high beta frequency range (25-30 Hz), a significant increase of coherence mainly confined to low beta frequencies (19-20 Hz) was observed with force of 16% MVC. Furthermore, this increase was associated with better performance, as reflected in decreased relative error in force during 16% MVC. We additionally show that periods of good motor performance within each condition were associated with higher values of EEG-EMG coherence and spectral power. In conclusion, our results suggest a role for beta-range corticomuscular coherence in effective sensorimotor integration, thus stabilizing corticospinal communication.

  4. Facilitation of Learning by Social-Emotional Feedback in Humans Is Beta-Noradrenergic-Dependent

    ERIC Educational Resources Information Center

    Mihov, Yoan; Mayer, Simon; Musshoff, Frank; Maier, Wolfgang; Kendrick, Keith M.; Hurlemann, Rene

    2010-01-01

    Adaptive behavior in dynamic environments critically depends on the ability to learn rapidly and flexibly from the outcomes of prior choices. In social environments, facial expressions of emotion often serve as performance feedback and thereby guide declarative learning. Abundant evidence implicates beta-noradrenergic signaling in the modulatory…

  5. A pseudodeficiency allele (D152N) of the human {beta}-glucuronidase gene

    SciTech Connect

    Vervoort, R.; Liebaers, I.; Lissens, W.

    1995-10-01

    We present evidence that a 480G{r_arrow}A transition in the coding region of the {Beta}glucuronidase gene, which results in an aspartic-acid-to-asparagine substitution at amino acid position 152 (D152N), produces a pseudodeficiency allele (GUSBp) that leads to greatly reduced levels of {Beta}-glucuronidase activity without apparent deleterious consequences. The 48OG{r_arrow}A mutation was found initially in the pseudodeficient mother of a child with mucopolysaccharidosis VII (MPSVII), but it was not on her disease-causing allele, which carried the L176F mutation. The 480G{r_arrow}A change was also present in an unrelated individual with another MPSVII allele who had unusually low {Beta}-glucuronidase activity, but whose clinical symptoms were probably unrelated to {Beta}-glucuronidase deficiency. This individual also had an R357X mutation, probably on his second allele. We screened 100 unrelated normal individuals for the 480G{r_arrow}A mutation with a PCR method and detected one carrier. Reduced {Beta}-glucuronidase activity following transfection of COS cells with the D152N cDNA supported the causal relationship between the D152N allele and pseudodeficiency. The mutation reduced the fraction of expressed enzyme that was secreted. Pulse-chase experiments indicated that the reduced activity in COS cells was due to accelerated intracellular turnover of the D152N enzyme. They also suggested that a potential glycosylation site created by the mutation is utilized in {approximately}50% of the enzyme expressed. 25 refs., 3 figs., 3 tabs.

  6. Purification, crystallization and preliminary X-ray diffraction analysis of the human mismatch repair protein MutS[beta

    SciTech Connect

    Tseng, Quincy; Orans, Jillian; Hast, Michael A.; Iyer, Ravi R.; Changela, Anita; Modrich, Paul L.; Beese, Lorena S.

    2012-03-16

    MutS{beta} is a eukaryotic mismatch repair protein that preferentially targets extrahelical unpaired nucleotides and shares partial functional redundancy with MutS{alpha} (MSH2-MSH6). Although mismatch recognition by MutS{alpha} has been shown to involve a conserved Phe-X-Glu motif, little is known about the lesion-binding mechanism of MutS{beta}. Combined MSH3/MSH6 deficiency triggers a strong predisposition to cancer in mice and defects in msh2 and msh6 account for roughly half of hereditary nonpolyposis colorectal cancer mutations. These three MutS homologs are also believed to play a role in trinucleotide repeat instability, which is a hallmark of many neurodegenerative disorders. The baculovirus overexpression and purification of recombinant human MutS{beta} and three truncation mutants are presented here. Binding assays with heteroduplex DNA were carried out for biochemical characterization. Crystallization and preliminary X-ray diffraction analysis of the protein bound to a heteroduplex DNA substrate are also reported.

  7. Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies.

    PubMed

    Jin, H; Uddin, M S; Huang, Y L; Teo, W K

    1994-01-01

    High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.

  8. Muscarinic receptor modulation of basal and beta-adrenergic stimulated function of the failing human left ventricle.

    PubMed

    Newton, G E; Parker, A B; Landzberg, J S; Colucci, W S; Parker, J D

    1996-12-15

    The objective of this study was to evaluate the effect of muscarinic receptor modulation on basal and beta-adrenergic stimulated left ventricular function in patients with heart failure. 21 heart failure patients and 14 subjects with normal ventricular function were studied. In Protocol 1 intracoronary acetylcholine resulted in a 60+/-8% inhibition of the left ventricular +dP/dt response to intracoronary dobutamine in the normal group, and a similar 70+/-13% inhibition in the heart failure group. Acetylcholine also attenuated the dobutamine-mediated acceleration of isovolumic relaxation (Tau) in both groups. Acetylcholine alone had no effect on Tau in the normal group, while it prolonged Tau in the heart failure group. In Protocol 2 intracoronary atropine resulted in a 35+/-10% augmentation of the inotropic response to dobutamine in the normal group, versus a non-significant 12+/-15% augmentation of the dobutamine response in the heart failure group. In Protocol 3, in 6 heart failure patients, both effects of acetylcholine, the slowing of ventricular relaxation and the inhibition of beta-adrenergic responses, were reversed by the addition of atropine. Therefore, in the failing human left ventricle muscarinic stimulation has an independent negative lusitropic effect and antagonizes the effects of beta-adrenergic stimulation.

  9. Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

    PubMed

    Rastaldi, M P; Candiano, G; Musante, L; Bruschi, M; Armelloni, S; Rimoldi, L; Tardanico, R; Sanna-Cherchi, S; Cherchi, S Sanna; Ferrario, F; Montinaro, V; Haupt, R; Parodi, S; Carnevali, M L; Allegri, L; Camussi, G; Gesualdo, L; Scolari, F; Ghiggeri, G M

    2006-08-01

    Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

  10. Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

    PubMed Central

    Hartwig, N. R.; Kalmbach, N.; Klietz, M.; Anlauf, M.; Eiden, L. E.; Weihe, E.

    2014-01-01

    Aims/hypothesis Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. Methods We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. Results The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. Conclusions/interpretation Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas. PMID:23404442

  11. Separation of beta-human chorionic gonadotropin and immunoglobulin G by a miniaturized size exclusion chromatography column

    NASA Astrophysics Data System (ADS)

    Yang, Yongmo; Chae, Junseok

    2009-04-01

    This report describes a miniaturized size exclusion chromatography column that effectively preseparates raw samples for medical point-of-care testing (POCT) devices. The minicolumn is constructed of polydimethylsiloxane fabricated on a glass slide. The minicolumn separates 300 ng/ml of beta-human chorionic gonadotropin (β-hCG) from an immunoglobulin G (IgG)-rich solution (100 μg/ml) in 7.7 min, with 2.23 resolution and 0.018 mm plate height. The complete analyte discrimination shows potential for the sample preparation stage of POCT devices for cancer screening, prognosis, and monitoring.

  12. beta-Hexachlorocyclohexane (beta-HCH)

    Integrated Risk Information System (IRIS)

    beta - Hexachlorocyclohexane ( beta - HCH ) ; CASRN 319 - 85 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Asses

  13. Upregulation of steroidogenic enzymes and ovarian 17beta-estradiol in human granulosa-lutein cells by Cordyceps sinensis mycelium.

    PubMed

    Huang, Bu-Miin; Hsiao, Kuei-Yang; Chuang, Pei-Chin; Wu, Meng-Hsing; Pan, Hsien-An; Tsai, Shaw-Jenq

    2004-05-01

    There is increasing evidence that 17beta-estradiol (E2) directly influences the quality of maturing oocytes and thus the outcome of assisted reproduction treatment. Although Cordyceps sinensis (CS) mycelium, a Chinese herbal medicine, is believed to enhance libido and fertility in both sexes, the mechanism of its effect in women has not been determined. The aim of the present study was to evaluate the effects of CS on steroidogenic enzyme expression and E2 biosynthesis in human granulosa-lutein cells (GLC). We found that CS induced E2 production by GLC in a dose- and time-dependent manner and that a 3-h treatment with CS induced increased levels of mRNAs coding for the P450 side chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and aromatase. Western blot analysis demonstrated that, after treatment with CS for 3 h, protein levels of steroidogenic acute regulatory protein (StAR) and aromatase were upregulated while P450scc and 3beta-HSD levels showed no substantial change. New protein synthesis was required for CS-induced E2 production because it was abrogated by cycloheximide pretreatment. Addition of 22(R)-hydroxycholesterol, thus bypassing the need for StAR protein, did not induce as much E2 production as CS treatment, indicating that upregulation of StAR protein was not the only factor contributing to CS-induced steroidogenesis. Cotreatment of GLCs with CS and aminoglutethimide, an aromatase inhibitor, completely abolished CS-induced E2 production. In conclusion, treatment of GLCs with CS results in increased E2 production due, at least in part, to increased StAR and aromatase expression. These data may help in the development of treatment regimens to improve the success rate of in vitro fertilization. PMID:14711788

  14. Induction of cytochrome P450IA1 gene expression in rat epidermis and human keratinocytes by. beta. -napthoflavone and benzanthracene

    SciTech Connect

    Khan, I.U.; Mukhtar, H.; Bickers, D.R.; Haqqi, T.M. )

    1991-03-15

    Cytochrome P450IA1 (P450IA1) plays a major role in the bioactivation of procarcinogens in various tissues including skin. However, factors controlling the expression of P450IA1 gene message in mammalian skin are unknown. In this study, the polymerase chain reaction (PCR) using specific primers was employed to study the expression of P450IA1 mRNA transcripts in rat epidermis and human keratinocytes (HK) treated with {beta}-napthoflavone ({beta}NF) and benzanthracene (BA). Total RNA was extracted from the epidermis of control and inducer-treated 4-day-old and adult Sprague Dawley rats, and from control and inducer-treated HL. cDNAs were synthesized using random primers and reverse transcriptase. PCR products were analyzed on agarose gel and quantitated by densitometry. Inducer treatment of rats and HK resulted in several-fold increases in aryl hydrocarbon hydroxylase (AHH) activity. The level of P450IA1 gene message increased 2-5-fold in treated animals as compared to controls; higher basal level and inducibility in adult than in 4-day-old rats. This induction occurred as early as 4 h after {beta}NF application, reached a maximum at 16 h and returned to basal levels by 36 h. Exposure to {beta}NF and BA resulted in 2-3-fold increase in gene message in HK. Northern blot analysis complemented PCR data. These results indicate that in mammalian skin P450IA1 gene expression is increased by the inducers of epidermal AHH activity.

  15. Case-control study of genus-beta human papillomaviruses in plucked eyebrow hairs and cutaneous squamous cell carcinoma.

    PubMed

    Iannacone, Michelle R; Gheit, Tarik; Pfister, Herbert; Giuliano, Anna R; Messina, Jane L; Fenske, Neil A; Cherpelis, Basil S; Sondak, Vernon K; Roetzheim, Richard G; Silling, Steffi; Pawlita, Michael; Tommasino, Massimo; Rollison, Dana E

    2014-05-01

    Cutaneous human papillomaviruses (HPV) have been reported in cutaneous squamous cell carcinoma (SCC). We conducted a clinic-based case-control study to investigate the association between genus-beta HPV DNA in eyebrow hairs (EBH) and SCC. EBH from 168 SCC cases and 290 controls were genotyped for genus-beta HPV DNA. SCC tumors from a subset of cases (n = 142) were also genotyped. Viral load was determined in a subset of specimens positive for a single HPV type. Associations with SCC were estimated by odds ratios (OR) and 95% confidence intervals (CI) adjusted for age and sex using logistic regression. Statistical tests were two-sided. EBH DNA prevalence was greater in cases (87%) than controls (73%) (p < 0.05), and the association with SCC increased with the number of HPV types present, (≥ 4 types vs. HPV-negative: OR = 2.02, 95% CI = 1.07-3.80; p(trend) = 0.02). Type-specific associations were observed between SCC and DNA in EBH for HPV23 (OR = 1.90, 95% CI = 1.10-3.30) and HPV38 (OR = 1.84, 95% CI = 1.04-3.24). Additionally, when compared with the controls, the DNA prevalence in EBH was significantly higher among cases for 11 of the 25 genus-beta types tested, when accounting for DNA for the same HPV type in the tumor (ORs = 3.44-76.50). Compared to controls, the mean viral DNA load in EBH among the selected cases was greater for HPV5, HPV8 and HPV24, but lower for HPV38. SCC cases were more likely than controls to have HPV DNA+ EBH for single and multiple HPV types, providing additional support for the potential role of genus-beta HPV infections in SCC development.

  16. Bone morphogenetic protein-7 inhibits constitutive and interleukin-1 beta-induced monocyte chemoattractant protein-1 expression in human mesangial cells: role for JNK/AP-1 pathway.

    PubMed

    Lee, Myung-Ja; Yang, Chul Woo; Jin, Dong Chan; Chang, Yoon Sik; Bang, Byung Kee; Kim, Yong-Soo

    2003-03-01

    Bone morphogenetic protein-7 (BMP-7), which belongs to the TGF-beta superfamily, has been shown to reduce macrophage infiltration and tissue injury in animal models of inflammatory renal disease. To explore the mechanism involved in the anti-inflammatory effect, we investigated the effect of BMP-7 on monocyte chemoattractant protein-1 (MCP-1) expression in cultured human mesangial cells. BMP- 7 significantly inhibited constitutive and IL-1 beta-induced MCP-1 protein production and MCP-1 mRNA expression by mesangial cells in a time- and concentration-dependent manner. BMP-7 also inhibited IL-1 beta-induced monocyte chemotactic activity released from the mesangial cells. We examined the role of transcription factors NF-kappa B and AP-1 in BMP-7 inhibition of IL-1 beta-induced MCP-1 expression. IL-1 beta increased NF-kappa B and AP-1 activity and both transcription factors mediated IL-1 beta-induced MCP-1 expression in mesangial cells. BMP-7 inhibited IL-1 beta-induced AP-1 activity in a concentration-dependent manner. In contrast, IL-1 beta-induced NF-kappa B activity and I kappa B alpha degradation were not affected by BMP-7. Furthermore, IL-1 beta-induced phosphorylation of c-Jun N-terminal kinase was inhibited by BMP-7. These data suggest that BMP-7 inhibits constitutive and IL-1 beta-induced MCP-1 expression in human mesangial cells partly by inhibiting c-Jun N-terminal kinase activity and subsequent AP-1 activity, and provide new insight into the therapeutic potential of BMP-7 in the inflammatory renal diseases.

  17. Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

    PubMed Central

    Caterina, J J; Ciavatta, D J; Donze, D; Behringer, R R; Townes, T M

    1994-01-01

    The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression. PMID:8152905

  18. Induction of apoptosis by penta-O-galloyl-beta-D-glucose through activation of caspase-3 in human leukemia HL-60 cells.

    PubMed

    Pan, M H; Lin, J H; Lin-Shiau, S Y; Lin, J K

    1999-09-24

    Penta-O-galloyl-beta-D-glucose is structurally related to (-)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta-O-galloyl-beta-D-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta-O-galloyl-beta-D-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta-O-galloyl-beta-D-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta-O-galloyl-beta-D-glucose-induced apoptosis. Treatment with penta-O-galloyl-beta-D-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) inhibited penta-O-galloyl-beta-D-glucose-induced DNA fragmentation. Furthermore, treatment with penta-O-galloyl-beta-D-glucose (50 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta-O-galloyl-beta-D-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta-O-galloyl-beta-D-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta-O-galloyl-beta-D-glucose may provide a pivotal mechanism for its cancer chemopreventive action.

  19. Beta-2-glycoprotein specificity of human anti-phospholipid antibody resides on the light chain: a novel mechanism for acquisition of cross-reactivity by an autoantibody.

    PubMed

    Kumar, Sanjeev; Nagl, Sylvia; Kalsi, Jatinderpal K; Ravirajan, Chelliah T; Athwal, Dee; Latchman, David S; Pearl, Laurence H; Isenberg, David A

    2005-01-01

    We have recently shown that the anti-cardiolipin activity of human anti-phospholipid antibody UK4 (lambda) resides on its heavy chain. We now show that UK4 possesses strong reactivity to the plasma-protein beta2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 (kappa) with no reactivity to beta2-GPI, we now demonstrate that both the constructs possessing the auto-antibody-derived light chain exhibited significant binding to beta2-GPI. However, the construct possessing UK4 heavy chain in association with 4D5 light chain, exhibited no anti-beta2-GPI activity. Furthermore, there was a low increase (approximately 10%) in the binding of UK4 to cardiolipin in the presence of beta2-GPI. The results demonstrate that anti-beta2-GPI activity resides on UK4 light chain and, importantly, this activity could be transferred to a novel antibody construct via the light chain alone. Computer-generated models of the three-dimensional structures of UK4 and its hybrids, suggest predominant interaction of UK4 light chain with domain IV of beta2-GPI. Molecular docking experiments highlight a number of potential sites on beta2-GPI for interaction of UK4 and indicate as to how beta2-GPI recognition may occur primarily via the autoantibody light chain. The study provides first demonstration of the occurrence of anti-phospholipid and anti-beta2-GPI activities separately on heavy and light chains of an autoantibody. The possible mechanisms that such antibodies may employ to recognise their antigens, are discussed.

  20. Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

    PubMed

    Dykxhoorn, Derek M; Schlehuber, Lisa D; London, Irving M; Lieberman, Judy

    2006-04-11

    A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

  1. Activation of a beta-galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV-infected cells.

    PubMed

    Rocancourt, D; Bonnerot, C; Jouin, H; Emerman, M; Nicolas, J F

    1990-06-01

    A quantitative bioassay for human immunodeficiency viruses has been developed on the basis of the ability of the tat gene to transactivate the expression of an integrated beta-galactosidase gene in a HeLa-CD4+ cell line. Infection by a single virion of HIV-1 or HIV-2 corresponds to a unique blue syncytium or a cell cluster detected after fixation and addition of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (a beta-galactosidase substrate). The number of infected lymphoid cells in a culture (stimulated human peripheral blood lymphocytes and cell lines) can also be quantified by cell-to-cell transmission of HIV into the HeLa-CD4(+)-beta-galactosidase monolayer. Infections by simian immunodeficiency viruses are similarly detected. This assay has been used to determine the dose response of drugs, the half-life of HIV at 37 degrees C, and the appearance of infectious particles after virus infection.

  2. beta. -endorphin-like and. alpha. -MSH-like immunoreactivities in human milk

    SciTech Connect

    Ferrando, T.; Rainero, I.; De Gennaro, T.; Oggero, R.; Mostert, M.; Dattola, P.; Pinessi, L. )

    1990-01-01

    We measured with radioimmunoassay the {beta}-endorphin-like and {alpha}-MSH-like immunoreactivities in milk and plasma of 8 lactating women. Mean {beta}-endorphin concentrations ({plus minus} SD) were 16.6 {plus minus} 6.7 fmol/ml in milk and 9.9 {plus minus} 4.1 fmol/ml in plasma. {alpha}-MSH concentrations were 39.4 {plus minus} 15.5 pg/ml in milk and 18.2 {plus minus} 8.4 pg/ml in plasma. The concentrations of both peptides in milk were significantly higher than in plasma. No significant correlation between milk and plasma concentrations of these peptides was found.

  3. Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

    NASA Technical Reports Server (NTRS)

    Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

    2006-01-01

    The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

  4. Towards an automated monitoring of human settlements in South Africa using high resolution SPOT satellite imagery

    NASA Astrophysics Data System (ADS)

    Kemper, T.; Mudau, N.; Mangara, P.; Pesaresi, M.

    2015-04-01

    Urban areas in sub-Saharan Africa are growing at an unprecedented pace. Much of this growth is taking place in informal settlements. In South Africa more than 10% of the population live in urban informal settlements. South Africa has established a National Informal Settlement Development Programme (NUSP) to respond to these challenges. This programme is designed to support the National Department of Human Settlement (NDHS) in its implementation of the Upgrading Informal Settlements Programme (UISP) with the objective of eventually upgrading all informal settlements in the country. Currently, the NDHS does not have access to an updated national dataset captured at the same scale using source data that can be used to understand the status of informal settlements in the country. This pilot study is developing a fully automated workflow for the wall-to-wall processing of SPOT-5 satellite imagery of South Africa. The workflow includes an automatic image information extraction based on multiscale textural and morphological image features extraction. The advanced image feature compression and optimization together with innovative learning and classification techniques allow a processing of the SPOT-5 images using the Landsat-based National Land Cover (NLC) of South Africa from the year 2000 as low-resolution thematic reference layers as. The workflow was tested on 42 SPOT scenes based on a stratified sampling. The derived building information was validated against a visually interpreted building point data set and produced an accuracy of 97 per cent. Given this positive result, is planned to process the most recent wall-to-wall coverage as well as the archived imagery available since 2007 in the near future.

  5. The Human-Technological Interface: An Analysis of a Satellite Communication Learning Environment.

    ERIC Educational Resources Information Center

    Collins, Valerie A. C.; Murphy, Peter J.

    1987-01-01

    The effectiveness of distance education methods that involve direct interaction between the student and communications satellite technology is discussed, drawing on experiences in British Columbia adult distance education programs using interactive instructional television and other modern technologies. (MSE)

  6. Facilitation of learning by social-emotional feedback in humans is beta-noradrenergic-dependent.

    PubMed

    Mihov, Yoan; Mayer, Simon; Musshoff, Frank; Maier, Wolfgang; Kendrick, Keith M; Hurlemann, René

    2010-08-01

    Adaptive behavior in dynamic environments critically depends on the ability to learn rapidly and flexibly from the outcomes of prior choices. In social environments, facial expressions of emotion often serve as performance feedback and thereby guide declarative learning. Abundant evidence implicates beta-noradrenergic signaling in the modulatory influence of emotion on declarative learning. It is currently unclear whether a similar mechanism also mediates a guidance of declarative learning by social-emotional feedback administered in the form of facial expressions. We therefore conducted a double-blind randomized placebo-controlled trial to test the effects of a 40-mg single oral dose of the nonspecific beta-noradrenergic antagonist propranolol in a behavioral task that required gradual declarative learning of item-category associations from either social-emotional (happy vs. angry faces) or nonsocial (green vs. red color signals) trial-by-trial feedback. As predicted on the basis of our previous experiments, learning from social-emotional feedback was more effective than learning from nonsocial feedback in placebo-treated subjects. This advantage of social-emotional over nonsocial feedback was abolished by propranolol treatment. Propranolol had no effect on learning during the nonsocial feedback condition. Our findings suggest that a facilitation of declarative learning by social-emotional feedback critically involves signaling via beta-noradrenergic receptors. PMID:20457167

  7. Neurofilament heavy polypeptide regulates the Akt-beta-catenin pathway in human esophageal squamous cell carcinoma.

    PubMed

    Kim, Myoung Sook; Chang, Xiaofei; LeBron, Cynthia; Nagpal, Jatin K; Lee, Juna; Huang, Yiping; Yamashita, Keishi; Trink, Barry; Ratovitski, Edward A; Sidransky, David

    2010-01-01

    Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.

  8. Satellite cell activity is differentially affected by contraction mode in human muscle following a work-matched bout of exercise.

    PubMed

    Hyldahl, Robert D; Olson, Ty; Welling, Tyson; Groscost, Logan; Parcell, Allen C

    2014-01-01

    Optimal repair and adaptation of skeletal muscle is facilitated by resident stem cells (satellite cells). To understand how different exercise modes influence satellite cell dynamics, we measured satellite cell activity in conjunction with markers of muscle damage and inflammation in human skeletal muscle following a single work- and intensity-matched bout of eccentric (ECC) or concentric contractions (CON). Participants completed a single bout of ECC (n = 7) or CON (n = 7) of the knee extensors. A muscle biopsy was obtained before and 24 h after exercise. Functional measures and immunohistochemical analyses were used to determine the extent of muscle damage and indices of satellite cell activity. Cytokine concentrations were measured using a multiplexed magnetic bead assay. Isokinetic peak torque decreased following ECC (p < 0.05) but not CON. Greater histological staining of the damage marker Xin was observed in muscle samples of ECC vs. CON. Tenasin C immunoreactivity increased 15 fold (p < 0.01) following ECC and was unchanged following CON. The inflammatory cytokines interferon gamma-induced protein 10 (IP-10) and monocyte chemotactic protein 1 (MCP-1) increased pre- to post-ECC (4.26 ± 1.4 vs. 10.49 ± 5.8 pg/ml, and 3.06 ± 0.7 vs. 6.25 ± 4.6 pg/ml, respectively; p < 0.05). There was no change in any cytokine post-CON. Satellite cell content increased 27% pre- to post-ECC (0.10 ± 0.031 vs. 0.127 ± 0.041, respectively; p < 0.05). There was no change in satellite cell number in CON (0.099 ± 0.027 vs. 0.102 ± 0.029, respectively). There was no fiber type-specific satellite cell response following either exercise mode. ECC but not CON resulted in an increase in MyoD positive nuclei per myofiber pre- to post-exercise (p < 0.05), but there was no change in MyoD DNA binding activity in either condition. In conclusion, ECC but not CON results in functional and histological evidence of muscle damage that is accompanied by increased satellite cell activity 24 h

  9. Satellite cell activity is differentially affected by contraction mode in human muscle following a work-matched bout of exercise

    PubMed Central

    Hyldahl, Robert D.; Olson, Ty; Welling, Tyson; Groscost, Logan; Parcell, Allen C.

    2014-01-01

    Optimal repair and adaptation of skeletal muscle is facilitated by resident stem cells (satellite cells). To understand how different exercise modes influence satellite cell dynamics, we measured satellite cell activity in conjunction with markers of muscle damage and inflammation in human skeletal muscle following a single work- and intensity-matched bout of eccentric (ECC) or concentric contractions (CON). Participants completed a single bout of ECC (n = 7) or CON (n = 7) of the knee extensors. A muscle biopsy was obtained before and 24 h after exercise. Functional measures and immunohistochemical analyses were used to determine the extent of muscle damage and indices of satellite cell activity. Cytokine concentrations were measured using a multiplexed magnetic bead assay. Isokinetic peak torque decreased following ECC (p < 0.05) but not CON. Greater histological staining of the damage marker Xin was observed in muscle samples of ECC vs. CON. Tenasin C immunoreactivity increased 15 fold (p < 0.01) following ECC and was unchanged following CON. The inflammatory cytokines interferon gamma-induced protein 10 (IP-10) and monocyte chemotactic protein 1 (MCP-1) increased pre- to post-ECC (4.26 ± 1.4 vs. 10.49 ± 5.8 pg/ml, and 3.06 ± 0.7 vs. 6.25 ± 4.6 pg/ml, respectively; p < 0.05). There was no change in any cytokine post-CON. Satellite cell content increased 27% pre- to post-ECC (0.10 ± 0.031 vs. 0.127 ± 0.041, respectively; p < 0.05). There was no change in satellite cell number in CON (0.099 ± 0.027 vs. 0.102 ± 0.029, respectively). There was no fiber type-specific satellite cell response following either exercise mode. ECC but not CON resulted in an increase in MyoD positive nuclei per myofiber pre- to post-exercise (p < 0.05), but there was no change in MyoD DNA binding activity in either condition. In conclusion, ECC but not CON results in functional and histological evidence of muscle damage that is accompanied by increased satellite cell activity 24 h

  10. Differential expression of laminin isoforms and alpha 6-beta 4 integrin subunits in the developing human and mouse intestine.

    PubMed

    Simon-Assmann, P; Duclos, B; Orian-Rousseau, V; Arnold, C; Mathelin, C; Engvall, E; Kedinger, M

    1994-09-01

    The intestinal tissue is characterized by important morphogenetic movements during development as well as by a continuous dynamic crypt to villus epithelial cell migration leading to differentiation of specialized cells. In this study, we have examined the spatio-temporal distribution of laminin A and M chains as well as of alpha 6 and beta 4 integrin subunits in adult and developing human and mouse intestine by indirect immunofluorescence. Selective expression of the constituent polypeptides of laminin isoforms (A and M chains) was demonstrated. In the mature human intestine, A and M chains were found to be complementary, the M chain being restricted to the base of crypts and the A chain lining the villus basement membrane. In the developing human intestine, M chain expression was delayed as compared to that of A chain; as soon as the M chain was visualized, it exhibited the typical localization in the crypt basement membrane. A somewhat different situation was found in the adult mouse intestine, since both M and A chains were found in the crypts. During mouse intestinal development the delayed expression of the M chain as compared to that of the A chain was also obvious. The absence of M chain expression in mutant dy mouse did not impair intestinal morphogenesis nor cell differentiation. The expression of alpha 6 and beta 4 subunits was not coordinated. In both species the alpha 6 expression preceded that of beta 4. Furthermore, while beta 4 staining in adult mouse intestine was detected at the basal surface of all cells lining the crypt-villus, that of alpha 6 was mainly confined to the crypt cell compartment. An overall similarity of location between alpha 6 integrin subunit and laminin A chain at the epithelial/stromal interface was noted. These data indicate that the spatial and temporal distribution of laminin variants in the developing intestine may be characteristic for each species and that interactions of laminin variants with particular receptors may be

  11. Validation of a self-excising marker in the human pathogen Aspergillus fumigatus by employing the beta-rec/six site-specific recombination system.

    PubMed

    Hartmann, Thomas; Dümig, Michaela; Jaber, Basem M; Szewczyk, Edyta; Olbermann, Patrick; Morschhäuser, Joachim; Krappmann, Sven

    2010-09-01

    Recyclable markers based on site-specific recombination allow repetitive gene targeting in filamentous fungi. Here we describe for the first time functionality of the bacterial recombination system employing beta serine recombinase acting on six recognition sequences (beta-rec/six) in a fungal host, the human pathogen Aspergillus fumigatus, and its use in establishing a self-excising resistance marker cassette for serial gene replacement.

  12. The human papillomavirus18 E7 protein inhibits CENP-C binding to α-satellite DNA.

    PubMed

    Yaginuma, Yuji; Yoshimoto, Masafumi; Eguchi, Ayami; Tokuda, Aoi; Takahashi, Shoko

    2015-07-01

    Human papillomavirus (HPV) infection leads to aneuploidy, a numerical chromosomal aberration that is caused by dysregulation of chromosomal segregation. We previously found that the E7 proteins of high-risk HPVs, but not of low-risk HPVs, could bind to centromere protein-C (CENP-C). In this study, we first found that CENP-C could bind centromere α-satellite DNAs using ChIP analysis and HA-tagged CENP-C/nuc transfected 293T cells. We then investigated if HA-CENP-C/nuc binding to α-satellite DNAs was affected by the E7 proteins of high- or low-risk HPVs. We found that transfection of the FLAG tagged HPV18 E7 inhibited the binding of HA-CENP-C/nuc to α-satellite DNAs. This finding was confirmed in HeLa S3 cells transfected with siRNA targeted to HPV18 E7 expression. We therefore speculate that altered function of kinetochores as a result of inhibition of CENP-C and α-satellite DNAs binding may be associated with the chromosomal abnormalities observed in HPV18-positive cancers.

  13. Distribution of beta-adrenergic receptors in failing human myocardium. Implications for mechanisms of down-regulation

    SciTech Connect

    Murphree, S.S.; Saffitz, J.E.

    1989-06-01

    The density of beta-adrenergic receptors is reduced in crude membranes prepared from failing human myocardium. We used quantitative autoradiography of radioligand binding sites in intact tissue slices to determine whether the total tissue content of receptors is reduced and to characterize the transmural distribution of receptors in cardiac myocytes and the coronary vasculature in hearts obtained from nine cardiac transplant patients with severe congestive failure. Binding of (125Iodo)cyanopindolol to transmural slices of human myocardium was rapid, saturable, stereoselective, and displaceable by agonists and antagonists with an appropriate rank order of potency. Binding isotherms in four normal and nine failing ventricles showed a significant reduction in the total tissue content of beta-receptors in failing myocardium (38.3 +/- 2.0 fmol/mg protein) compared with normal tissue (52.4 +/- 1.7 fmol/mg protein, p = 0.038). In the normal ventricles, the greatest receptor density was observed autoradiographically in myocytic regions of the subendocardium. Receptor density of the coronary arterioles was approximately 70% of that in adjacent myocytic regions. The density of binding sites in both myocytic regions and arterioles was diminished in all regions of the failing ventricles, but down-regulation was due primarily to a selective reduction of beta-receptors of subendocardial myocytes (63 +/- 5% of subepicardial receptor density vs. 115 +/- 6% in controls, p less than 0.0001). These observations indicate that down-regulation occurs nonuniformly in the transmural distribution and thus is likely not related simply to elevated circulating catecholamine levels.

  14. Differential cell cycle response of nontumorigenic and tumorigenic human papillomavirus-positive keratinocytes towards transforming growth factor-beta1.

    PubMed

    Hasskarl, J; Butz, K; Whitaker, N; Ullmann, A; Dürst, M; Hoppe-Seyler, F

    2000-01-01

    Human papillomaviruses (HPVs) are causative agents of a number of malignancies in humans, including cervical cancer. Their tumorigenic potential is linked to expression of the viral E6/E7 genes which can interfere with normal cell cycle control by targeting p53, p21WAF1, p27KIP1, and pRb. We show here that nontumorigenic and tumorigenic HPV-positive keratinocytes (HPK) exhibit striking differences in the response of cell cycle regulatory genes towards transforming growth factor beta-beta1. Treatment with this agent led to an efficient induction of p53 and the growth-inhibitory p15INK4 and p21WAF1 genes only in nontumorigenic HPKs and was linked to an efficient reduction in viral E6/E7 oncogene expression. This was associated with increased pRb levels, exhibiting sustained hypophosphorylation, and a permanent growth arrest in the G1 phase of the cell cycle. In contrast, tumorigenic HPKs exhibited only a modest rise in p53 protein levels and a substantially reduced induction of the p15INK4 and p21WAF1 genes, which was linked to a lesser degree of viral oncogene repression. In addition, tumorigenic HPKs rapidly resumed cell growth after a transient G1 arrest, concomitantly with the reappearance of hyperphosphorylated pRb. These results support the notion that the progression of HPV-positive cells to a malignant phenotype is associated with increased resistance to growth inhibition by transforming growth factor-beta1. This is linked in the tumorigenic cells to a lack of persistent G1 arrest, inefficient induction of several cell cycle control genes involved in growth inhibition, and inefficient repression of the growth-promoting viral E6/E7 oncogenes. PMID:10794545

  15. A high performance sensorimotor beta rhythm-based brain computer interface associated with human natural motor behavior

    NASA Astrophysics Data System (ADS)

    Bai, Ou; Lin, Peter; Vorbach, Sherry; Floeter, Mary Kay; Hattori, Noriaki; Hallett, Mark

    2008-03-01

    To explore the reliability of a high performance brain-computer interface (BCI) using non-invasive EEG signals associated with human natural motor behavior does not require extensive training. We propose a new BCI method, where users perform either sustaining or stopping a motor task with time locking to a predefined time window. Nine healthy volunteers, one stroke survivor with right-sided hemiparesis and one patient with amyotrophic lateral sclerosis (ALS) participated in this study. Subjects did not receive BCI training before participating in this study. We investigated tasks of both physical movement and motor imagery. The surface Laplacian derivation was used for enhancing EEG spatial resolution. A model-free threshold setting method was used for the classification of motor intentions. The performance of the proposed BCI was validated by an online sequential binary-cursor-control game for two-dimensional cursor movement. Event-related desynchronization and synchronization were observed when subjects sustained or stopped either motor execution or motor imagery. Feature analysis showed that EEG beta band activity over sensorimotor area provided the largest discrimination. With simple model-free classification of beta band EEG activity from a single electrode (with surface Laplacian derivation), the online classifications of the EEG activity with motor execution/motor imagery were: >90%/~80% for six healthy volunteers, >80%/~80% for the stroke patient and ~90%/~80% for the ALS patient. The EEG activities of the other three healthy volunteers were not classifiable. The sensorimotor beta rhythm of EEG associated with human natural motor behavior can be used for a reliable and high performance BCI for both healthy subjects and patients with neurological disorders. Significance: The proposed new non-invasive BCI method highlights a practical BCI for clinical applications, where the user does not require extensive training.

  16. Gross alpha and beta activity analyses in urine-a routine laboratory method for internal human radioactivity detection.

    PubMed

    Chen, Xiaowen; Zhao, Luqian; Qin, Hongran; Zhao, Meijia; Zhou, Yirui; Yang, Shuqiang; Su, Xu; Xu, Xiaohua

    2014-05-01

    The aim of this work was to develop a method to provide rapid results for humans with internal radioactive contamination. The authors hypothesized that valuable information could be obtained from gas proportional counter techniques by screening urine samples from potentially exposed individuals rapidly. Recommended gross alpha and beta activity screening methods generally employ gas proportional counting techniques. Based on International Standards Organization (ISO) methods, improvements were made in the evaporation process to develop a method to provide rapid results, adequate sensitivity, and minimum sample preparation and operator intervention for humans with internal radioactive contamination. The method described by an American National Standards Institute publication was used to calibrate the gas proportional counter, and urine samples from patients with or without radionuclide treatment were measured to validate the method. By improving the evaporation process, the time required to perform the assay was reduced dramatically. Compared with the reference data, the results of the validation samples were very satisfactory with respect to gross-alpha and gross-beta activities. The gas flow proportional counting method described here has the potential for radioactivity monitoring in the body. This method was easy, efficient, and fast, and its application is of great utility in determining whether a sample should be analyzed by a more complicated method, for example radiochemical and/or γ-spectroscopy. In the future, it may be used commonly in medical examination and nuclear emergency treatment.Health Phys. 106(5):000-000; 2014.

  17. Transforming growth factor beta 1 enhances expression of 50 kDa protein related to 2'-5' oligoadenylate synthetase in human sperm cells.

    PubMed

    Naz, R K; Kumar, R

    1991-01-01

    Human cellular polypeptide factors, namely interferon-alpha, interferon-gamma transforming growth factor (TGF)-alpha, and TGF-beta 1, were analyzed for their effect on motility of human sperm cells. Both interferons caused an inhibition of sperm cell motility due to direct cytotoxic effects without inducing 2'-5' oligoadenylate [2-5(A)]synthetase activity. TGF-alpha affected neither motility nor the levels of 2-5(A) synthetase in sperm cells. TGF-beta 1 had no affect on sperm motility, yet it caused an induction of 2-5(A)synthetase activity. Western immunoblot analysis of TGF-beta 1-treated sperm indicated an enhancement of a 50 kDa protein. Metabolic labeling of sperm cells revealed biosynthesis of one major protein of 50 kDa and at least five minor proteins in the range of 30-92 kDa; the level of 50 kDa protein increased after treatment with TGF-beta 1. The treatment of sperm cells with TGF-beta 1 did not affect their penetration in zona-free hamster eggs (SPA). These results indicate that TGF-beta 1 enhances expression of a 50 kDa protein related to 2-5(A) synthetase in human sperm cells along with other minor proteins, and this increase does not affect sperm motility and SPA.

  18. Cellular growth and survival are mediated by beta 1 integrins in normal human breast epithelium but not in breast carcinoma

    SciTech Connect

    Howlett, Anthony R; Bailey, Nina; Damsky, Caroline; Petersen, Ole W; Bissell, Mina J

    1994-11-28

    We previously established a rapid three-dimensional assay for discrimination of normal and malignant human breast epithelial cells using a laminin-rich reconstituted basement membrane. In this assay, normal epithelial cells differentiate into well-organized acinar structures whereas tumor cells fail to recapitulate this process and produce large, disordered colonies. The data suggest that breast acinar morphogenesis and differentiation is regulated by cell-extracellular matrix (ECM) interactions and that these interactions are altered in malignancy. Here, we investigated the role of ECM receptors (integrins) in these processes and report on the expression and function of potential laminin receptors in normal and tumorigenic breast epithelial cells. Immmunocytochemical analysis showed that normal and carcinoma cells in a three-dimensional substratum express profiles of integrins similar to normal and malignant breast tissues in situ. Normal cells express {alpha}1, {alpha}2, {alpha}3, {alpha}6, {beta}1 and {beta}4 integrin subunits, whereas breast carcinoma cells show variable losses, disordered expression, or down regulation of these subunits. Function-blocking experiments using inhibitory antiintegrin subunit antibodies showed a >5-fold inhibition of the formation of acinar structures by normal cells in the presence of either anti-{beta}1 or anti-{alpha}3 antibodies, whereas anti-{alpha}2 or -{alpha}6 had little or no effect. In experiments where collagen type I gels were used instead of basement membrane, acinar morphogenesis was blocked by anti-{beta}1 and -{alpha}2 antibodies but not by anti-{alpha}3. These data suggest a specificity of integrin utilization dependent on the ECM ligands encountered by the cell. The interruption of normal acinar morphogenesis by anti-integrin antibodies was associated with an inhibition of cell growth and induction of apoptosis. Function-blocking antibodies had no inhibitory effect on the rate of tumor cell growth, survival or

  19. Impact of Inhibitors and L2 Antibodies upon the Infectivity of Diverse Alpha and Beta Human Papillomavirus Types

    PubMed Central

    Kwak, Kihyuck; Jiang, Rosie; Wang, Joshua W.; Jagu, Subhashini; Kirnbauer, Reinhard; Roden, Richard B. S.

    2014-01-01

    The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families

  20. Genetic mapping of the beta 1 GABA receptor gene to human chromosome 4, using a tetranucleotide repeat polymorphism.

    PubMed Central

    Dean, M; Lucas-Derse, S; Bolos, A; O'Brien, S J; Kirkness, E F; Fraser, C M; Goldman, D

    1991-01-01

    As more coding loci for functional human genes are described, there is a growing need to identify DNA polymorphisms in specific genes. By examining DNA sequences within the introns of the beta 1 subunit of the gamma-aminobutyric acid receptor gene, GABARB1, we found a tetranucleotide repeat sequence (GATA). Amplification of this region by using PCR revealed seven alleles and a high degree of polymorphism (PIC = .75) in human populations. DNAs from the CEPH families were typed for the GABARB1 intron polymorphism and were analyzed with respect to 20 linked markers on chromosome 4. The results permit placement of GABARB1 on the linkage map of chromosome 4, between D4S104 and ALB. These results affirm that sequence analysis of noncoding segments included within or adjacent to functional genes has value as a strategy to detect highly informative polymorphisms. Images Figure 2 PMID:1652891

  1. Alpha 4 beta 7 integrin expression is associated with the leukemic evolution of human and murine T-cell lymphoblastic lymphomas.

    PubMed Central

    Dolcetti, R.; Giardini, R.; Doglioni, C.; Cariati, R.; Pomponi, F.; D'Orazi, C.; Rao, S.; Lazarovits, A. I.; Butcher, E. C.; Boiocchi, M.

    1997-01-01

    We have previously shown that the in vivo coordinated expression of individual alpha 4 and beta 7 integrin chains correlated with the leukemic potential displayed by cell lines derived from murine lymphoblastic T-cell lymphomas (T-LBLs) when transplanted subcutaneously into syngeneic AKR mice. In the present study, by using immunofluorescence and immunocytochemical analyses, we have confirmed that the in vivo up-regulation of the alpha 4 beta 7 heterodimeric complex is associated with the leukemic behavior of AKR T-LBLs. In addition, when compared with the parental, highly leukemic NQ22 cells, the variant cell line NQ22V exhibited a reduced leukemic potential that was invariably associated with a delayed alpha 4 beta 7 up-regulation in vivo Moreover, the leukemic cell line SJ-1, derived from a spontaneous T-LBL of the SJL strain, also displayed high levels of alpha 4 beta 7 expression with a pattern of tissue distribution similar to that of NQ22 cells from leukemic AKR animals. Of note, in most of the tissues involved by murine T-LBL dissemination, and particularly in liver, kidney, and lung, alpha 4 beta 7-positive leukemic cells were always located around strongly VCAM-1-positive vascular spaces. These findings are consistent with a possible role of alpha 4 beta 7/VCAM-1 interactions in the extravasation and, consequently, in the leukemic dissemination of murine T-LBL cells. Immunocytochemical analysis carried out in 11 human T-LBLs showed that pathological lymph nodes from all 7 cases with bone marrow infiltration at presentation carried alpha 4 beta 7-positive cells, whereas all 4 aleukemic T-LBLs were repeatedly alpha 4 beta 7 negative, also in metachronous lesions. These findings suggest that alpha 4 beta 7-positive human T-LBLs may represent a distinct clinicopathological entity. In addition, alpha 4 beta 7 expression was significantly more prevalent in younger patients (< 11 years; P = 0.02), further supporting such a hypothesis. Moreover, as in murine T

  2. Isocholic acid formation from 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid with human liver enzyme.

    PubMed

    Amuro, Y; Yamade, W; Yamamoto, T; Kudo, K; Fujikura, M; Maebo, A; Hada, T; Higashino, K

    1986-12-01

    The formation of isocholic acid from 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7 alpha, 12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid at pH 7.4 in a phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography/mass spectrometry. Results showed that 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid was reduced mainly to isocholic acid and to cholic acid in a smaller amount in the presence of NADPH, while it was reduced only to cholic acid in the presence of NADH. The reducing enzyme participating in the formation of isocholic acid was localized largely in the cytosol and had more specificity to the unconjugated form as substrate than to the conjugated forms. 3-Keto bile acid analogues, 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids were not reduced to the corresponding iso-bile acids by the cytosol in the same conditions used in the isocholic acid formation and the activity of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was not inhibited by the addition of 3-keto-5 beta-cholanoic acid or 7 alpha-hydroxy-3-keto-5 beta-cholanoic acid to the reaction mixture. Furthermore, on column chromatography of Affi-Gel Blue, the peak of the enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid was clearly distinguished from that of the enzyme catalyzing the reduction of 3-keto-5 beta-cholanoic acid to isolithocholic acid and that of alcohol dehydrogenase. These results indicate that this enzyme catalyzing the reduction of 7 alpha,12 alpha-dihydroxy-3-keto-5 beta-cholanoic acid to isocholic acid is different from the enzyme(s) catalyzing the reduction 3-keto-5 beta-cholanoic and 7 alpha-hydroxy-3-keto-5 beta-cholanoic acids to the corresponding iso-bile acids

  3. Absorption and excretion of conjugated flavonols, including quercetin-4'-O-beta-glucoside and isorhamnetin-4'-O-beta-glucoside by human volunteers after the consumption of onions.

    PubMed

    Aziz, A A; Edwards, C A; Lean, M E; Crozier, A

    1998-09-01

    Flavonols are polyphenols found ubiquitously in plants and plant-products. Flavonols, particularly quercetin, are potent antioxidants in vitro and their intake has been associated inversely with the incidence of coronary heart disease. The aim of this study was to investigate the accumulation in plasma and excretion in urine of flavonol glucosides following ingestion of lightly fried onions. Five healthy volunteers followed a low-flavonoid diet for 3 days. On day 4, after an overnight fast, subjects were given 300 g of lightly fried yellow onions which contain conjugates of quercetin and isorhamnetin, including quercetin-3,4 '-diO-beta-glucoside, isorhamnetin-4'-O-beta-glucoside and quercetin-4'-O-beta-glucoside. Blood collection was carried out at 0 min, 0.5, 1.0, 1.5, 2, 3, 4, 5 and 24h after the supplement. In addition, subjects collected all their urine for 24h following the onion supplement. Isorhamnetin-4'-O-beta-glucoside and quercetin-4 '-O-beta-glucoside accumulated in plasma with maximum levels, defined as proportion of intake, of 10.7+/-2.6% and 0.13+/-0.03% respectively. The time of the quercetin-4'glucoside peak plasma concentration was 1.3+/-0.2 h after the ingestion of onions while a value of 1.8+/-0.7 h was obtained for isorhamnetin-4'-glucoside. Excretion in urine, as a proportion of intake, was 17.4+/-8.3% for isorhamnetin-4'-O-beta-glucoside and 0.2+/-0.1% for quercetin-4'-O-beta-glucoside. Possible reasons for the accumulation and excretion of isorhamnetin-4'-glucoside in proportionally much higher amounts than quercetin-4'-glucoside are discussed. It is concluded that flavonols are absorbed into the bloodstream as glucosides and minor structural differences affect markedly both the level of accumulation and the extent to which the conjugates are excreted. PMID:9802557

  4. Human interleukin 1. beta. (IL-1. beta. ), a more powerful inducer of bone demineralization than interleukin 1. cap alpha. IL-1. cap alpha. ), parathyroid hormone (PTH) or prostaglandin E/sub 2/ (PGE/sub 2/) in vitro

    SciTech Connect

    Chin, R.C.; Hodges, Y.C.; Allison, A.C.

    1986-03-01

    Effects of human IL-1..cap alpha.. and IL-1..beta.., prepared by recombinant DNA technology on cultures of rat fetal long bones, prelabelled with /sup 45/Ca were studied. IL-1..beta.. was found to be the most powerful inducer of bone calcium loss so far known. Maximal activity (2.5 times the control rate of calcium loss) was induced by IL-1..beta.. at concentrations between 1 x 10/sup -10/ M to 6 x 10/sup -12/ M. With IL-1..cap alpha.. maximal activity (1.5 times the control rate of calcium loss) was obtained at 6 x 10/sup -10/ M. With bovine PTH (1-34) maximal activity (1.8 times the control rate of calcium loss) was obtained at 1 x 10/sup -8/ M. With PGE/sub 2/ maximal activity (1.6 times the control rate of calcium loss) was obtained at 1 x 10/sup -7/ M. The calcium loss induced by IL-1..beta.. was inhibited in the presence of 1 x 10/sup -7/ M indomethacin, 5 x 10/sup -5/ M naproxen or ketorolac, or 5 x 10/sup -6/ M cyclohexamide. These findings suggest that protein synthesis and prostaglandin formation are required to mediate bone demineralization induced by IL-1..beta...

  5. Activating point mutations in the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of beta subunit dimerization and cell type-specific molecules in signalling.

    PubMed Central

    Jenkins, B J; D'Andrea, R; Gonda, T J

    1995-01-01

    We have combined retroviral expression cloning with random mutagenesis to identify two activating point mutations in the common signal-transducing subunit (h beta c) of the receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 by virtue of their ability to confer factor independence on the haemopoietic cell line, FDC-P1. One mutation (V449E) is located within the transmembrane domain and, by analogy with a similar mutation in the neu oncogene, may act by inducing dimerization of h beta c. The other mutation (I374N) lies in the extracellular, membrane-proximal portion of h beta c. Neither of these mutants, nor a previously described mutant of h beta c (FI delta, which has a small duplication in the extracellular region), was capable of inducing factor independence in CTLL-2 cells, while only V449E could induce factor independence in BAF-B03 cells. These results imply that the extracellular and transmembrane mutations act by different mechanisms. Furthermore, they imply that the mutants, and hence also wild-type h beta c, interact with cell type-specific signalling molecules. Models are presented which illustrate how these mutations may act and predict some of the characteristics of the putative receptor-associated signalling molecules. Images PMID:7556069

  6. Ccdc13 is a novel human centriolar satellite protein required for ciliogenesis and genome stability.

    PubMed

    Staples, Christopher J; Myers, Katie N; Beveridge, Ryan D D; Patil, Abhijit A; Howard, Anna E; Barone, Giancarlo; Lee, Alvin J X; Swanton, Charles; Howell, Michael; Maslen, Sarah; Skehel, J Mark; Boulton, Simon J; Collis, Spencer J

    2014-07-01

    Here, we identify coiled-coil domain-containing protein 13 (Ccdc13) in a genome-wide RNA interference screen for regulators of genome stability. We establish that Ccdc13 is a newly identified centriolar satellite protein that interacts with PCM1, Cep290 and pericentrin and prevents the accumulation of DNA damage during mitotic transit. Depletion of Ccdc13 results in the loss of microtubule organisation in a manner similar to PCM1 and Cep290 depletion, although Ccdc13 is not required for satellite integrity. We show that microtubule regrowth is enhanced in Ccdc13-depleted cells, but slowed in cells that overexpress Ccdc13. Furthermore, in serum-starved cells, Ccdc13 localises to the basal body, is required for primary cilia formation and promotes the localisation of the ciliopathy protein BBS4 to both centriolar satellites and cilia. These data highlight the emerging link between DNA damage response factors, centriolar and peri-centriolar satellites and cilia-associated proteins and implicate Ccdc13 as a centriolar satellite protein that functions to promote both genome stability and cilia formation. PMID:24816561

  7. Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor. beta. 1 and by the retinoblastoma gene product

    SciTech Connect

    Pietenpol, J.A.; Stein, R.W.; Moses, H.L. ); Muenger, K.; Howley, P.M. )

    1991-11-15

    Previous studies have shown that transforming growth factor {beta}1 (TGF-{beta}1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter region was required for regulation by both TGF-{beta}1 and pRB. These sequences, termed the TGF-{beta} control element (TCE), lie between positions {minus}86 and {minus}63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-{beta}1 treatment of TGF-{beta}-sensitive but not TGF-{beta}-insensitive cells. These data indicate that pRB can suppress c-myc transcription and growth inhibition.

  8. Neogenesis and proliferation of {beta}-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

    SciTech Connect

    Tokui, Yae . E-mail: ytokui@imed2.med.osaka-u.ac.jp; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro |

    2006-12-01

    Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of {beta}-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a {beta}-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of {beta}-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of {beta}-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted {beta}-cell differentiation (mainly from duct cells) and proliferation of pre-existing {beta}-cells, resulting in an increase of the {beta}-cell mass that improved glucose tolerance in diabetic mice.

  9. A benzothiophene-carboxamide is a potent inhibitor of IL-1beta induced VCAM-1 gene expression in human endothelial cells.

    PubMed

    Cobb, R R; Felts, K A; McKenzie, T C; Parry, G C; Mackman, N

    1996-03-18

    Vascular endothelial cells respond to cytokines such as IL-1 beta or TNF-alpha by undergoing a number of functional alterations. Among these alterations is the induction of cell surface adhesion molecules, including VCAM-1. In this report, we investigated the effects of a 3-alkoxybenzo[beta]thiophene-2-carboxamide (BZT) on the cytokine induction of VCAM-1 expression and activation of the transcription factor NF-kappa B in human endothelial cells. BZT blocked the IL-1 beta induced cell surface expression of VCAM-1 in human endothelial cells but did not prevent nuclear translocation of NF-kappa B. This study demonstrates that BZT is a potent inhibitor of VCAM-1 expression in human endothelial cells.

  10. Mouse model of human ovarian endometrioid adenocarcinoma based on somatic defects in the Wnt/beta-catenin and PI3K/Pten signaling pathways.

    PubMed

    Wu, Rong; Hendrix-Lucas, Neali; Kuick, Rork; Zhai, Yali; Schwartz, Donald R; Akyol, Aytekin; Hanash, Samir; Misek, David E; Katabuchi, Hidetaka; Williams, Bart O; Fearon, Eric R; Cho, Kathleen R

    2007-04-01

    One histologic subtype of ovarian carcinoma, ovarian endometrioid adenocarcinoma (OEA), frequently harbors mutations that constitutively activate Wnt/beta-catenin-dependent signaling. We now show that defects in the PI3K/Pten and Wnt/beta-catenin signaling pathways often occur together in a subset of human OEAs, suggesting their cooperation during OEA pathogenesis. Deregulation of these two pathways in the murine ovarian surface epithelium by conditional inactivation of the Pten and Apc tumor suppressor genes results in the formation of adenocarcinomas morphologically similar to human OEAs with 100% penetrance, short latency, and rapid progression to metastatic disease in upwards of 75% of mice. The biological behavior and gene expression patterns of the murine cancers resemble those of human OEAs with defects in the Wnt/beta-catenin and PI3K/Pten pathways.

  11. Modeling risk of ignition as a function of landscape and human presence using satellite observations

    NASA Astrophysics Data System (ADS)

    Loboda, T. V.; Hight-Harf, C.; French, N. H.

    2012-12-01

    Wildland fire is an integral component of many ecosystems worldwide. However, relatively little is known about the patterns of fire ignitions as a function of landscape and how they influence regional fire occurrence. Conventional methods of identifying the patterns of ignition are limited especially in areas where field-based data are not available. Satellite observations of fire activity from the Moderate Resolution Imaging Spectroradiometer (MODIS) now provide a 12-year record of fire activity globally. In this study we show how Fire Spread Reconstruction approach is applied to the MODIS active fire detections to analyze the spatial patterns and drivers of fire ignition in three different ecoregions: 1) boreal forests the Russian Far East (RFE), 2) Mediterranean ecosystems of Southern California, and 3) North American tundra. The resultant spatio-temporal patterns are used to develop a Risk of Ignition (ROI) coefficient within the Fire Occurrence modeling framework. Our results show pronounced differences in the spatial patterns of the ROI between the three regions. Only in tundra's remote and unpopulated regions temporal patterns of ROI coincide with the known patterns of fire occurrence and spread. In Southern California and the RFE ROI is more closely linked to spatial and temporal patterns of human activity on the landscape and thus does not correlate well with seasons when large burns occur. Fig. 1 shows the spatial and temporal distribution of ROI in the Russian Far East. The success rate of this method is limited by the amount of observations and fire occurrence within the ecosystems of interest: ROI is established with higher accuracy in the RFE for a single month of April when more than 4000 individual fire events were observed between 2001 and 2010. In tundra and in the winter months within the RFE, where relative amount of fire occurrence is low, the results are less robust. However, the accuracy of the ROI modeling will grow as the MODIS era

  12. Effect of xanthine derivates and dexamethasone on Streptococcus pneumoniae-stimulated production of tumor necrosis factor alpha, interleukin-1 beta (IL-1 beta), and IL-10 by human leukocytes.

    PubMed Central

    van Furth, A M; Seijmonsbergen, E M; Langermans, J A; van der Meide, P H; van Furth, R

    1995-01-01

    The present study concerns the release of the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha and of the anti-inflammatory cytokine IL-10 by human leukocytes in whole blood during stimulation with Streptococcus pneumoniae and the effects of various xanthine derivates, i.e., pentoxifylline (PTX), caffeine, and theofylline, and of dexamethasone (DXM). All three xanthine derivates and DXM inhibited the release of tumor necrosis factor alpha, PTX being the most effective. PTX, theofylline, and DXM inhibited the release of IL-1 beta, but caffeine did not affect IL-1 beta release. The release of IL-10 was significantly reduced by PTX at 24 h and by caffeine at 48 h, but DXM increased the release of this cytokine. In sum, the results of this study demonstrate that DXM inhibits only the release of proinflammatory cytokines but not of the anti-inflammatory cytokine IL-10 by human leukocytes, while PTX is the most potent inhibitor of both proinflammatory and anti-inflammatory cytokines. PMID:8574830

  13. The effects of dexamethasone and chlorpromazine on tumour necrosis factor-alpha, interleukin-1 beta, interleukin-1 receptor antagonist and interleukin-10 in human volunteers.

    PubMed Central

    Bleeker, M W; Netea, M G; Kullberg, B J; Van der Ven-Jongekrijg, J; Van der Meer, J W

    1997-01-01

    Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are pro-inflammatory cytokines that play an important role in severe infections, whereas IL-1 receptor antagonist (IL-1ra) and IL-10 are anti-inflammatory cytokines that counteract their effects. Chlorpromazine and dexamethasone protect mice against lethal endotoxaemia by decreasing circulating concentrations of TNF-alpha and IL-1 beta. We investigated whether administration of chlorpromazine or dexamethasone to human volunteers is able to modulate the lipopolysaccharide (LPS)-stimulated cytokine production capacity in whole blood. Blood samples were taken before and several time-points after medication. Circulating cytokine concentrations were low in all samples. LPS-induced TNF-alpha and IL-1 beta production in whole blood was inhibited by dexamethasone treatment, while chlorpromazine had no effect. When peripheral blood mononuclear cells were stimulated in vitro with LPS, the addition of chlorpromazine (1-100 ng/ml) had no modulatory action on TNF-alpha, IL-1 beta, IL-1ra or IL-10 synthesis. The chlorpromazine concentrations measured in circulation of volunteers were eight to 40 times lower than the concentrations shown to be effective in mice. In conclusion, chlorpromazine inhibits TNF-alpha and IL-1 beta production in mice at concentrations that cannot be reached in humans, thus precluding its usage in clinical anti-cytokine strategies. In contrast, dexamethasone is an effective inhibitor of pro-inflammatory cytokine production. PMID:9378493

  14. Non-invasive in vivo determination of the carotenoids beta-carotene and lycopene concentrations in the human skin using the Raman spectroscopic method

    NASA Astrophysics Data System (ADS)

    Darvin, M. E.; Gersonde, I.; Meinke, M.; Sterry, W.; Lademann, J.

    2005-08-01

    Resonance Raman spectroscopy was used as a fast and non-invasive optical method of measuring the absolute concentrations of beta-carotene and lycopene in living human skin. Beta-carotene and lycopene have different absorption values at 488 and 514.5 nm and, consequently, the Raman lines for beta-carotene and lycopene have different scattering efficiencies at 488 and 514.5 nm excitations. These differences were used for the determination of the concentrations of beta-carotene and lycopene. Using multiline Ar+ laser excitation, clearly distinguishable carotenoid Raman spectra can be obtained which are superimposed on a large fluorescence background. The Raman signals are characterized by two prominent Stokes lines at 1160 and 1525 cm-1, which have nearly identical relative intensities. Both substances were detected simultaneously. The Raman spectra are obtained rapidly, i.e. within about 10 s, and the required laser light exposure level is well within safety standards. The disturbance of the measurements by non-homogeneous skin pigmentation was avoided by using a relatively large measuring area of 35 mm2. It was shown that beta-carotene and lycopene distribution in human skin strongly depends upon the skin region studied and drastically changed inter-individually. Skin beta-carotene and lycopene concentrations are lower in smokers than in non-smokers and higher in the vegetarian group.

  15. Structure of the iSH2 domain of Human phosphatidylinositol 3-kinase p85 beta Subunit Reveals Conformational Plasticity in the Interhelical Turn Region

    SciTech Connect

    C Schauder; L Ma; R Krug; G Montelione; R Guan

    2011-12-31

    Phosphatidylinositol 3-kinase (PI3K) proteins actively trigger signaling pathways leading to cell growth, proliferation and survival. These proteins have multiple isoforms and consist of a catalytic p110 subunit and a regulatory p85 subunit. The iSH2 domain of the p85 {beta} isoform has been implicated in the binding of nonstructural protein 1 (NS1) of influenza A viruses. Here, the crystal structure of human p85 {beta} iSH2 determined to 3.3 {angstrom} resolution is reported. The structure reveals that this domain mainly consists of a coiled-coil motif. Comparison with the published structure of the bovine p85 {beta} iSH2 domain bound to the influenza A virus nonstructural protein 1 indicates that little or no structural change occurs upon complex formation. By comparing this human p85 {beta} iSH2 structure with the bovine p85 {beta} iSH2 domain, which shares 99% sequence identity, and by comparing the multiple conformations observed within the asymmetric unit of the bovine iSH2 structure, it was found that this coiled-coil domain exhibits a certain degree of conformational variability or 'plasticity' in the interhelical turn region. It is speculated that this plasticity of p85 {beta} iSH2 may play a role in regulating its functional and molecular-recognition properties.

  16. Transforming growth factor-beta and response to anticancer therapies in human liver and gastric tumors in vitro and in vivo.

    PubMed

    Liu, P; Menon, K; Alvarez, E; Lu, K; Teicher, B A

    2000-03-01

    Liver cancer and gastric cancer are the most common solid tumors worldwide. Transforming growth factor-beta (TGF-beta) production and lack of response to TGF-beta growth inhibitory effects have been associated with tumor progression and therapeutic resistance. HepG2, Hep3B, and SK-HEP-1 human liver cancer lines produce 3, 5.7, and 2.5 ng TGF-beta1; 1.4, 2, and 4 ng TGF-beta2 and 0.15, 0.2 and 0.22 ng TGF-beta3 per 107 cells (24 h). Expression of the TGF-beta type I receptor is 20x, 1x, and 0.6x the level in mink lung MvLu1 cells in the HepG2, Hep3B, and SK-HEP-1 cells, respectively. HepG2 and Hep3B cells do not express the TGF-beta type II receptor while SK-HEP-1 cells express 7x the level found in mink lung MvLu1 cells. Hs 746T, KATO III, RF-1, and RF-48 human gastric cancer cell lines produce 12. 5, 0.35, 0.4, and 0.4 ng TGF-beta1; 2.6, 0.95, 0.5, and 0.52 ng TGF-beta2 and 0.42, 0.17, 0.12, and 0.14 ng TGF-beta3 per 107 cells (24 h). Expression of TGF-beta type I receptor is 0.7x, 0.7x, 0.8x, 0.6x the level in mink lung MvLu1 cells in the Hs 746T, KATO III, RF-1 and RF-48 cells, respectively. KATO III cells are lacking in the TGF-beta type II receptor while Hs 746T, RF-1 and RF-48 cells express 10x, 0.8x, and 1x the levels in mink lung MvLu1 cells. The IC50 for TGF-beta1 is >10 ng/ml in all of these lines except RF-48 where TGF-beta1 is mitogenic. The response of the cell lines to radiation, doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, methotrexate, and gemcitabine showed that SK-HEP-1 was the most drug resistant liver cancer cell line and KATO III was the most drug resistant gastric cancer cell line. Overall, there was no correlation between TGF-beta secretion in cell culture and sensitivity of the cells to anticancer agents. Increased TGF-beta1 levels were detectable in the plasma of nude mice bearing Hep3B and Hs 746T xenografts. Those tumors which secreted greater amounts of TGF-beta were more therapeutically resistant in vivo.

  17. [Implication of integrin alpha5beta1 signal pathways in proliferation and apoptosis of MCF-7/Dox human breast carcinoma cells].

    PubMed

    Kozlova, N I; Morozevich, G E; Ushakova, N A; Gevorkian, N M; Berman, A E

    2016-03-01

    In MCF-7/Dox human breast carcinoma cells, down-regulation of integrin alpha5beta1 and inhibition of epidermal growth factor receptor (EGFR) markedly reduced rates of cell proliferation. Mitotic cycle analysis showed that alpha5beta1 down-regulation resulted in cell cycle arrest at the S phase, followed by a significant increase in the population of apoptotic cells (subG1 population). Inhibition of EGFR activity also caused cell cycle arrest at the S-phase but without any increase in the subG1 population. Down-regulation of alpha5beta1 and EGFR inhibition resulted in a significant decrease of cell content of the active (phosphorylated) forms of FAK and Erk protein kinases. The data obtained suggest that alpha5beta1 integrin is implicated in cell growth control via inhibition of apoptotic cell death and through EGFR activation. PMID:27420618

  18. PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor.

    PubMed

    Wakshull, E; Brunke-Reese, D; Lindermuth, J; Fisette, L; Nathans, R S; Crowley, J J; Tufts, J C; Zimmerman, J; Mackin, W; Adams, D S

    1999-02-01

    PGG-Glucan, a soluble beta-(1,6)-branched beta-(1,3)-linked glucose homopolymer derived from the cell wall of the yeast Saccharomyces cerevisiae, is an immunomodulator which enhances leukocyte anti-infective activity and enhances myeloid and megakaryocyte progenitor proliferation. Incubation of human whole blood with PGG-Glucan significantly enhanced the oxidative burst response of subsequently isolated blood leukocytes to both soluble and particulate activators in a dose-dependent manner, and increased leukocyte microbicidal activity. No evidence for inflammatory cytokine production was obtained under these conditions. Electrophoretic mobility shift assays demonstrated that PGG-Glucan induced the activation of an NF-kappaB-like nuclear transcription factor in purified human neutrophils. The binding of 3H-PGG-Glucan to human leukocyte membranes was specific, concentration-dependent, saturable, and high affinity (Kd approximately 6 nM). A monoclonal antibody specific to the glycosphingolipid lactosylceramide was able to inhibit activation of the NF-kappaB-like factor by PGG-Glucan, and ligand binding data, including polysaccharide specificity, suggested that the PGG-Glucan binding moiety was lactosylceramide. These results indicate that PGG-Glucan enhances neutrophil anti-microbial functions and that interaction between this beta-glucan and human neutrophils is mediated by the glycosphingolipid lactosylceramide present at the cell surface.

  19. Novel amyloid-beta specific scFv and VH antibody fragments from human and mouse phage display antibody libraries.

    PubMed

    Medecigo, M; Manoutcharian, K; Vasilevko, V; Govezensky, T; Munguia, M E; Becerril, B; Luz-Madrigal, A; Vaca, L; Cribbs, D H; Gevorkian, G

    2010-06-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications.

  20. In vitro studies of human and rat osteoclast activity on hydroxyapatite, beta-tricalcium phosphate, calcium carbonate.

    PubMed

    Monchau, F; Lefèvre, A; Descamps, M; Belquin-myrdycz, A; Laffargue, P; Hildebrand, H F

    2002-08-01

    Investigations on the ceramic degradation caused by osteoclasts are designed to assess osteoclast-ceramic interactions and to determine which ceramics are more suitable for use as bone substitute. This study investigated the resorptive activity of osteoclasts on ceramics presenting different solubility rates. Osteoclasts isolated from new-born rat and from human giant cell tumour were cultured on different bioceramics: hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and calcium carbonate (calcite). Cytoskeletal was revealed by actin labelling and ceramic surfaces were observed by scanning electron microscopy (SEM). On all materials, the distribution of actin in typical ring was revealed. SEM examinations showed a clear difference in the shape and the depth of resorption lacunae on different ceramics. On pure HA, a superficial attack, clearly visible but very little extended. Numerous resorption lacunae, deep and well-delimited were observed on pure beta-TCP, but attacks less punctually were detected too. On pure calcite, an attack with form of spikes, very widespread but superficial was revealed. Degradation measurements revealed a significant increase of P release from the phosphocalcic ceramics and of Ca from all ceramics in the presence of osteoclasts. The both cell models found these characteristics, the rat osteoclasts were also an excellent model to study the ceramic resorption.

  1. NOVEL AMYLOID-BETA SPECIFIC scFv and VH ANTIBODY FRAGMENTS FROM HUMAN AND MOUSE PHAGE DISPLAY ANTIBODY LIBRARIES

    PubMed Central

    Medecigo, M.; Manoutcharian, K.; Vasilevko, V.; Govezensky, T.; Munguia, M. E.; Becerril, B.; Luz-Madrigal, A.; Vaca, L.; Cribbs, D. H.; Gevorkian, G.

    2010-01-01

    Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer’s disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Aβ1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single domain (VH) formats. We demonstrated that these antibody fragments recognize in a specific manner amyloid beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Aβ1-42 in neuroblastoma cell cultures in a concentration-dependently manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Aβ, which makes them strong therapeutic candidates due to the fact that most of the Aβ species found in the brains of AD patients display extensive N-terminus truncations/modifications. PMID:20451261

  2. Effect of myrrh oil on IL-1beta stimulation of NF-kappaB activation and PGE(2) production in human gingival fibroblasts and epithelial cells.

    PubMed

    Tipton, D A; Hamman, N R; Dabbous, M Kh

    2006-03-01

    Anecdotal and scientific evidence suggest that myrrh oil (MO) has anti-inflammatory properties. Subtoxic MO levels decrease interleukin (IL)-1beta-stimulated production of the inflammatory cytokine IL-6 by human gingival fibroblasts, but not epithelial cells. IL-1beta upregulates IL-6 via PGE(2), and via NF-kappaB, a transcription factor for many inflammatory mediator genes. NF-kappaB is inhibited by sesquiterpene compounds (from plants other than myrrh). This study determined MO effect on IL-1beta-stimulated PGE(2) production and NF-kappaB activation in gingival fibroblasts and epithelial cells. Cells were preincubated with MO, exposed to IL-1beta, cytoplasmic and nuclear fractions were isolated, and activated NF-kappaB was measured using an ELISA-based assay. IL-1beta increased nuclear activated NF-kappaB levels in fibroblasts and epithelial cells [10- and 2.5-fold over controls, respectively (p=0.0001)], and these increases were not significantly affected by MO. PGE(2) was measured in cell supernatants by ELISA, after preincubation with MO and exposure to IL-1beta. MO inhibited IL-1beta-stimulated PGE(2) production by fibroblasts (p=0.001), but not epithelial cells. The data suggest that gingival epithelial cells and fibroblasts may differ in the magnitude of NF-kappaB activation after IL-1beta stimulation, and that MO inhibition of IL-1beta-stimulated IL-6 production in fibroblasts is due in part to inhibition of PGE(2), but not NF-kappaB activation. (Supported by NIDCR DE-0725.). PMID:16112536

  3. Expression of two human beta-adrenergic receptors in Escherichia coli: functional interaction with two forms of the stimulatory G protein.

    PubMed Central

    Freissmuth, M; Selzer, E; Marullo, S; Schütz, W; Strosberg, A D

    1991-01-01

    When expressed in Escherichia coli, the human beta 1- and beta 2-adrenergic receptors retain their ligand binding specificity. Their functional integrity was investigated by analyzing receptor-guanine nucleotide-binding regulatory (G) protein coupling by using two splice variants of the alpha subunit of the stimulatory G protein Gs synthesized in E. coli (rGs alpha-S and rGs alpha-L) and the beta gamma subunits of G protein purified from bovine brain. In competition binding experiments with (-)-[125I]iodocyanopindolol and (-)-isoproterenol, rGs alpha-S.beta gamma and rGs alpha-L.beta gamma reconstituted guanine nucleotide-sensitive high-affinity agonist binding with comparable affinities, whereas rGs alpha PT, a mutant of rGs alpha-L with an altered carboxyl terminus, and a recombinant subtype of the alpha subunit of the inhibitory G protein, rGi alpha-1, were approximately 20- and approximately 200-fold less potent, respectively. A comparison of the beta 1- and beta 2-adrenergic receptor expressed in E. coli with the beta 2-receptor in S49 murine lymphoma cyc- cell membranes revealed a similar affinity of rGs alpha-S and rGs alpha-L for the recombinant and native receptors. After stable incorporation of rGs alpha-S.beta gamma into E. coli membranes, receptor-G protein coupling was also verified by determining the isoproterenol-mediated acceleration of the rate for guanine 5'-[gamma-[35S]thio]triphosphate binding. These results show that (i) receptor-G protein coupling can be reconstituted in E. coli using recombinant components and that (ii) such an approach may be more generally used to evaluate coupling preferences between defined molecular species of receptors and G-protein subunits. PMID:1656450

  4. Delta-subunit confers novel biophysical features to alpha beta gamma-human epithelial sodium channel (ENaC) via a physical interaction.

    PubMed

    Ji, Hong-Long; Su, Xue-Feng; Kedar, Shrestha; Li, Jie; Barbry, Pascal; Smith, Peter R; Matalon, Sadis; Benos, Dale J

    2006-03-24

    Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of delta-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the delta-subunit may form multimeric channels with alpha beta gamma-ENaC. Reverse transcription-PCR revealed that delta-ENaC is co-expressed with alpha beta gamma-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that delta-ENaC is co-expressed with alpha-, beta-, and gamma-ENaC in H441 cells at the protein level. Measurement of current-voltage that cation selectivity ratios for the revealed relationships Na+/Li+/K+/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for delta alpha beta gamma-ENaC differed from those of both alpha beta gamma- and delta beta gamma-ENaC (n = 6). The contribution of the delta subunit to P(Li)/P(Na) ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of delta alpha beta gamma-ENaC differed from those of alpha beta gamma- and delta beta gamma-ENaC. Co-immunoprecipitation of delta-ENaC with alpha- and gamma-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of delta-ENaC with other subunits could account for heterogeneity of native epithelial channels. PMID:16423824

  5. Regulation of growth and gene expression in human papillomavirus-transformed keratinocytes by transforming growth factor-beta: implications for the control of papillomavirus infection.

    PubMed

    Braun, L; Dürst, M; Mikumo, R; Crowley, A; Robinson, M

    1992-01-01

    Cervical carcinogenesis is a multistep process that appears to be initiated by infection of squamous epithelial cells in the cervix with one of a limited number of human papillomavirus (HPV) types. However, the mechanisms involved in the evolution of benign, HPV-induced lesions to malignancy have not yet been fully elucidated. Transforming growth factor-beta (TGF-beta), a multifunctional growth factor produced by cells in the skin, inhibits the proliferation of foreskin and cervical keratinocytes in vitro. We examined the effects of TGF-beta on growth and virus early-gene expression in cell lines immortalized by two HPV types associated with cervical carcinogenesis as well as the expression of TGF-beta 1 mRNA transcripts in normal and HPV-positive cells in vivo and in vitro. We found that normal and HPV-positive cells expressed similar levels of TGF-beta 1 mRNAs and exhibited similar patterns of responsiveness to three isoforms of TGF-beta in both monolayer and modified organotypic cultures. Of particular interest is our finding that the expression of the E6 and E7 early viral transforming regions of both HPV16 and HPV18 was reversibly and rapidly inhibited by TGF-beta. In one HPV16-positive cell line examined in detail, inhibition of HPV expression required protein synthesis and occurred at the level of transcription. HPV-immortalized cells selected for resistance to in vitro differentiation signals remained sensitive to TGF-beta-mediated growth inhibition. These results, showing that both growth and virus gene expression in HPV-transformed cells were responsive to TGF-beta, suggest that endogenous growth factors produced by different cell types in squamous epithelium may play a role in the progression of cervical neoplasia. PMID:1326988

  6. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells.

    PubMed Central

    Goldgaber, D; Harris, H W; Hla, T; Maciag, T; Donnelly, R J; Jacobsen, J S; Vitek, M P; Gajdusek, D C

    1989-01-01

    We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter. Images PMID:2508093

  7. Human studies on abecarnil a new beta-carboline anxiolytic: safety, tolerability and preliminary pharmacological profile.

    PubMed Central

    Duka, T; Schütt, B; Krause, W; Dorow, R; McDonald, S; Fichte, K

    1993-01-01

    1. Abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-beta-carboline-3-carboxylate), a beta-carboline with high affinity for benzodiazepine receptors, was tested in healthy male subjects; single doses of abecarnil were given in five dosage levels (1 mg, 5 mg, 10 mg, 20 mg, 40 mg) and in a multiple dose study in four dosage levels (15 mg, 30 mg, 60 mg, 90 mg day-1) for 7 days. On two days following multiple dose treatment, placebo was given in single-blind conditions (follow-up). In each dosage level, in both studies drug was given to 10 subjects (7: verum, 3: placebo). 2. Safety and tolerability were evaluated by changes in vital signs, incidence and severity of adverse reactions and biochemical and haematological screening. Drug effects were estimated utilizing a bipolar visual analogue scale (poles: 'sleepy'-'alert') and a psychomotor task, the digit symbol substitution task. The pharmacokinetics of single and multiple doses were also determined in the multiple dose study. 3. Abecarnil was generally well tolerated. In the single dose study the most frequently reported side effects associated with abecarnil at high doses (20 and 40 mg) were dizziness, unsteady gait, and lack of concentration. A decrement in performance on the digit symbol substitution task was also observed in the two high dosage groups 20 mg and 40 mg. Evaluation of visual analogue scale ratings did not reveal a sedative effect even at higher doses. 4. In the multiple dose study the most frequently reported side effects during the treatment period were dizziness, unsteady gait, and lack of concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8097921

  8. Molecular targets of a human HNF1 alpha mutation responsible for pancreatic beta-cell dysfunction.

    PubMed

    Wang, H; Antinozzi, P A; Hagenfeldt, K A; Maechler, P; Wollheim, C B

    2000-08-15

    The reverse tetracycline-dependent transactivator system was employed in insulinoma INS-1 cells to achieve controlled inducible expression of hepatocyte nuclear factor-1 alpha (HNF1 alpha)-P291fsinsC, the most common mutation associated with subtype 3 of maturity-onset diabetes of the young (MODY3). Nuclear localized HNF1 alpha-P291fsinsC protein exerts its dominant-negative effects by competing with endogenous HNF1 alpha for the cognate DNA-binding site. HNF1 alpha controls multiple genes implicated in pancreatic beta-cell function and notably in metabolism- secretion coupling. In addition to reduced expression of the genes encoding insulin, glucose transporter-2, L-pyruvate kinase, aldolase B and 3-hydroxy-3-methylglutaryl coenzyme A reductase, induction of HNF1 alpha-P291fsinsC also significantly inhibits expression of mitochondrial 2-oxoglutarate dehydrogenase (OGDH) E1 subunit mRNA and protein. OGDH enzyme activity and [(14)C]pyruvate oxidation were also reduced. In contrast, the mRNA and protein levels of mitochondrial uncoupling protein-2 were dramatically increased by HNF1 alpha-P291fsinsC induction. As predicted from this altered gene expression profile, HNF1 alpha-P291fsinsC also inhibits insulin secretory responses to glucose and leucine, correlated with impaired nutrient-evoked mitochondrial ATP production and mitochondrial membrane hyperpolarization. These unprecedented results suggest the molecular mechanism of HNF1 alpha-P291fsinsC causing beta-cell dysfunction. PMID:10944108

  9. Using satellite remote sensing and household survey data to assess human health and nutrition response to environmental change.

    PubMed

    Brown, Molly E; Grace, Kathryn; Shively, Gerald; Johnson, Kiersten B; Carroll, Mark

    2014-01-01

    Climate change and degradation of ecosystem services functioning may threaten the ability of current agricultural systems to keep up with demand for adequate and inexpensive food and for clean water, waste disposal and other broader ecosystem services. Human health is likely to be affected by changes occurring across multiple geographic and time scales. Impacts range from increasing transmissibility and the range of vectorborne diseases, such as malaria and yellow fever, to undermining nutrition through deleterious impacts on food production and concomitant increases in food prices. This paper uses case studies to describe methods that make use of satellite remote sensing and Demographic and Health Survey data to better understand individual-level human health and nutrition outcomes. By bringing these diverse datasets together, the connection between environmental change and human health outcomes can be described through new research and analysis. PMID:25132700

  10. Using Satellite Remote Sensing and Household Survey Data to Assess Human Health and Nutrition Response to Environmental Change

    NASA Technical Reports Server (NTRS)

    Brown, Molly E.; Grace, Kathryn; Shively, Gerald; Johnson, Kiersten B.; Carroll, Mark

    2014-01-01

    Climate change and degradation of ecosystem services functioning may threaten the ability of current agricultural systems to keep up with demand for adequate and inexpensive food and for clean water, waste disposal and other broader ecosystem services. Human health is likely to be affected by changes occurring across multiple geographic and time scales. Impacts range from increasing transmissibility and the range of vector-borne diseases, such as malaria and yellow fever, to undermining nutrition through deleterious impacts on food production and concomitant increases in food prices. This paper uses case studies to describe methods that make use of satellite remote sensing and Demographic and Health Survey data to better understand individual-level human health and nutrition outcomes. By bringing these diverse datasets together, the connection between environmental change and human health outcomes can be described through new research and analysis.

  11. CD133 expression correlates with membrane beta-catenin and E-cadherin loss from human hair follicle placodes during morphogenesis.

    PubMed

    Gay, Denise L; Yang, Chao-Chun; Plikus, Maksim V; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E; Cotsarelis, George

    2015-01-01

    Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode "budding" is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.

  12. CD133 expression correlates with membrane beta-catenin and e-cadherin loss from human hair follicle placodes during morphogenesis

    PubMed Central

    Gay, Denise; Yang, Chao-Chun; Plikus, Maksim; Ito, Mayumi; Rivera, Charlotte; Treffeisen, Elsa; Doherty, Laura; Spata, Michelle; Millar, Sarah E.; Cotsarelis, George

    2014-01-01

    Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode ‘budding’ is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions is clearly required for budding. Snail-mediated downregulation of adherens junction component E-cadherin is important for placode budding in mice. Beta-catenin, another adherens junction component, has been more difficult to study due to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent adherens junction dissolution. PMID:25010141

  13. Galectin-1 as a fusion partner for the production of soluble and folded human {beta}-1,4-galactosyltransferase-T7 in E. coli

    SciTech Connect

    Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy; Qasba, Pradman K.

    2010-04-09

    The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of a large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.

  14. Exploring Global Patterns in Human Appropriation of Net Primary Production Using Earth Observation Satellites and Statistical Data

    NASA Astrophysics Data System (ADS)

    Imhoff, M.; Bounoua, L.

    2004-12-01

    A unique combination of satellite and socio-economic data were used to explore the relationship between human consumption and the carbon cycle. Biophysical models were applied to consumption data to estimate the annual amount of Earth's terrestrial net primary production humans require for food, fiber and fuel using the same modeling architecture as satellite-supported NPP measurements. The amount of Earth's NPP required to support human activities is a powerful measure of the aggregate human impacts on the biosphere and indicator of societal vulnerability to climate change. Equations were developed estimating the amount of landscape-level NPP required to generate all the products consumed by 230 countries including; vegetal foods, meat, milk, eggs, wood, fuel-wood, paper and fiber. The amount of NPP required was calculated on a per capita basis and projected onto a global map of population to create a spatially explicit map of NPP-carbon demand in units of elemental carbon. NPP demand was compared to a map of Earth's average annual net primary production or supply created using 17 years (1982-1998) of AVHRR vegetation index to produce a geographically accurate balance sheet of terrestrial NPP-carbon supply and demand. Globally, humans consume 20 percent of Earth's total net primary production on land. Regionally the NPP-carbon balance percentage varies from 6 to over 70 percent and locally from near 0 to over 30,000 percent in major urban areas. The uneven distribution of NPP-carbon supply and demand, indicate the degree to which various human populations rely on NPP imports, are vulnerable to climate change and suggest policy options for slowing future growth in NPP demand.

  15. Novel dihydro-beta-agarofuran sesquiterpenes as potent modulators of human P-glycoprotein dependent multidrug resistance.

    PubMed

    Torres-Romero, David; Muñoz-Martínez, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Gamarro, Francisco; Bazzocchi, Isabel L

    2009-12-21

    P-Glycoprotein (Pgp) overexpression is one factor contributing to multidrug resistance (MDR) in cancer cells and represents one drawback in the treatment of cancer. In an attempt to find more specific and less toxic anticancer MDR-reversal agents, we report herein the isolation, structure elucidation and biological activity of nine new (, and ) and seven known (, and ) dihydro-beta-agarofuran sesquiterpenes from the leaves of Celastrus vulcanicola. Their stereostructures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies and biogenetic means. All the compounds were assayed on human MDR1-transfected NIH-3T3 cells, in order to determine their ability to reverse the MDR phenotype due to Pgp overexpression. Six compounds from these series (, , , , and ) showed an effectiveness that was similar to (or higher than) the classical Pgp reversal agent verapamil for the reversal of resistance to daunomycin and vinblastine. The structure-activity relationships are discussed. PMID:20024113

  16. Novel dihydro-beta-agarofuran sesquiterpenes as potent modulators of human P-glycoprotein dependent multidrug resistance.

    PubMed

    Torres-Romero, David; Muñoz-Martínez, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Gamarro, Francisco; Bazzocchi, Isabel L

    2009-12-21

    P-Glycoprotein (Pgp) overexpression is one factor contributing to multidrug resistance (MDR) in cancer cells and represents one drawback in the treatment of cancer. In an attempt to find more specific and less toxic anticancer MDR-reversal agents, we report herein the isolation, structure elucidation and biological activity of nine new (, and ) and seven known (, and ) dihydro-beta-agarofuran sesquiterpenes from the leaves of Celastrus vulcanicola. Their stereostructures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies and biogenetic means. All the compounds were assayed on human MDR1-transfected NIH-3T3 cells, in order to determine their ability to reverse the MDR phenotype due to Pgp overexpression. Six compounds from these series (, , , , and ) showed an effectiveness that was similar to (or higher than) the classical Pgp reversal agent verapamil for the reversal of resistance to daunomycin and vinblastine. The structure-activity relationships are discussed.

  17. Digoxigenin-tagged anti-GFAP and multiple labelling of human glia, vessels and beta-amyloid.

    PubMed

    Härtig, W; Hausen, D; Brauer, K; Arendt, T; Bigl, V; Brückner, G

    1994-01-31

    Cytochemical procedures for the combined demonstration of astroglia, microglia, blood vessels and beta-amyloid in the human brain were developed. These multiple label experiments include the first adaptation of the digoxigenin-antidigoxigenin technique to immunocytochemistry by using digoxigenylated antibodies directed against the glial fibrillary acidic protein and its visualization in astrocytes with an antidigoxigenin-peroxidase conjugate and diaminobenzidine as chromogen. Furthermore, the specific labelling of microglial cells was performed with the novel enhanced polymer one-step staining technique, non-interfering with conventional detection systems. The demonstration of spatial relationships between glial and vascular components in normal tissue and their alterations, e.g. in Alzheimer's disease, might provide insights into the time course and localization of pathological events.

  18. Effects of 1-beta-D-arabinofuranosylcytosine and phorbol ester on differentiation of human K562 erythroleukemia cells.

    PubMed

    Watanabe, T; Mitchell, T; Sariban, E; Sabbath, K; Griffin, J; Kufe, D

    1985-06-01

    We have previously demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces hemoglobin synthesis in human K562 erythroleukemia cells. The present study extends these findings by demonstrating that ara-C treatment of K562 cells results in both increased heme synthesis and accumulation of alpha-, gamma-, epsilon-, and zeta-globin RNA. The results also demonstrate that ara-C enhances K562 cell surface expression of glycophorin. Furthermore, we demonstrate that phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) inhibits the effects of ara-C on heme production, accumulation of globin RNA, and glycophorin expression. The inhibitory effect occurs maximally when K562 cells are treated with TPA before undergoing ara-C-induced commitment to erythroid differentiation. These findings suggest that TPA inhibits an early step in the process required for ara-C to enhance expression of genes involved in the erythroid program.

  19. In vitro antiviral efficacy of the ganciclovir complexed with beta-cyclodextrin on human cytomegalovirus clinical strains.

    PubMed

    Nicolazzi, Céline; Venard, Véronique; Le Faou, Alain; Finance, Chantal

    2002-05-01

    The toxicity of the compounds currently used in the treatment of human cytomegalovirus (HCMV) infections in immunocompromised hosts may force the treatment to be discontinued. The aim of this study was to improve the antiviral activity of ganciclovir (GCV), one the most widely used drug, by complexing it with beta-cyclodextrin. Cyclodextrins (cds) have the property to form inclusion complexes with a great number of molecules and to enhance bioavailability and biological properties of these molecules. In this study, we investigated the in vitro antiviral activity of complexed GCV against several strains of HCMV: AD169, a reference strain, RCL-1, a laboratory mutant resistant to GCV, and four clinical isolates. The complexed GCV was more effective than free GCV against all HCMV strains tested. Cds as carriers for antiviral drugs would represent a useful adjunct to classical treatment procedures. They may make it possible to administer lower doses, thus reducing the toxic side effects of the drugs. PMID:12062397

  20. Intestinal alpha beta T cells differentiate and rearrange antigen receptor genes in situ in the human infant.

    PubMed

    Williams, Amanda M; Bland, Paul W; Phillips, Anne C; Turner, Susan; Brooklyn, Trevor; Shaya, Gabriel; Spicer, Richard D; Probert, Christopher S J

    2004-12-15

    Intestinal Ag exposure during neonatal life influences appropriate adult immune responses. To define the mechanisms shaping the T cell repertoire during this period, we examined T cell differentiation and receptor diversity in the intestine of human infants. Developmental phenotypes of intraepithelial and lamina propria intestinal T cells from infants aged 1 day to 2 years were assessed ex vivo by flow cytometry and in situ by triple-fluorescent immunohistochemistry. Gene recombination-specific enzymes were assessed by PCR. TCR beta-chain V region gene diversity was determined by sequencing. Several different early lineage T cell populations were present neonatally: CD3(+)4(-)8(-) T cells were present at birth and numbers decreased during the neonatal period; CD3(+)4(+)8(+) T cells were present in low numbers throughout infancy; and CD3(+)4(+)8(-) or CD3(+)4(-)8(+) T cells increased with age. Very early lineage T cells, CD3(-)2(-)7(+) and CD3(-)2(+)7(+), were present neonatally, but were essentially absent at 1 year. Most lamina propria T cells differentiated rapidly after birth, but maturation of intraepithelial T cells took place over 1 year. Intestinal samples from infants less than 6 mo old contained transcripts of T early alpha and TdT, and 15 of 19 infant samples contained mRNA for RAG-1, some coexpressing RAG-2. TCR beta-chain repertoires were polyclonal in infants. Immature T cells, pre-T cells, and genes involved in T cell recombination were found in the intestine during infancy. T cell differentiation occurs within the neonatal human intestine, and the TCR repertoire of these developing immature T cells is likely to be influenced by luminal Ags. Thus, mucosal T cell responsiveness to environmental Ag is shaped in situ during early life.

  1. Creatine supplementation augments the increase in satellite cell and myonuclei number in human skeletal muscle induced by strength training

    PubMed Central

    Olsen, Steen; Aagaard, Per; Kadi, Fawzi; Tufekovic, Goran; Verney, Julien; Olesen, Jens L; Suetta, Charlotte; Kjær, Michael

    2006-01-01

    The present study investigated the influence of creatine and protein supplementation on satellite cell frequency and number of myonuclei in human skeletal muscle during 16 weeks of heavy-resistance training. In a double-blinded design 32 healthy, male subjects (19–26 years) were assigned to strength training (STR) while receiving a timed intake of creatine (STR-CRE) (n = 9), protein (STR-PRO) (n = 8) or placebo (STR-CON) (n = 8), or serving as a non-training control group (CON) (n = 7). Supplementation was given daily (STR-CRE: 6–24 g creatine monohydrate, STR-PRO: 20 g protein, STR-CON: placebo). Furthermore, timed protein/placebo intake were administered at all training sessions. Muscle biopsies were obtained at week 0, 4, 8 (week 8 not CON) and 16 of resistance training (3 days per week). Satellite cells were identified by immunohistochemistry. Muscle mean fibre (MFA) area was determined after histochemical analysis. All training regimes were found to increase the proportion of satellite cells, but significantly greater enhancements were observed with creatine supplementation at week 4 (compared to STR-CON) and at week 8 (compared to STR-PRO and STR-CON) (P < 0.01–0.05). At week 16, satellite cell number was no longer elevated in STR-CRE, while it remained elevated in STR-PRO and STR-CON. Furthermore, creatine supplementation resulted in an increased number of myonuclei per fibre and increases of 14–17% in MFA at week 4, 8 and 16 (P < 0.01). In contrast, STR-PRO showed increase in MFA only in the later (16 week, +8%) and STR-CON only in the early (week 4, +14%) phases of training, respectively (P < 0.05). In STR-CRE a positive relationship was found between the percentage increases in MFA and myonuclei from baseline to week 16, respectively (r = 0.67, P < 0.05). No changes were observed in the control group (CON). In conclusion, the present study demonstrates for the first time that creatine supplementation in combination with strength training amplifies

  2. Islet distribution of Peptide YY and its regulatory role in primary mouse islets and immortalised rodent and human beta-cell function and survival.

    PubMed

    Khan, Dawood; Vasu, Srividya; Moffett, R Charlotte; Irwin, Nigel; Flatt, Peter R

    2016-11-15

    Recent evidence suggests that the classic gut peptide, Peptide YY (PYY), could play a fundamental role in endocrine pancreatic function. In the present study expression of PYY and its NPY receptors on mouse islets and immortalised rodent and human beta-cells was examined together with the effects of both major circulating forms of PYY, namely PYY(1-36) and PYY(3-36), on beta-cell function, murine islet adaptions to insulin deficiency/resistance, as well as direct effects on cultured beta-cell proliferation and apoptosis. In vivo administration of PYY(3-36), but not PYY(1-36), markedly (p < 0.05) decreased food intake in overnight fasted mice. Neither form of PYY affected glucose disposal or insulin secretion following an i.p. glucose challenge. However, in vitro, PYY(1-36) and PYY(3-36) inhibited (p < 0.05 to p < 0.001) glucose, alanine and GLP-1 stimulated insulin secretion from immortalised rodent and human beta-cells, as well as isolated mouse islets, by impeding alterations in membrane potential, [Ca(2+)]i and elevations of cAMP. Mice treated with multiple low dose streptozotocin presented with severe (p < 0.01) loss of beta-cell mass accompanied by notable increases (p < 0.001) in alpha and PP cell numbers. In contrast, hydrocortisone-induced insulin resistance increased islet number (p < 0.01) and beta-cell mass (p < 0.001). PYY expression was consistently observed in alpha-, PP- and delta-, but not beta-cells. Streptozotocin decreased islet PYY co-localisation with PP (p < 0.05) and somatostatin (p < 0.001), whilst hydrocortisone increased PYY co-localisation with glucagon (p < 0.05) in mice. More detailed in vitro investigations revealed that both forms of PYY augmented (p < 0.05 to p < 0.01) immortalised human and rodent beta-cell proliferation and protected against streptozotocin-induced cytotoxicity, to a similar or superior extent as the well characterised beta-cell proliferative and anti-apoptotic agent GLP-1. Taken together

  3. Beta-palmitate - a natural component of human milk in supplemental milk formulas.

    PubMed

    Havlicekova, Zuzana; Jesenak, Milos; Banovcin, Peter; Kuchta, Milan

    2016-03-17

    The composition and function of human milk is unique and gives a basis for the development of modern artificial milk formulas that can provide an appropriate substitute for non-breastfed infants. Although human milk is not fully substitutable, modern milk formulas are attempting to mimic human milk and partially substitute its complex biological positive effects on infants. Besides the immunomodulatory factors from human milk, research has been focused on the composition and structure of human milk fat with a high content of β-palmitic acid (sn-2 palmitic acid, β-palmitate). According to the available studies, increasing the content of β-palmitate added to milk formulas promotes several beneficial physiological functions. β-palmitate positively influences fatty acid metabolism, increases calcium absorption, improves bone matrix quality and the stool consistency, and has a positive effect on the development of the intestinal microbiome.

  4. Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

    PubMed

    Bayer, Andreas; Lammel, Justus; Rademacher, Franziska; Groß, Justus; Siggelkow, Markus; Lippross, Sebastian; Klüter, Tim; Varoga, Deike; Tohidnezhad, Mersedeh; Pufe, Thomas; Cremer, Jochen; Gläser, Regine; Harder, Jürgen

    2016-06-01

    Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds. PMID:26843467

  5. Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK-3{beta} signaling

    SciTech Connect

    Tateishi, Kento |; Ashihara, Eishi; Honsho, Shoken |; Takehara, Naofumi; Nomura, Tetsuyaital |; Takahashi, Tomosaburo; Ueyama, Tomomi; Yamagishi, Masaaki; Yaku, Hitoshi; Matsubara, Hiroaki |. E-mail: matsubah@koto.kpu-m.ac.jp; Oh, Hidemasa . E-mail: hidemasa@kuhp.kyoto-u.ac.jp

    2007-01-19

    Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant > adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3{beta} may be crucial modulators for hCSC maintenance in human heart.

  6. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    PubMed Central

    Wang, Yuan-Yi; Zhu, Qing-San; Wang, Yi-Wei; Yin, Ruo-Feng

    2015-01-01

    Background: Thymosin beta-4 (TB-4) is considered key roles in tissue development, maintenance and pathological processes. The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation. Methods: TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells. Cell of same group were cultured without gene modification as controlled group. Proliferation capacity and cell apoptosis were observed during 6 passages of the cells. Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage. Results: NP cells with TB-4 transfection has normal TB-4 expression and exocytosis. NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation. TB-4 recombinant AAV-transfected human NP cells also show slower cell aging, lower cell apoptosis and higher cell proliferation than control group. Conclusions: TB-4 can prevent NP cell apoptosis, slow NP cell aging and promote NP cell proliferation. AAV transfection technique was able to highly and stably express TB-4 in human NP cells, which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases. PMID:26021512

  7. TGF-alpha and TGF-beta expression during sodium-N-butyrate-induced differentiation of human keratinocytes: evidence for subpopulation-specific up-regulation of TGF-beta mRNA in suprabasal cells.

    PubMed

    Staiano-Coico, L; Khandke, L; Krane, J F; Sharif, S; Gottlieb, A B; Krueger, J G; Heim, L; Rigas, B; Higgins, P J

    1990-12-01

    Sodium-N-butyrate (NaB) induces terminal differentiation and cornified envelope formation in cultured human keratinocytes. In the present study we explored the question of whether NaB-induced maturation could be mediated through changes in TGF-alpha and TGF-beta expression in normal keratinocytes. NaB induced a four-fold increase in TGF-beta mRNA transcript levels. This increase in TGF-beta mRNA abundance occurred only within the nonbasal keratinocyte subpopulation which maximally responds to NaB treatment by progression to cornified envelopes. Basal keratinocytes, which are relatively refractive to cornified envelope formation, did not show any increase in TGF-beta mRNA abundance after NaB treatment. By comparison, TGF-alpha mRNA transcript and extracellular TGF-alpha protein levels were unaffected by NaB treatment. A 50% decrease in EGF receptor binding was observed in NaB-treated keratinocyte cultures, rendering the cells less responsive to proliferation induction.

  8. Differential expression and upregulation of interleukin-1alpha, interleukin-1beta and interleukin-6 by freshly isolated human small intestinal epithelial cells.

    PubMed Central

    Madrigal-Estebas, Laura; Doherty, Derek G; O'Donoghue, Diarmuid P; Feighery, Conleth; O'Farrelly, Cliona

    2002-01-01

    BACKGROUND: Small intestinal epithelial cells (SIEC) may contribute to local immune regulation. AIM: To examine production of interleukin (IL)-1alpha, IL-1beta and IL-6 by freshly isolated human SIEC. METHODS: IL-1alpha and IL-1beta mRNA in epithelial layers (EL) prepared from small intestine and in intestinal epithelial cell (EC) lines were examined by reverse transcription-polymerase chain reaction. IL-1alpha, IL-1beta and IL-6 protein expression by SIEC was examined by flow cytometry before and after activation with lipopolysaccharide and epithelial growth factor. RESULTS: IL-1alpha and IL-1beta mRNA was detected in EL and EC lines. Background expression of IL-1alpha and IL-1beta protein by SIEC was observed, which did not increase even following activation. IL-6 protein was expressed by SIEC, in a proportion that increased in two out of three samples following activation. CONCLUSIONS: IL-6 expression and the presence of IL-1alpha and IL-1beta mRNA suggest a role for SIEC in the regulation of local inflammation. PMID:12467524

  9. Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit.

    PubMed

    Vendel, Andrew C; Rithner, Christopher D; Lyons, Barbara A; Horne, William A

    2006-02-01

    Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating. PMID:16385006

  10. Cross-talk between Smad and p38 MAPK signalling in transforming growth factor {beta} signal transduction in human glioblastoma cells

    SciTech Connect

    Dziembowska, Magdalena; Danilkiewicz, Malgorzata; Wesolowska, Aleksandra; Zupanska, Agata; Chouaib, Salem; Kaminska, Bozena . E-mail: bozenakk@nencki.gov.pl

    2007-03-23

    Transforming growth factor-beta (TGF-{beta}) is a multifunctional cytokine involved in the regulation of cell proliferation, differentiation, and survival. Malignant tumour cells often do not respond to TGF-{beta} by growth inhibition, but retain responsiveness to cytokine in regulating extracellular matrix deposition, cell adhesion, and migration. We demonstrated that TGF-{beta}1 does not affect viability or proliferation of human glioblastoma T98G, but increases transcriptional responses exemplified by induction of MMP-9 expression. TGF-{beta} receptors were functional in T98G glioblastoma cells leading to SMAD3/SMAD4 nuclear translocation and activation of SMAD-dependent promoter. In parallel, a selective activation of p38 MAPK, and phosphorylation of its substrates: ATF2 and c-Jun proteins were followed by a transient activation of AP-1 transcription factor. Surprisingly, an inhibition of p38 MAPK with a specific inhibitor, SB202190, abolished TGF-inducible activation of Smad-dependent promoter and decreased Smad2 phosphorylation. It suggests an unexpected interaction between Smad and p38 MAPK pathways in TGF-{beta}1-induced signalling.

  11. Expression of CD44 isoforms and beta 1,6-branched oligosaccharides in human malignant melanoma is correlated with tumor progression but not with metastatic potential.

    PubMed

    Seelentag, W K; Böni, R; Günthert, U; Futo, E; Burg, G; Heitz, P U; Roth, J

    1997-04-01

    CD44, a family of closely related glycoproteins generated by alternative splicing, as well as the increased beta 1,6-branching of Asn-linked oligosaccharides (beta 1,6-branches), have been implicated in tumor progression and metastasis. We have investigated the expression of CD44 standard (CD44s), various CD44 splice variants (CD44v3, -v4, -v5, -v6 and -v9), and of beta 1,6-branches in a total of 37 paraffin-embedded human primary melanomas and metastases. Out of the 28 studied primary melanomas, 27 were positive for CD44s, 21 for CD44v5 (cytoplasmic staining) and 26 for beta 1,6 branches. Furthermore, superficial spreading melanomas showed a significant (p = 0.004) stronger staining for CD44s than the thick (> 1.5 mm) nodular melanomas, whereas no significant difference was found with regard to staining for CD44v5 and beta 1,6-branches. Eight of the 9 studied melanoma metastases were positive for CD44s, 6 for CD44v5 (cytoplasmic staining) and 7 for beta 1,6-branches. No CD44v3, -v4, -v6 and -v9 could be detected in any of the tumors. On average, metastases as compared to primary tumors, exhibited a significant (p = 0.002) weaker staining for CD44s. However, metastasizing melanomas could not be distinguished from non-metastasizing ones based on CD44 immunostaining. PMID:9138110

  12. Variation in the binding of /sup 125/I-labeled interferon-beta ser to cellular receptors during growth of human renal and bladder carcinoma cells in vitro

    SciTech Connect

    Ruzicka, F.J.; Schmid, S.M.; Groveman, D.S.; Cummings, K.B.; Borden, E.C.

    1987-09-01

    Studies of various established human bladder and renal carcinoma cell lines cultured in vitro demonstrated the presence of specific, saturable, high affinity binding sites for /sup 125/I-labeled human interferon Beta ser IFN-beta ser). This recombinant produced interferon labeled with approximately one atom of /sup 125/I/molecule of IFN expressed minimal or no loss of antiviral activity. A single class of binding sites (1000-2000/cell) with an affinity constant of 10(10)-10(11) L/M was measured at 4 degrees C for cells exhibiting widely different sensitivity to the antiproliferative effect of IFN-beta ser. Major fluctuations in the binding of /sup 125/I-labeled IFN-beta ser to cellular receptors were observed during in vitro proliferation of four of five cell lines examined. A significant decrease (P less than 0.001) in specific binding was observed 48 h after cultures were established. Cell cycle analysis suggested that within the first 24 h and in the very late log and stationary phase of growth of ACHN (human renal carcinoma) cells, variations in the binding of /sup 125/I-labeled IFN-beta ser were partially attributable to binding fluctuations during the mitotic cycle. The 2- to 3-fold decline 24 h following plating of ACHN cells corresponded to a 70% decrease in the number of cells in G0-G1. T24 (human transitional cell carcinoma) and ACHN cells, synchronized by serum starvation, demonstrated increased binding of /sup 125/I-labeled IFN-beta ser 4-16 h following serum replenishment. This increase in receptor binding occurred prior to the onset of DNA and protein synthesis and was followed by a decline immediately prior to cell division. Binding site analysis indicated that the increased binding prior to DNA synthesis was due to a 5- to 6-fold increase in receptor affinity for the radiolabeled ligand.

  13. ATS-6 - A satellite for human needs. [Health, Education, Telecommunications Experiment

    NASA Technical Reports Server (NTRS)

    Whalen, A. A.; Johnston, W. A., Jr.

    1975-01-01

    On May 30, 1974, NASA launched the ATS-6 experimental communications satellite into a geosynchronous orbit at a station centered over the United States. The 1400 kg satellite was designed to be body-stabilized with a 3-axis control system capable of precision offset pointing. It deployed a 9.1 meter (30 foot) parabolic reflector antenna with a transponder that covered a frequency range from VHF through C-band. The high RF gains obtained with the antenna were to be used for many dramatic communications experiments, one of which was the Health/Education Telecommunications Experiment (HET), a demonstration of direct broadcast of color television to low cost terminals in remote regions of the United States. More than 120 terminals with 3-meter antennas were deployed in Alaska, Washington, the Rocky Mountains, and Appalachia to provide educational and health services to selected community centers. After 11 months of nearly continuous service, the performance of both the satellite and the experiment have exceeded all expectations.

  14. Human alpha- and beta-NRXN1 isoforms rescue behavioral impairments of Caenorhabditis elegans neurexin-deficient mutants.

    PubMed

    Calahorro, F; Ruiz-Rubio, M

    2013-06-01

    Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx-1 and nlg-1, respectively. Here we show that nrx-1-deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx-1 mutants was markedly different to nlg-1-deficient mutants; thus, while the former had a 'hyper-reversal' phenotype increasing the number of changes of direction with respect to the wild-type strain, the nlg-1 mutants presented a 'hypo-reversal' phenotype. On the other hand, the nrx-1- and nlg-1-defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma-aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx-1-deficient mutants were rescued in transgenic strains expressing either human alpha- or beta-NRXN-1 isoforms under the worm nrx-1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.

  15. beta-Dystroglycan modulates the interplay between actin and microtubules in human-adhered platelets.

    PubMed

    Cerecedo, Doris; Cisneros, Bulmaro; Suárez-Sánchez, Rocío; Hernández-González, Enrique; Galván, Iván

    2008-05-01

    To maintain the continuity of an injured blood vessel, platelets change shape, secrete granule contents, adhere, aggregate, and retract in a haemostatic plug. Ordered arrays of microtubules, microfilaments, and associated proteins are responsible for these platelet responses. In full-spread platelets, microfilament bundles in association with other cytoskeleton proteins are anchored in focal contacts. Recent studies in migrating cells suggest that co-ordination and direct physical interaction of microtubules and actin network modulate adhesion development. In platelets, we have proposed a feasible association between these two cytoskeletal systems, as well as the participation of the dystrophin-associated protein complex, as part of the focal adhesion complex. The present study analysed the participation of microtubules and actin during the platelet adhesion process. Confocal microscopy, fluorescence resonance transfer energy and immunoprecipitation assays were used to provide evidence of a cross-talk between these two cytoskeletal systems. Interestingly, beta-dystroglycan was found to act as an interplay protein between actin and microtubules and an additional communication between these two cytoskeleton networks was maintained through proteins of focal adhesion complex. Altogether our data are indicative of a dynamic co-participation of actin filaments and microtubules in modulating focal contacts to achieve platelet function.

  16. The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein.

    PubMed

    Khrustalev, Vladislav Victorovich; Khrustaleva, Tatyana Aleksandrovna; Szpotkowski, Kamil; Poboinev, Victor Vitoldovich; Kakhanouskaya, Katsiaryna Yurieuna

    2016-10-01

    Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C-half of the second helix and N-half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179-Cys214) failed because of the beta hairpin formation in the region 206-MERVVEQMC-214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn(2+) cations, which causes small temperature-associated structural shifts at concentrations of 2 - 10·10(-6) M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184-IKQHTVT-190 and 197-TETDVKM-205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462-1479. © 2016 Wiley Periodicals, Inc. PMID:27317933

  17. The effects of folic acid application on IL-1beta levels of human gingival fibroblasts stimulated by phenytoin and TNFalpha in vitro: a preliminary study.

    PubMed

    Doğan, A; Tunca, Y; Ozdemir, A; Sengül, A; Imirzalioğlu, N

    2001-12-01

    Folic acid (FA), that is required for the integrity of gingival tissues, was found to decrease in patients using phenytoin (PHT). Interleukin-1 beta (IL-1beta) was reported to enhance the extracellular matrix synthesis in fibroblasts. The purpose of this study was to assess the role of FA supplementation on PHT-induced overgrowth by investigating its effect on IL-1beta production of human gingival fibroblasts induced by tumor necrosis factor alfa (TNFalpha) in cell culture. PHT (20 microg/ml), FA (20 or 40 ng/ml) + PHT, PHT + TNF (10 ng/ml), FA (20 or 40 ng/ml) + PHT + TNF, or only culture medium (control) was added to 24-well plates containing fibroblasts. After an incubation period of 72 h, culture medium and cells were harvested separately. Then, IL-1beta levels in cell lysate were measured using enzyme-linked immunosorbent assay. The cellular IL-1beta level in the PHT group was 1 pg/ml. In PHT + 20 or 40 ng/ml FA-added cultures, the results obtained respectively were 0.8 and 0.7 pg/ml, whereas the control group value was 0.7 pg/ml. IL-1beta level was 4 pg/ml in the cultures that PHT and TNFalpha were applied simultaneously (P < 0.05). When PHT and either 20 or 40 ng/ml FA were simultaneously added into TNFalpha-induced cultures, the IL-1beta levels were 1.8 and 1.3 pg/ml, respectively. IL-1beta level in gingival tissues might play a role in PHT-induced overgrowth by increasing in the gingival tissues, and FA application might play a role in decreasing gingival tissues. However, further studies are needed for a more complete understanding of PHT-induced gingival overgrowth at the cellular level.

  18. Tissue localization of beta receptors for platelet-derived growth factor and platelet-derived growth factor B chain during wound repair in humans.

    PubMed Central

    Reuterdahl, C; Sundberg, C; Rubin, K; Funa, K; Gerdin, B

    1993-01-01

    The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled. Images PMID:8486774

  19. Splicing and 3' end formation in the definition of nonsense-mediated decay-competent human beta-globin mRNPs.

    PubMed

    Neu-Yilik, G; Gehring, N H; Thermann, R; Frede, U; Hentze, M W; Kulozik, A E

    2001-02-01

    Premature translation termination codons are common causes of genetic disorders. mRNAs with such mutations are degraded by a surveillance mechanism termed nonsense-mediated decay (NMD), which represents a phylogenetically widely conserved post-transcriptional mechanism for the quality control of gene expression. How NMD-competent mRNPs are formed and specified remains a central question. Here, we have used human beta-globin mRNA as a model system to address the role of splicing and polyadenylation for human NMD. We show that (i) splicing is an indispensable component of the human beta-globin NMD pathway, which cannot be compensated for by exonic beta-globin 'failsafe' sequences; (ii) the spatial requirements of human beta-globin NMD, as signified by the maximal distance of the nonsense mutation to the final exon-exon junction, are less constrained than in yeast; and (iii) non-polyadenylated mRNAs with a histone 3' end are NMD competent. Thus, the formation of NMD-competent mRNP particles critically depends on splicing but does not require the presence of a poly(A) tail.

  20. The human beta-subunit of rod photoreceptor cGMP phosphodiesterase: complete retinal cDNA sequence and evidence for expression in brain.

    PubMed

    Collins, C; Hutchinson, G; Kowbel, D; Riess, O; Weber, B; Hayden, M R

    1992-07-01

    We have identified and sequenced cDNA clones that encode for the human beta-subunit of rod cGMP phosphodiesterase (PDEB). A single 2565-bp open reading frame that codes for an 854-amino-acid protein was identified. The human beta-subunit protein is 90% identical to the bovine beta-subunit and 91% identical to the mouse protein. Northern blot analysis indicates that the gene is expressed as an abundant 3.5-kb transcript in retina and as a rare 2.9-kb transcript in brain. The isolation of cDNAs from human brain cDNA libraries confirms the brain as a site of expression for this gene. The molecular defect underlying retinal degeneration in the rd mouse has been found to be a nonsense mutation in the beta-subunit of the mouse cGMP PDE, resulting in a truncated protein (Pittler et al., 1991b, Proc. Natl. Acad. Sci. USA. 88: 8322-8326). The molecular cloning of the cDNA encoding for the PDEB represents the first step in establishing whether this gene plays a causative role in any one of the several human hereditary retinopathies or, based on its localization to chromosome 4p 16.3, in the pathogenesis of Huntington disease.

  1. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    SciTech Connect

    Bouhlel, Mohamed Amine; Brozek, John; Derudas, Bruno; Zawadzki, Christophe; Jude, Brigitte; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  2. Suppression of natural killer cell activity by rabbit antibody to human beta 2-microglobulin (beta 2m) is an Fc-mediated phenomenon and is not beta 2m specific.

    PubMed

    Jones, R A; Richards, S J; Patel, D; Scott, C S

    1991-07-01

    Natural killer (NK) cell cytotoxicity constitutes an important component of the host immune defence system. The NK effector cell has been relatively well defined in terms of immunophenotypic characteristics, but in contrast to the functional T-cell receptor molecule associated with major histocompatibility complex (MHC)-restricted cytotoxic activity, the NK cell receptor has not to date been defined. However, several studies have suggested that the beta 2-microglobulin (beta 2m) molecule is functionally associated with NK cell activity. Using various heterospecific and monoclonal antibodies, this study has shown that intact rabbit IgG antibody bound either directly or indirectly to peripheral mononuclear cell (PMNC) effector populations significantly reduced their lytic activity against K562 targets. Substitution of F(ab)2 fragments for rabbit IgG antibodies, or the use of monoclonal antibodies alone, failed to reduce peripheral blood mononuclear cell (PMNC) lytic activity. Addition of non-NK cell components labelled with rabbit anti-beta 2m to purified NK-enriched effector cell populations also suppressed K562 lysis. In contrast, pre-treatment of a NK-enriched PMNC fraction with rabbit anti-beta 2m enhanced target lysis. These results strongly suggest that antibody-induced suppression of PMNC NK activity is mediated via rabbit Fc attached to co-existing non-NK cells in the mononuclear fraction, and are inconsistent with the previously suggested functional association between NK activity and membrane beta 2m.

  3. Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter.

    PubMed

    Naruse, Kenji; Yoo, Seung Kwon; Kim, Sun Myoung; Choi, Yun Jaie; Lee, Hong Mie; Jin, Dong Il

    2006-01-01

    To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.

  4. Proteasome inhibitors prevent the degradation of familial Alzheimer's disease-linked presenilin 1 and potentiate A beta 42 recovery from human cells.

    PubMed Central

    Marambaud, P.; Ancolio, K.; Lopez-Perez, E.; Checler, F.

    1998-01-01

    BACKGROUND: Several lines of evidence suggest that most of the early-onset forms of familial Alzheimer's disease (FAD) are due to inherited mutations borne by a chromosome 14-encoded protein, presenilin 1 (PS1). This is likely related to an increased production of amyloid beta-peptide (A beta) 42, one of the main components of the extracellular deposits called senile plaques that invade human cortical areas during the disease. MATERIALS AND METHODS: We set up stably transfected HEK293 cells overexpressing wild-type (wt) and various FAD-linked mutated PS1. By Western blot analysis, we examined the influence of specific proteasome inhibitors on PS1-like immunoreactivities. Furthermore, by means of metabolic labeling and immunoprecipitation with A beta 40 and A beta 42-directed specific antibodies, we assessed the effect of the inhibitors on the production of A beta s by wt and mutated PS1-expressing cells transiently transfected with beta APP751. RESULTS: We show that two distinct proteasome inhibitors, Z-IE (Ot-Bu)A-Leucinal and lactacystin, increase in a time- and dose-dependent manner the immunoreactivities of both wt and mutated PS1. Furthermore, we demonstrate that PS1 is polyubiquitinated in these cells. Other inhibitors, ineffective on the proteasome, fail to protect wt and mutated PS1-like immunoreactivities. We also establish that the FAD-linked mutations of PS1 trigger a selective increased formation of A beta 42 as reflected by higher A beta 42 over total A beta ratios when compared with wtPS1-expressing cells. Interestingly, this augmentation was further amplified by proteasome inhibitors in cells expressing mutated but not wtPS1. CONCLUSION: Altogether, our data indicate that PS1 undergoes polyubiquitination in HEK293 cells and that the proteasome contributes to the degradation of wt and FAD-linked PS1, thereby directly influencing the A beta production in human cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:9562973

  5. Definition of a Skp2-c-Myc Pathway to Expand Human Beta-cells

    PubMed Central

    Tiwari, Shiwani; Roel, Chris; Tanwir, Mansoor; Wills, Rachel; Perianayagam, Nidhi; Wang, Peng; Fiaschi-Taesch, Nathalie M.

    2016-01-01

    Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional β-cell mass. Developmental differences, failure of adaptive expansion and loss of β-cells via β-cell death or de-differentiation have emerged as the possible causes of this reduced β-cell mass. We hypothesized that the proliferative response to mitogens of human β-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human β-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27kip1. Increasing nuclear abundance of p27kip1 by adenoviral delivery decreases the proliferative response of β-cells from non-diabetic donors, mimicking T2D β-cells. However, while both p27kip1 gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human β-cells, only Skp2 was capable of inducing a significant human β-cell expansion. Skp2 was also able to double the proliferative response of T2D β-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D β-cells. PMID:27380896

  6. Definition of a Skp2-c-Myc Pathway to Expand Human Beta-cells.

    PubMed

    Tiwari, Shiwani; Roel, Chris; Tanwir, Mansoor; Wills, Rachel; Perianayagam, Nidhi; Wang, Peng; Fiaschi-Taesch, Nathalie M

    2016-01-01

    Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional β-cell mass. Developmental differences, failure of adaptive expansion and loss of β-cells via β-cell death or de-differentiation have emerged as the possible causes of this reduced β-cell mass. We hypothesized that the proliferative response to mitogens of human β-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human β-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27(kip1). Increasing nuclear abundance of p27(kip1) by adenoviral delivery decreases the proliferative response of β-cells from non-diabetic donors, mimicking T2D β-cells. However, while both p27(kip1) gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human β-cells, only Skp2 was capable of inducing a significant human β-cell expansion. Skp2 was also able to double the proliferative response of T2D β-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D β-cells.

  7. Release, biological potency, and biochemical integrity of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) combined with Augment(TM) Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP).

    PubMed

    Young, C S; Ladd, P A; Browning, C F; Thompson, A; Bonomo, J; Shockley, K; Hart, C E

    2009-12-16

    Over 10 million surgical procedures are performed annually in the United States to treat musculoskeletal injuries, and a significant portion of these involve orthopedic bone grafting. The goals of the study were to evaluate the in vitro and in vivo release kinetics, biological potency and biochemical integrity of rhPDGF-BB combined with large (1000-2000 microm) and small (250-1000 microm) beta-TCP particles. Recombinant human platelet-derived growth factor B homodimer (rhPDGF-BB) is a protein growth factor under development as a therapeutic for accelerating bone healing. Release of the protein was monitored in vitro by ELISA, and in vivo by measurement of radioactive rhPDGF-BB implanted in rat calvarial defects. Biological activity was measured using a cell-based bioassay, and biochemical integrity was determined by SDS-PAGE and high pressure size exclusion chromatography (HPSEC). Release of rhPDGF-BB occurred rapidly from beta-TCP both in vitro and in vivo. Almost 100% of the rhPDGF-BB was recovered from large and small beta-TCP after 90 min in vitro. Approximately 90% of the rhPDGF-BB was depleted from calvarial defect sites within 72 h of implantation. RhPDGF-BB retained 100% of its biological potency compared to reference standard rhPDGF-BB, manifested as a single band at ~30 kDa by SDS-PAGE and a single peak eluted after 13 min by HPSEC following release from beta-TCP. RhPDGF-BB is rapidly released from large and small beta-TCP particles and is biochemically unaltered following release. PMID:19577598

  8. Reactive oxygen species mediate arsenic induced cell transformation and tumorigenesis through Wnt/{beta}-catenin pathway in human colorectal adenocarcinoma DLD1 cells

    SciTech Connect

    Zhang Zhuo; Wang Xin; Cheng Senping; Sun Lijuan; Son, Young-Ok; Yao Hua; Li Wenqi; Budhraja, Amit; Li Li; Shelton, Brent J.; Tucker, Thomas; Arnold, Susanne M.; Shi Xianglin

    2011-10-15

    Long term exposure to arsenic can increase incidence of human cancers, such as skin, lung, and colon rectum. The mechanism of arsenic induced carcinogenesis is still unclear. It is generally believed that reactive oxygen species (ROS) may play an important role in this process. In the present study, we investigate the possible linkage between ROS, {beta}-catenin and arsenic induced transformation and tumorigenesis in human colorectal adenocarcinoma cell line, DLD1 cells. Our results show that arsenic was able to activate p47{sup phox} and p67{sup phox}, two key proteins for activation of NADPH oxidase. Arsenic was also able to generate ROS in DLD1 cells. Arsenic increased {beta}-catenin expression level and its promoter activity. ROS played a major role in arsenic-induced {beta}-catenin activation. Treatment of DLD1 cells by arsenic enhanced both transformation and tumorigenesis of these cells. The tumor volumes of arsenic treated group were much larger than those without arsenic treatment. Addition of either superoxide dismutase (SOD) or catalase reduced arsenic induced cell transformation and tumor formation. The results indicate that ROS are involved in arsenic induced cell transformation and tumor formation possible through Wnt/{beta}-catenin pathway in human colorectal adenocarcinoma cell line DLD1 cells. - Highlights: > Arsenic activates NADPH oxidase and increases reactive oxygen species generation in DLD1 cells. > Arsenic increases {beta}-catenin expression. > Inhibition of ROS induced by arsenic reduce {beta}-catenin expression. > Arsenic increases cell transformation in DLD1 cells and tumorigenesis in nude mice. > Blockage of ROS decrease cell transformation and tumorigenesis induced by arsenic.

  9. Spironolactone and canrenone inhibit UGT2B7-catalyzed human liver and kidney microsomal aldosterone 18beta-glucuronidation: a potential drug interaction.

    PubMed

    Knights, Kathleen M; Bowalgaha, Kushari; Miners, John O

    2010-07-01

    Elevated plasma concentrations of aldosterone (ALDO) are observed in patients treated with spironolactone. Because ALDO is eliminated via UGT2B7-catalyzed 18beta-glucuronidation, this study aimed to determine whether spironolactone and its primary metabolites, canrenone and canrenoic acid, inhibit ALDO 18beta-glucuronidation by recombinant UGT2B7 and by human liver (HLM) and human kidney cortical (HKCM) microsomes. Initial experiments characterized the effects of all three compounds on 4-methylumbelliferone and ALDO glucuronidation by recombinant human UGT2B7. IC(50) values for spironolactone and canrenone ranged from 26 to 50 microM, whereas canrenoic acid was a weak inhibitor. Inhibitor constant (K(i)) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation were subsequently determined with HLM, HKCM, and UGT2B7 as the enzyme sources. Spironolactone and canrenone were competitive inhibitors of ALDO 18beta-glucuronidation by HLM, HKCM, and UGT2B7. Mean (+/-) K(i) values for spironolactone were 52 +/- 22 (HLM) and 34 +/- 4 microM (HKCM), and mean (+/-) K(i) values for canrenone were 41 +/- 19 (HLM) and 23 +/- 2 microM (HKCM). K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation by recombinant UGT2B7 were 23 and 11 microM, respectively. "Actual" K(i) values for spironolactone and canrenone inhibition of ALDO 18beta-glucuronidation, which take into account the role of endogenous microsomal inhibitors, are predicted to be 3 to 5 and 2 to 4 microM, respectively. The data indicate that the elevated ALDO concentrations observed in patients treated with spironolactone may be due, at least in part, to a pharmacokinetic interaction, and spironolactone and canrenone should be considered to be potential inhibitors of the UGT2B7-mediated metabolic clearance of drugs in both liver and kidney. PMID:20304966

  10. Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11{beta}-hydroxysteroid dehydrogenase

    SciTech Connect

    Agarwal, A.K.; Rogerson, F.M.; Mune, T.; White, P.C.

    1995-09-01

    11{beta}-hydroxysteroid dehydrogenase (11{beta}HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11{beta}HSD. In vitro, the NAD{sup +}-dependent kidney (type 2) isozyme catalyzes 11{beta}-dehydrogenase but not reductase reactions, whereas the NADP{sup +}-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11{beta}HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11{beta}HSD. 25 refs., 5 figs.

  11. Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-{beta}1-mediated gene activation

    SciTech Connect

    Zhuang, Yan; Nguyen, Hong T.; Lasky, Joseph A.; Cao, Subing; Li, Cui; Hu, Jiyao; Guo, Xinyue; Burow, Matthew E.; Shan, Bin

    2010-02-19

    Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of {alpha}-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-{beta}1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114 kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against {alpha}-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-{beta}1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

  12. Constitutive and inflammatory induction of alpha and beta chemokines in human first trimester forebrain astrocytes and neurons.

    PubMed

    Bakhiet, Moiz; Mousa, Alyaa; Seiger, Ake; Andersson, Jan

    2002-05-01

    Chemokine effects on leukocyte infiltration into the central nervous system (CNS) are key events in the inflammatory processes of neuroimmunologic and neuroinfectious diseases. Because, chemokines may play important roles in proliferation and differentiation of brain cells and in the initiation and progression of CNS inflammatory disorders, we analyzed constitutive and inflammatory-induced expression of alpha and beta chemokines in human first trimester forebrain cells. Constitutive induction of IL-8, MIP-1alpha, MIP-1beta, MCP-1 and regulated on activation, normal T-cell expressed, and secreted (Rantes) was detected in cryostat sections of embryonic forebrains in an age-dependent manner. Dissociated cell cultures were studied for spontaneous chemokine induction and after stimulation with the trypanosome lymphocyte triggering factor (TLTF), a novel trypanokine secreted by African trypanosomes that triggers a complex of immune responses. LPS and variant surface glycoprotein (VSG) were used as controls. In cultures, unstimulated cells expressed minimal chemokine levels except for Rantes. In response to TLTF and LPS, but not VSG, all chemokines were highly induced at the mRNA and protein levels in a dose- and age-dependent manner. Combined assays (in situ hybridization and immunohistochemistry) revealed that astrocytes and neurons are major sources for chemokines. These results illustrate the ability of resident brain cells to constitutively express chemokine genes, which may suggest an important role for chemokines during brain development. Furthermore, TLTF-induced chemokine expression in astrocytes and neurons indicate the capacity of TLTF to provoke neuroinflammation in the brain, which may have important therapeutic implications for the neurological manifestations of African trypanosomiasis. PMID:12009570

  13. Effects of bioactive compounds from carrots (Daucus carota L.), polyacetylenes, beta-carotene and lutein on human lymphoid leukaemia cells.

    PubMed

    Zaini, Rana G; Brandt, Kirsten; Clench, Malcolm R; Le Maitre, Christine L

    2012-07-01

    New therapies for leukaemia are urgently needed. Carrots have been suggested as a potential treatment for leukaemia in traditional medicine and have previously been studied in other contexts as potential sources of anticancer agents. Indicating that carrots may contain bioactive compounds, which may show potential in leukaemia therapies. This study investigated the effects of five fractions from carrot juice extract (CJE) on human lymphoid leukaemia cell lines, together with five purified bioactive compounds found in Daucus carota L, including: three polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) and two carotenoids (beta-carotene and lutein). Their effects on induction of apoptosis using Annexin V/PI and Caspase 3 activity assays analysed via flow cytometry and inhibition of cellular proliferation using Cell Titer Glo assay and cell cycle analysis were investigated. Treatment of all three lymphoid leukaemia cell lines with the fraction from carrot extracts which contained polyacetylenes and carotenoids was significantly more cytotoxic than the 4 other fractions. Treatments with purified polyacetylenes also induced apoptosis in a dose and time responsive manner. Moreover, falcarinol and falcarindiol-3-acetate isolated from Daucus carota L were more cytotoxic than falcarindiol. In contrast, the carotenoids showed no significant effect on either apoptosis or cell proliferation in any of the cells investigated. This suggests that polyacetylenes rather than beta-carotene or lutein are the bioactive components found in Daucus carota L and could be useful in the development of new leukemic therapies. Here, for the first time, the cytotoxic effects of polyacetylenes have been shown to be exerted via induction of apoptosis and arrest of cell cycle.

  14. beta-2 Microglobulin values among human immunodeficiency virus (HIV)-negative, HIV-positive asymptomatic, and HIV-positive symptomatic Ugandans.

    PubMed Central

    Piwowar, E M; Tugume, S B; Grant, R M; Lutalo, T; Pattishall, K; Katongole-Mbidde, E

    1995-01-01

    Mean serum beta-2 microglobulin levels among healthy human immunodeficiency virus-seronegative and asymptomatic and symptomatic human immunodeficiency virus-seropositive Ugandans were found to be 2.35, 3.75, and 5.06 mg/liter, respectively (P < 0.001). The upper limit of the normal range (3.5 mg/liter) is higher in this African population than that reported elsewhere. PMID:7697536

  15. Infundibular neurons of the human hypothalamus simultaneously reactive with antisera against endorphins, ACTH, MSH and beta-LPH.

    PubMed

    Bugnon, C; Bloch, B; Lenys, D; Fellmann, D

    1979-06-27

    In man, discrete neurons of the infundibular (arcuate) nucleus contain compounds that can be stained with anti-endorphin (alpha and beta), anti-ACTH, anti-MSH (alpha and beta) and anti-beta-LPH immune sera (I.S.). In the fetus, certain neurons stain with anti-beta-endorphin or anti((17--39)ACTH starting from the 11th week of fetal life. At the ultrastructural level, these neurons contain elementary granules that are immunoreactive with anti-beta-endorphin. In the adult, neurons immunoreactive with anti-beta-endorphin are found in the infundibular nucleus. Their axonal fibers terminate around blood vessels in the neurovascular zone and in the pituitary stalk, or establish contacts with non-immunoreactive perikarya of the infundibular nucleus. These neurons can be stained with anti(17--39)ACTH and anti-beta-endorphin I.S. The most reactive are also stained moderately with anti-alpha-MSH, anti-beta-MSH, anti-beta-LPH, anti-alpha-endorphin, or anti(1--24)ACTH I.S. These results indicate that, in man, compound(s) identical with or immunologically related to endorphins, beta-LPH, ACTH and MSH are secreted by certain hypothalamic neurons. These agents probably originate from a common precursor molecula similar to the so-called pro-opiocortin.

  16. Sialylation of Integrin beta1 is Involved in Radiation-Induced Adhesion and Migration in Human Colon Cancer Cells

    SciTech Connect

    Lee, Minyoung; Lee, Hae-June; Seo, Woo Duck; Park, Ki Hun; Lee, Yun-Sil

    2010-04-15

    Purpose: Previously, we reported that radiation-induced ST6 Gal I gene expression was responsible for an increase of integrin beta1 sialylation. In this study, we have further investigated the function of radiation-mediated integrin beta1 sialylation in colon cancer cells. Methods and Materials: We performed Western blotting and lectin affinity assay to analyze the expression and level of sialylated integrin beta1. After exposure to ionizing radiation (IR), adhesion and migration of cells were measured by in vitro adhesion and migration assay. Results: IR increased sialylation of integrin beta1 responsible for its increased protein stability and adhesion and migration of colon cancer cells. However, for cells with an N-glycosylation site mutant of integrin beta1 located on the I-like domain (Mu3), these effects were dramatically inhibited. In addition, integrin beta1-mediated radioresistance was not observed in cells containing this mutant. When sialylation of integrin beta1 was targeted with a sulfonamide chalcone compound, inhibition of radiation-induced sialylation of integrin beta1 and inhibition of radiation-induced adhesion and migration occurred. Conclusion: The increase of integrin beta1 sialylation by ST6 Gal I is critically involved in radiation-mediated adhesion and migration of colon cancer cells. From these findings, integrin beta1 sialylation may be a novel target for overcoming radiation-induced survival, especially radiation-induced adhesion and migration.

  17. Conversion of Human Steroid 5[beta]-Reductase (AKR1D1) into 3[beta]-Hydroxysteroid Dehydrogenase by Single Point Mutation E120H: Example of Perfect Enzyme Engineering

    SciTech Connect

    Chen, Mo; Drury, Jason E.; Christianson, David W.; Penning, Trevor M.

    2012-10-10

    Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5{beta}-reduction of {Delta}{sup 4}-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His120 (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5{beta}-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5{alpha}-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3{beta}-HSD as opposed to a 3{alpha}-HSD. The catalytic efficiency achieved for 3{beta}-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5{beta}-dihydrotestosterone, and {Delta}{sup 4}-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the {Delta}{sup 4}-double bond and confers 3{beta}-HSD activity on the 5{beta}-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its {alpha}-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.

  18. Daidzein-sulfate metabolites affect transcriptional and antiproliferative activities of estrogen receptor-beta in cultured human cancer cells.

    PubMed

    Totta, Pierangela; Acconcia, Filippo; Virgili, Fabio; Cassidy, Aedin; Weinberg, Peter D; Rimbach, Gerald; Marino, Maria

    2005-11-01

    Daidzein (D), a soy isoflavone, is almost completely metabolized in the gut and liver. This biotransformation converts D to more water-soluble products and may affect its biological activity. The ability of daidzein metabolites to modulate 17beta-estradiol (E2)-sensitive gene transcription, cell growth, and a proapoptotic cascade was determined in human cancer cells devoid of any estrogen receptor (ER) and rendered E2 sensitive after transfection with ERbeta. The data show that D and some but not all of its metabolites 1) induce promoter activity, 2) reduce proliferation, 3) promote p38/mitogen-activated protein kinase (MAPK) phosphorylation, and 4) activate a proapoptotic cascade involving the cleavage of caspase-3 and its substrate poly(ADP-ribose)polymerase (PARP) in human cancer cells in an ERbeta-dependent manner. Pretreatment of cells with ICI 182,780, a pure antiestrogen, completely prevented the actions of D and its metabolites. These findings highlight the important and complex influence of metabolic transformation on key physiological effects of isoflavones and demonstrate the need to take biotransformation into account when assessing the potential health benefits of consuming soy isoflavones. PMID:16251631

  19. Structural basis for the killing of human beta cells by CD8(+) T cells in type 1 diabetes.

    PubMed

    Bulek, Anna M; Cole, David K; Skowera, Ania; Dolton, Garry; Gras, Stephanie; Madura, Florian; Fuller, Anna; Miles, John J; Gostick, Emma; Price, David A; Drijfhout, Jan W; Knight, Robin R; Huang, Guo C; Lissin, Nikolai; Molloy, Peter E; Wooldridge, Linda; Jakobsen, Bent K; Rossjohn, Jamie; Peakman, Mark; Rizkallah, Pierre J; Sewell, Andrew K

    2012-03-01

    The structural characteristics of the engagement of major histocompatibility complex (MHC) class II-restricted self antigens by autoreactive T cell antigen receptors (TCRs) is established, but how autoimmune TCRs interact with complexes of self peptide and MHC class I has been unclear. Here we examined how CD8(+) T cells kill human islet beta cells in type 1 diabetes via recognition of a human leukocyte antigen HLA-A*0201-restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHC class I-restricted TCRs. However, this interaction was extraordinarily weak because of limited contacts with MHC class I. TCR binding was highly peptide centric, dominated by two residues of the complementarity-determining region 3 (CDR3) loops that acted as an 'aromatic-cap' over the complex of peptide and MHC class I (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8(+) T cell-mediated autoreactivity. PMID:22245737

  20. Amyloid-beta leads to impaired cellular respiration, energy production and mitochondrial electron chain complex activities in human neuroblastoma cells.

    PubMed

    Rhein, V; Baysang, G; Rao, S; Meier, F; Bonert, A; Müller-Spahn, F; Eckert, A

    2009-09-01

    Evidence suggests that amyloid-beta (Abeta) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Abeta can lead to neuronal dysfunction. Here we investigated the specific effects of Abeta on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Abeta protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.

  1. Non-identity of human plasma lysozyme and 4-methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside hydrolase.

    PubMed

    Den Tandt, W R; Inaba, T; Verhamme, I; Overdyk, B; Brouwer, J; Prieur, D

    1988-01-01

    1. Using 4-methylumbelliferyl-tetra-N-acetyl-beta-D-chitotetraoside (MU-TACT) as substrate, it is possible to measure the activity of purified lysozyme and to demonstrate lysozyme activity in the urine of patients with acute monocytic leukemia, characterized by massive lysozymuria. 2. Notwithstanding this observation, we present evidence that in normal human plasma another acid endoglucosaminidase is hydrolyzing the substrate. 3. The following data support the hypothesis of the existence of a separate hydrolase: (a) Thermoinactivation is different for MU-TACT hydrolase and lysozyme. (b) In plasma and many other biological samples, the concentration of lysozyme is too low to be measured with the artificial substrate and there is no correlation between MU-TACT hydrolase and lysozyme. (c) Serum of lysozyme deficient rabbits has normal MU-TACT hydrolase activity. (d) On Sephadex G-200 and DEAE cellulose chromatography, lysozyme and MU-TACT hydrolase are eluted separately. (e) Immunoremoval of lysozyme from human plasma does not affect the activity towards MU-TACT. (f) The effect of N-acetylglucosamine and N-acetylmuramic acid on the activity of lysozyme and MU-TACT hydrolase is different. PMID:3181601

  2. Human beta-defensin-3 producing cells in septic implant loosening.

    PubMed

    Levón, Jaakko; Al-Samadi, Ahmed; Mackiewicz, Zygmunt; Coer, Andrej; Trebse, Rihard; Waris, Eero; Konttinen, Yrjö T

    2015-02-01

    Human β-defensin-3 (hBD-3) has been found in synovial fluid and later in periprosthetic tissues in septic joint implant loosening. The aim of the present study was to identify its cellular sources. Tissue samples from 12 patients were analyzed. A fully automatic Leica BOND MAX staining robot was used. Affinity-purified rabbit anti-human hBD-3 IgG was applied in a two-layer horse radish peroxidase/anti-rabbit-labeled polymer method. Double immunofluorescence of hBD3 together with CD68, CD31, heat shock protein 47 (HSP47) and mast cell tryptase (MCT) staining was done. Human BD-3 was found in monocyte/macrophage-like cells, vascular endothelial cells and fibroblasts-like cells, but was weakly expressed in foreign body giant cells and negative in neutrophils. Human BD-3 was found in CD68 and CD31 immunoreactive cells, whereas HSP47 and MCT positive cells were hBD-3 negative. Immunostaining of hBD-3 was strong in some tissue areas but weak or absent in others. Monocyte/macrophages and endothelial cells were established in this study as the major cellular sources of hBD-3 in septic loosening, but fibroblasts and foreign body giant cells can also contribute to its production. The heterogeneous topological staining of hBD-3 suggests local regulation, possibly by bacterial products, damage-associated molecular patterns and cytokines. The results explain the increased synovial fluid/tissue concentrations of hBD-3 in septic loosening. PMID:25655501

  3. Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

    PubMed Central

    Esposito, G.; Michelutti, R.; Verdone, G.; Viglino, P.; Hernández, H.; Robinson, C. V.; Amoresano, A.; Dal Piaz, F.; Monti, M.; Pucci, P.; Mangione, P.; Stoppini, M.; Merlini, G.; Ferri, G.; Bellotti, V.

    2000-01-01

    The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed. PMID:10850793

  4. Satellite data regarding the eutrophication response to human activities in the plateau lake Dianchi in China from 1974 to 2009.

    PubMed

    Huang, Changchun; Wang, Xiaolei; Yang, Hao; Li, Yunmei; Wang, Yanhua; Chen, Xia; Xu, Liangjiang

    2014-07-01

    Human activities contribute highly to water eutrophication. In this study, the relationship between human activities and water eutrophication in Dianchi Lake in China was characterized using a combination of satellite imaging, sedimentary physicochemical and meteorological data analyses. The heavy eutrophic status and algal bloom in Dianchi Lake were first observed by satellite in 1977 and 1989, respectively. The C/N ratio, an indicator of organic sources in sediments, also showed that the planktonic organic source in the sediment significantly increased beginning in 1989. The land use cover in the Dianchi basin showed that both farm lands and forests, but particularly farmlands, were reduced during the period from 1974 to 2009. The urbanized land area increased from 1974 to 2009, particularly after 2000. The effects of human activities on water eutrophication were expressed by land use cover, population, gross domestic product (GDP; separated into primary, secondary and tertiary industries) and wastewater discharge. For land use cover, farm and urbanized lands were the main sources of water nutrients; forest contributed slightly to these nutrients. For GDP, primary (correlation coefficient=0.94, P<0.001) and tertiary (correlation coefficient=0.95, P<0.001) industries were highly correlated with total nitrogen. Secondary (correlation coefficient=0.95, P<0.001) and tertiary (correlation coefficient=0.96, P<0.001) industries were highly correlated with total phosphorus. The algal bloom area was significantly correlated with wastewater discharge (correlation coefficient=0.78, P<0.005) (except industrial wastewater), which was primarily led by the non-agricultural population, from 2000 to 2009. This study suggests that the protection of water environments requires a comprehensive protection policy in addition to a unilateral protection policy.

  5. Arsenic Exposure and Calpain-10 Polymorphisms Impair the Function of Pancreatic Beta-Cells in Humans: A Pilot Study of Risk Factors for T2DM

    PubMed Central

    Díaz-Villaseñor, Andrea; Cruz, Laura; Cebrián, Arturo; Hernández-Ramírez, Raúl U.; Hiriart, Marcia; García-Vargas, Gonzálo; Bassol, Susana; Sordo, Monserrat; Gandolfi, A. Jay; Klimecki, Walter T.; López-Carillo, Lizbeth; Cebrián, Mariano E.; Ostrosky-Wegman, Patricia

    2013-01-01

    The incidence of type 2 diabetes mellitus (T2DM) is increasing worldwide and diverse environmental and genetic risk factors are well recognized. Single nucleotide polymorphisms (SNPs) in the calpain-10 gene (CAPN-10), which encodes a protein involved in the secretion and action of insulin, and chronic exposure to inorganic arsenic (iAs) through drinking water have been independently associated with an increase in the risk for T2DM. In the present work we evaluated if CAPN-10 SNPs and iAs exposure jointly contribute to the outcome of T2DM. Insulin secretion (beta-cell function) and insulin sensitivity were evaluated indirectly through validated indexes (HOMA2) in subjects with and without T2DM who have been exposed to a gradient of iAs in their drinking water in northern Mexico. The results were analyzed taking into account the presence of the risk factor SNPs SNP-43 and -44 in CAPN-10. Subjects with T2DM had significantly lower beta-cell function and insulin sensitivity. An inverse association was found between beta-cell function and iAs exposure, the association being more pronounced in subjects with T2DM. Subjects without T2DM who were carriers of the at-risk genotype SNP-43 or -44, also had significantly lower beta-cell function. The association of SNP-43 with beta-cell function was dependent on iAs exposure, age, gender and BMI, whereas the association with SNP-44 was independent of all of these factors. Chronic exposure to iAs seems to be a risk factor for T2DM in humans through the reduction of beta-cell function, with an enhanced effect seen in the presence of the at-risk genotype of SNP-43 in CAPN-10. Carriers of CAPN-10 SNP-44 have also shown reduced beta-cell function. PMID:23349674

  6. Regulation of pigmentation in human epidermal melanocytes by functional high-affinity beta-melanocyte-stimulating hormone/melanocortin-4 receptor signaling.

    PubMed

    Spencer, J D; Schallreuter, K U

    2009-03-01

    To date, the principal receptor considered to regulate human pigmentation is the melanocortin-1 receptor (MC1-R) via induction of the cAMP/protein kinase A pathway by the melanocortins alpha-MSH and ACTH. In this context, it is noteworthy that beta-MSH can also induce melanogenesis, although it has a low affinity for the MC1-R, whereas the preferred receptor for this melanocortin is the MC4-R. Because beta-MSH is present in the epidermal compartment, it was of interest to ascertain whether functioning MC4-Rs are present in human epidermal keratinocytes and melanocytes. Our results provide evidence that the MC4-R is expressed in situ and in vitro throughout the human epidermis at the mRNA and protein level using RT-PCR, Western blotting, and double immunofluorescence staining. Moreover, radioligand binding studies yielded high-affinity receptors for beta-MSH on epidermal melanocytes (3600 receptors per cell), undifferentiated keratinocytes (7200 receptors per cell), and differentiated keratinocytes (72,700 receptors per cell), indicating that MC4-R expression correlates with epidermal differentiation. Importantly, increased melanogenesis after stimulation of the beta-MSH/cAMP/microphthalmia-associated transcription factor/tyrosinase cascade proved the functionality of this signal in melanocytes, which was attenuated in the presence of the specific MC4-R antagonist HS014. In summary, our results imply an important role for the beta-MSH/MC4-R cascade in human melanocyte biology, although the function and purpose of this signal in keratinocytes needs further elucidation.

  7. Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

    PubMed

    Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie

    2009-01-01

    The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha

  8. Synergistic cytotoxicity of red beetroot (Beta vulgaris L.) extract with doxorubicin in human pancreatic, breast and prostate cancer cell lines.

    PubMed

    Kapadia, Govind J; Rao, G Subba; Ramachandran, Cheppail; Iida, Akira; Suzuki, Nobutaka; Tokuda, Harukuni

    2013-06-26

    Although a wide variety of cytotoxic plant extracts and phytochemicals are known to act synergistically with anticancer drug doxorubicin (D), their clinical application is hindered by safety concerns of such combination therapy. Our earlier studies showed that red beetroot (Beta vulgaris L.) extract (B), approved by Food and Drug Administration and European Union as red food color E162, reduced multi-organ tumor formations in various animal models when administered in drinking water. This led us to postulate that a long-term daily exposure to low doses of B through diet might be safe and sufficient to produce cancer chemopreventive effect in humans. Further, our recent comparative cytotoxic investigation with B and D in several human cancer cell lines indicated their potential for synergistic activity. Since B is considered safe for human use with no known toxicity, we conducted the present study to evaluate its synergistic antiproliferative activity with D against pancreatic (PaCa), breast (MCF-7) and prostate (PC-3) tumor cells of human origin. Different concentrations of B and D (0.29-290 μg/ml) and in various combinations (B:D ratio = 1:0, 1:1, 5:1, 1:5 and 0:1) were tested for cytotoxic effects against the three cancer cells. The viability of cells was assessed after 72 h incubation with various combinations of B and D using the trypan-blue staining method. The cytotoxic data were analyzed by the combination index method of Chou and Talalay to establish synergy between B and D. The results indicated that an overall positive reduction in drug concentration was achieved by D when combined with B in its cytotoxicity profile in the three human cancer cells tested. The synergistic cytotoxicity was best when the B:D ratio of 1:5 was used in PaCa cells at IC50, IC75 and IC90 dose levels and in MCF-7 cells at IC90 dose level. These results warrant further studies on the potential of red beetroot extract-doxorubicin combination in treating human cancers.

  9. Synergistic cytotoxicity of red beetroot (Beta vulgaris L.) extract with doxorubicin in human pancreatic, breast and prostate cancer cell lines.

    PubMed

    Kapadia, Govind J; Rao, G Subba; Ramachandran, Cheppail; Iida, Akira; Suzuki, Nobutaka; Tokuda, Harukuni

    2013-01-01

    Although a wide variety of cytotoxic plant extracts and phytochemicals are known to act synergistically with anticancer drug doxorubicin (D), their clinical application is hindered by safety concerns of such combination therapy. Our earlier studies showed that red beetroot (Beta vulgaris L.) extract (B), approved by Food and Drug Administration and European Union as red food color E162, reduced multi-organ tumor formations in various animal models when administered in drinking water. This led us to postulate that a long-term daily exposure to low doses of B through diet might be safe and sufficient to produce cancer chemopreventive effect in humans. Further, our recent comparative cytotoxic investigation with B and D in several human cancer cell lines indicated their potential for synergistic activity. Since B is considered safe for human use with no known toxicity, we conducted the present study to evaluate its synergistic antiproliferative activity with D against pancreatic (PaCa), breast (MCF-7) and prostate (PC-3) tumor cells of human origin. Different concentrations of B and D (0.29-290 μg/ml) and in various combinations (B:D ratio = 1:0, 1:1, 5:1, 1:5 and 0:1) were tested for cytotoxic effects against the three cancer cells. The viability of cells was assessed after 72 h incubation with various combinations of B and D using the trypan-blue staining method. The cytotoxic data were analyzed by the combination index method of Chou and Talalay to establish synergy between B and D. The results indicated that an overall positive reduction in drug concentration was achieved by D when combined with B in its cytotoxicity profile in the three human cancer cells tested. The synergistic cytotoxicity was best when the B:D ratio of 1:5 was used in PaCa cells at IC50, IC75 and IC90 dose levels and in MCF-7 cells at IC90 dose level. These results warrant further studies on the potential of red beetroot extract-doxorubicin combination in treating human cancers. PMID

  10. Mass spectrometric measurement of [beta]-endorphin and methionine enkephalin in human pituitaries. Tumors and post-mortem controls

    NASA Astrophysics Data System (ADS)

    Kusmierz, Jozef J.; Dass, Chhabil; Robertson, James T.; Desiderio, Dominic M.

    1991-12-01

    Two opioid neuropeptides, [beta]-endorphin (BE), which derives from the proopiomelanocortin (POMC) precursor, and methionine enkephalin (DE), which derives from proenkephalin A, were quantified with fast atom bombardment mass spectrometry (FAB-MS) in individual human pituitaries (post-mortem) and in tumor pituitaries (post-surgery) in a study to clarify the molecular processes that occur in tumor formation. FAB-MS in the multiple reaction monitoring mode linked the precursor ion (the MH+ ion) of the peptide with a fragment ion that was unique to each neuropeptide to increase significantly the molecular specificity of these quantitative analytical measurements. The ME was quantified as the intact pentapeptide, whereas BE1-31 was quantified via its tryptic fragment BE20-24 (NAIIK). Two corresponding stable isotope-incorporated peptides, [2H5-4Phe]-ME and [2H4-22Ile]-BE1-31,human respectively, were used as the internal standards. The amount of each neuropeptide quantified in control post-mortem pituitaries (n = 8) was 75.2 ± 29.6(s.e.m.) pmol ME mg-1 protein, and in the pituitary tumor samples (n = 5), 25.0 ± 7.6 pmol ME mg-1 protein and 36.0 ± 14.8 pmol BE mg-1 protein. The difference in the BE content between the control and tumor pituitaries was significant (p = 0.004), and reflected an aberrant metabolism of the POMC system in those human pituitary tumor tissues.

  11. Down regulation and nuclear localization of human beta-defensin-1 in pleomorphic adenomas of salivary glands.

    PubMed

    Pantelis, Annette; Wenghoefer, Matthias; Haas, Susanne; Merkelbach-Bruse, Sabine; Pantelis, Dimitrios; Jepsen, Søren; Bootz, Friedrich; Winter, Jochen

    2009-06-01

    Although antimicrobial peptides (AMPs) appear to have diverse functional activities in innate immunity, a few reports suggest a potential role of human beta-defensin (hBD)-1 in tumor suppression. The aim of the present study was to compare the expression patterns of hBD-1, -2 and -3 in various features of human salivary gland tissues, such as healthy parenchyma, chronic sialadenitis and intraglandular pleomorphic adenomas, with their adjacent normal tissues. Twenty human salivary gland specimens (five healthy, five chronic sialadenitis, five pleomorphic adenomas and five adenoma adjacent normal tissues (AANTs)) were investigated for mRNA expression levels of hBD-1, -2 and -3 by quantitative real-time RT-PCR. Additionally, immunohistochemistry for the hBD-1, -2 and -3 peptides was performed for analysis of localization. Considerably increased, 80-fold higher hBD-1 and increased hBD-3 mRNA expression levels compared to healthy salivary gland tissues were detected in chronic sialadenitis. In pleomorphic adenomas hBD-2 expression levels were lower, but hBD-1 expression levels were significant decreased (p=0.03) compared to healthy parenchyma. Interestingly, the AANTs showed a 48-fold higher expression of hBD-1 and increased hBD-3 expression compared to the healthy salivary gland. Immunohistochemistry of the tumors showed nuclear hBD-1 localization. For the first time, it was shown that hBD-1 gene expression is significantly decreased in pleomorphic adenomas, while simultaneously the protein is localized in the nucleus. Increased expression levels in glandular inflammation have been described previously albeit not in AANTs. These data support the hypothesis that hBD-1 might be a potential tumor suppressor also in benign salivary gland tumors in addition to other genetic alterations.

  12. Characterization of cDNAs of the human pregnancy-specific beta1-glycoprotein family, a new subfamily of the immunoglobulin gene superfamily

    SciTech Connect

    Zheng, Q.X.; Tease, L.A.; Shupert, W.L.; Chan, W.Y. )

    1990-03-20

    Three highly homologous cDNAs encoding human pregnancy-specific {beta}1-glycoprotein (SP1) were isolated from a human placental cDNA library. These cDNAs share >90% nucleotide homology in their coding sequences, and >79% of the encoded amino acids are homologous. Proteins encoded by these cDNAs are very similar to members of the carcinoembryonic antigen family and contain repeating domains, conserved disulfided bridges, and {beta}-sheet structure typical of the immunoglobulin gene superfamily. However, the high degree of sequence homology and relatively lesser degree of glycosylation among the SP1 proteins suggest that they exist as a unique family instead of being members of the CEA family. Both soluble and potentially membrane-bound forms of SP1 proteins were present in the placenta. Northern blot analysis using specific probes confirmed the expression of multiple mRNA species in human term placenta.

  13. Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin/TCF pathway.

    PubMed

    Wang, Yihua; Zhou, Xiaobo; Zhu, Hongxia; Liu, Shuang; Zhou, Cuiqi; Zhang, Guo; Xue, Liyan; Lu, Ning; Quan, Lanping; Bai, Jinfeng; Zhan, Qimin; Xu, Ningzhi

    2005-10-01

    Esophageal squamous cell carcinoma (ESCC) has a multifactorial etiology involving environmental and/or genetic factors. End-binding protein 1 (EB1), which was cloned as an interacting partner of the adenomatous polyposis coli (APC) tumor suppressor protein, was previously found overexpressed in ESCC. However, the precise role of EB1 in the development of this malignancy has not yet been elucidated. In this study, we analysed freshly resected ESCC specimens and demonstrated that EB1 was overexpressed in approximately 63% of tumor samples compared to matched normal tissue. We report that overexpression of EB1 in the ESCC line EC9706 significantly promotes cell growth, whereas suppression of EB1 protein level by RNA interference significantly inhibited growth of esophageal tumor cells. In addition, EB1 overexpression induced nuclear accumulation of beta-catenin and promoted the transcriptional activity of beta-catenin/T-cell factor (TCF). These effects were partially or completely abolished by coexpression of APC or DeltaN TCF4, respectively. Also, we found that EB1 affected the interaction between beta-catenin and APC. Furthermore, EB1 overexpression was correlated with cytoplasmic/nuclear accumulation of beta-catenin in primary human ESCC. Taken together, these results support the novel hypothesis that EB1 overexpression may play a role in the development of ESCC by affecting APC function and activating the beta-catenin/TCF pathway.

  14. Human Trifunctional Protein Alpha Links Cardiolipin Remodeling to Beta-Oxidation

    PubMed Central

    Taylor, William A.; Mejia, Edgard M.; Mitchell, Ryan W.; Choy, Patrick C.; Sparagna, Genevieve C.; Hatch, Grant M.

    2012-01-01

    Cardiolipin (CL) is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL) to CL and was identical to the alpha subunit of trifunctional protein (αTFP) lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling. PMID:23152787

  15. Generation of Functional Beta-Like Cells from Human Exocrine Pancreas

    PubMed Central

    Lima, Maria J.; Muir, Kenneth R.; Docherty, Hilary M.; McGowan, Neil W. A.; Forbes, Shareen; Heremans, Yves; Heimberg, Harry; Casey, John; Docherty, Kevin

    2016-01-01

    Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting β-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant β-like cells expressed insulin protein levels at ~15–30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants. PMID:27243814

  16. Angiogenic gene signature in human pancreatic cancer correlates with TGF-beta and inflammatory transcriptomes

    PubMed Central

    Wilson, Julie L.; Korc, Murray

    2016-01-01

    Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, but overexpress pro-angiogenic factors and exhibit regions of microvasculature. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we previously reported that ∼12% of PDACs have an angiogenesis gene signature with increased expression of multiple pro-angiogenic genes. By analyzing the recently expanded TCGA dataset, we now report that this signature is present in ∼35% of PDACs but that it is mostly distinct from an angiogenesis signature present in pancreatic neuroendocrine tumors (PNETs). These PDACs exhibit a transcriptome that reflects active TGF-β signaling, and up-regulation of several pro-inflammatory genes, and many members of JAK signaling pathways. Moreover, expression of SMAD4 and HDAC9 correlates with endothelial cell abundance in PDAC tissues. Concomitantly targeting the TGF-β type I receptor (TβRI) kinase with SB505124 and JAK1-2 with ruxolitinib suppresses JAK1 phosphorylation and blocks proliferative cross-talk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs), and these anti-proliferative effects were mimicked by JAK1 silencing in ECs. By contrast, either inhibitor alone does not suppress their enhanced proliferation in 3D co-cultures. These findings suggest that targeting both TGF-β and JAK1 signaling could be explored therapeutically in the 35% of PDAC patients whose cancers exhibit an angiogenesis gene signature. PMID:26586478

  17. Metabolic and febrile responses to typhoid vaccine in humans: effect of beta-adrenergic blockade.

    PubMed

    Cooper, A L; Horan, M A; Little, R A; Rothwell, N J

    1992-06-01

    Fever and activation of acute phase responses were induced in human volunteers by intramuscular injection of typhoid vaccine. Vaccine injection caused a rapid (within 1 h) and sustained rise in metabolic rate (peak response 16%, 6-8 h), followed by later increases in white blood cell count (3-4 h), skin temperature (4-5 h), oral temperature (5-6 h), heart rate (6-8 h), and plasma cortisol (5-8 h). A peak fever [1.2 +/- 0.2 degree C (SE) rise] was recorded 12 h after vaccine injection. The involvement of the sympathetic nervous system in the development of these responses was investigated by the oral administration of propranolol before (80 mg) and 3 h after (40 mg) vaccine injection. Propranolol prevented the increases in metabolic rate, heart rate, and skin temperature but did not inhibit the rise in oral temperature or white cell count after vaccine administration. These data indicate that the sympathetic nervous system is responsible for the rise in energy expenditure associated with fever in humans. However, the rise in body temperature can develop in the absence of this increase in metabolic rate possibly by changes in heat loss.

  18. Wi-Fi and Satellite-Based Location Techniques for Intelligent Agricultural Machinery Controlled by a Human Operator

    PubMed Central

    Drenjanac, Domagoj; Tomic, Slobodanka; Agüera, Juan; Perez-Ruiz, Manuel

    2014-01-01

    In the new agricultural scenarios, the interaction between autonomous tractors and a human operator is important when they jointly perform a task. Obtaining and exchanging accurate localization information between autonomous tractors and the human operator, working as a team, is a critical to maintaining safety, synchronization, and efficiency during the execution of a mission. An advanced localization system for both entities involved in the joint work, i.e., the autonomous tractors and the human operator, provides a basis for meeting the task requirements. In this paper, different localization techniques for a human operator and an autonomous tractor in a field environment were tested. First, we compared the localization performances of two global navigation satellite systems’ (GNSS) receivers carried by the human operator: (1) an internal GNSS receiver built into a handheld device; and (2) an external DGNSS receiver with centimeter-level accuracy. To investigate autonomous tractor localization, a real-time kinematic (RTK)-based localization system installed on autonomous tractor developed for agricultural applications was evaluated. Finally, a hybrid localization approach, which combines distance estimates obtained using a wireless scheme with the position of an autonomous tractor obtained using an RTK-GNSS system, is proposed. The hybrid solution is intended for user localization in unstructured environments in which the GNSS signal is obstructed. The hybrid localization approach has two components: (1) a localization algorithm based on the received signal strength indication (RSSI) from the wireless environment; and (2) the acquisition of the tractor RTK coordinates when the human operator is near the tractor. In five RSSI tests, the best result achieved was an average localization error of 4 m. In tests of real-time position correction between rows, RMS error of 2.4 cm demonstrated that the passes were straight, as was desired for the autonomous tractor

  19. Wi-Fi and satellite-based location techniques for intelligent agricultural machinery controlled by a human operator.

    PubMed

    Drenjanac, Domagoj; Tomic, Slobodanka; Agüera, Juan; Perez-Ruiz, Manuel

    2014-10-22

    In the new agricultural scenarios, the interaction between autonomous tractors and a human operator is important when they jointly perform a task. Obtaining and exchanging accurate localization information between autonomous tractors and the human operator, working as a team, is a critical to maintaining safety, synchronization, and efficiency during the execution of a mission. An advanced localization system for both entities involved in the joint work, i.e., the autonomous tractors and the human operator, provides a basis for meeting the task requirements. In this paper, different localization techniques for a human operator and an autonomous tractor in a field environment were tested. First, we compared the localization performances of two global navigation satellite systems' (GNSS) receivers carried by the human operator: (1) an internal GNSS receiver built into a handheld device; and (2) an external DGNSS receiver with centimeter-level accuracy. To investigate autonomous tractor localization, a real-time kinematic (RTK)-based localization system installed on autonomous tractor developed for agricultural applications was evaluated. Finally, a hybrid localization approach, which combines distance estimates obtained using a wireless scheme with the position of an autonomous tractor obtained using an RTK-GNSS system, is proposed. The hybrid solution is intended for user localization in unstructured environments in which the GNSS signal is obstructed. The hybrid localization approach has two components: (1) a localization algorithm based on the received signal strength indication (RSSI) from the wireless environment; and (2) the acquisition of the tractor RTK coordinates when the human operator is near the tractor. In five RSSI tests, the best result achieved was an average localization error of 4 m. In tests of real-time position correction between rows, RMS error of 2.4 cm demonstrated that the passes were straight, as was desired for the autonomous tractor. From

  20. Wi-Fi and satellite-based location techniques for intelligent agricultural machinery controlled by a human operator.

    PubMed

    Drenjanac, Domagoj; Tomic, Slobodanka; Agüera, Juan; Perez-Ruiz, Manuel

    2014-01-01

    In the new agricultural scenarios, the interaction between autonomous tractors and a human operator is important when they jointly perform a task. Obtaining and exchanging accurate localization information between autonomous tractors and the human operator, working as a team, is a critical to maintaining safety, synchronization, and efficiency during the execution of a mission. An advanced localization system for both entities involved in the joint work, i.e., the autonomous tractors and the human operator, provides a basis for meeting the task requirements. In this paper, different localization techniques for a human operator and an autonomous tractor in a field environment were tested. First, we compared the localization performances of two global navigation satellite systems' (GNSS) receivers carried by the human operator: (1) an internal GNSS receiver built into a handheld device; and (2) an external DGNSS receiver with centimeter-level accuracy. To investigate autonomous tractor localization, a real-time kinematic (RTK)-based localization system installed on autonomous tractor developed for agricultural applications was evaluated. Finally, a hybrid localization approach, which combines distance estimates obtained using a wireless scheme with the position of an autonomous tractor obtained using an RTK-GNSS system, is proposed. The hybrid solution is intended for user localization in unstructured environments in which the GNSS signal is obstructed. The hybrid localization approach has two components: (1) a localization algorithm based on the received signal strength indication (RSSI) from the wireless environment; and (2) the acquisition of the tractor RTK coordinates when the human operator is near the tractor. In five RSSI tests, the best result achieved was an average localization error of 4 m. In tests of real-time position correction between rows, RMS error of 2.4 cm demonstrated that the passes were straight, as was desired for the autonomous tractor. From

  1. Exploring functional cyclophellitol analogues as human retaining beta-glucosidase inhibitors.

    PubMed

    Li, Kah-Yee; Jiang, Jianbing; Witte, Martin D; Kallemeijn, Wouter W; Donker-Koopman, Wilma E; Boot, Rolf G; Aerts, Johannes M F G; Codée, Jeroen D C; van der Marel, Gijsbert A; Overkleeft, Herman S

    2014-10-21

    The natural product, cyclophellitol and its aziridine analogue are potent mechanism-based retaining β-glucosidase inhibitors. In this paper we explore the inhibitory potency of a number of cyclophellitol analogues against the three human retaining β-glucosidases, GBA, GBA2 and GBA3. We demonstrate that N-alkyl cyclophellitol aziridine is at least equally potent in inhibiting the enzymes evaluated as its N-acyl congener, whereas the N-sulfonyl analogue is a considerably weaker inhibitor. Our results complement the literature on the inhibitory potency of cyclophellitol analogues and hold promise for the future design of more effective activity-based retaining glycosidase probes with respect to probe stability in physiological media.

  2. Expression of Beta-Defensin 131 Promotes an Innate Immune Response in Human Prostate Epithelial Cells

    PubMed Central

    Kim, Hae Jong; Lee, Jaehyouk; Myung, Soon Chul

    2015-01-01

    Previously, using the Illumina HumanHT-12 microarray we found that β-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line RWPE-1 upon stimulation with lipoteichoic acid (LTA; a gram-positive bacterial component), than that in the untreated RWPE-1 cells. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. We examined the intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-κB directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-κB activation, which was evident in the phosphorylation of both p38MAPK and IκBα. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-κB, respectively, suppressed LTA-induced DEFB131 expression. The chromatin immunoprecipitation assay showed that NF-κB directly binds to the DEFB131 promoter, suggesting that NF-κB is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, with DEFB131 overexpression in RWPE-1 cells, the accumulation of mRNA and protein secretion of cytokines (IL-1α, IL-1β, IL-6, and IL-12α) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-κB signaling pathway in RWPE-1 cells during bacterial infection and promotes an innate immune response. PMID:26649771

  3. Comparative simulation studies of native and single-site mutant human beta-defensin-1 peptides.

    PubMed

    Toubar, Rabab A; Zhmurov, Artem; Barsegov, Valeri; Marx, Kenneth A

    2013-01-01

    Human defensins play important roles in a broad range of biological functions, such as microbial defense and immunity. Yet, little is known about their molecular properties, i.e. secondary structure stability, structural variability, important side chain interactions, surface charge distribution, and resistance to thermal fluctuations, and how these properties are related to their functions. To assess these factors, we studied the native human β-defensin-1 monomer and dimer as well as several single-site mutants using molecular dynamics simulations. The results showed that disulfide bonds are important determinants in maintaining the defensins' structural integrity, as no structural transitions were observed at 300 K and only minor structural unfolding was detected upon heating to 500 K. The α-helix was less thermally stable than the core β-sheet structure held together by hydrogen bonds and hydrophobic interactions. The monomer α-helix stability was directly correlated, whereas the end-to-end distance was inversely correlated to the experimentally measured β-defensin-1 chemotactic activity, in the order: mutant 2 (Gln24Glu) > mutant 3 (Lys31Ala) = wild type > mutant 1 (Asn4Ala). The structural stability of the β-defensin-1 dimer species exhibited an inverse correlation to their chemotactic activity. In dimers formed by mutants 2 and 3, we observed sliding of one monomer upon the surface of the other in the absence of unbinding. This dynamic sliding feature may enhance the molecular oligomerization of β-defensin-1 peptides contributing to their antibacterial activity. It could also help these peptides orient correctly in the CC chemokine receptor 6 binding site, thereby initiating their chemotactic activity. In agreement with this notion, the remarkable sliding behavior was observed only for the mutants with the highest chemotactic activity.

  4. Satellite image analysis of human caused changes in the tundra vegetation around the city of Vorkuta, north-European Russia.

    PubMed

    Virtanen, Tarmo; Mikkola, Kari; Patova, Elena; Nikula, Ari

    2002-01-01

    In this paper we present what kind of human impacted changes can be found in dwarf birch (Betula nana) dominated shrub tundra vegetation around the large industrial complex of Vorkuta in the north-European Russian tundra. Using fieldwork data and Landsat TM satellite image we could identify two impact zones: (1) Pollution zone (150-200 km2). In this zone most of the lichen species are absent. Changes in vegetation communities' species composition in all main plant groups are obvious. Willows especially are more dominant than in the unpolluted sites. (2) Slight pollution/disturbance zone (600-900 km2). Here vegetation changes are mainly similar but less so than the changes in the first zone. Particularly, the amount of herbs and grasses is increased when compared to unpolluted areas. The pollution zones are spatially connected to the main emission sources in the area. Zones spread furthest to the northeast, matching the prevailing winds during winter.

  5. Occurrence of Multidrug Resistant Extended Spectrum Beta-Lactamase-Producing Bacteria on Iceberg Lettuce Retailed for Human Consumption.

    PubMed

    Bhutani, Natasha; Muraleedharan, Chithra; Talreja, Deepa; Rana, Sonia Walia; Walia, Sandeep; Kumar, Ashok; Walia, Satish K

    2015-01-01

    Antibiotic resistance in bacteria is a global problem exacerbated by the dissemination of resistant bacteria via uncooked food, such as green leafy vegetables. New strains of bacteria are emerging on a daily basis with novel expanded antibiotic resistance profiles. In this pilot study, we examined the occurrence of antibiotic resistant bacteria against five classes of antibiotics on iceberg lettuce retailed in local convenience stores in Rochester, Michigan. In this study, 138 morphologically distinct bacterial colonies from 9 iceberg lettuce samples were randomly picked and tested for antibiotic resistance. Among these isolates, the vast majority (86%) demonstrated resistance to cefotaxime, and among the resistant bacteria, the majority showed multiple drug resistance, particularly against cefotaxime, chloramphenicol, and tetracycline. Three bacterial isolates (2.17%) out of 138 were extended spectrum beta-lactamase (ESBL) producers. Two ESBL producers (T1 and T5) were identified as Klebsiella pneumoniae, an opportunistic pathogen with transferable sulfhydryl variable- (SHV-) and TEM-type ESBLs, respectively. The DNA sequence analysis of the bla SHV detected in K. pneumoniae isolate T1 revealed 99% relatedness to bla SHV genes found in clinical isolates. This implies that iceberg lettuce is a potential reservoir of newly emerging and evolving antibiotic resistant bacteria and its consumption poses serious threat to human health. PMID:26064922

  6. Water-Soluble alpha,beta-unsaturated aldehydes of cigarette smoke induce carbonylation of human serum albumin.

    PubMed

    Colombo, Graziano; Aldini, Giancarlo; Orioli, Marica; Giustarini, Daniela; Gornati, Rosalba; Rossi, Ranieri; Colombo, Roberto; Carini, Marina; Milzani, Aldo; Dalle-Donne, Isabella

    2010-03-01

    Cigarette smoking is a major risk factor for developing pulmonary and cardiovascular diseases as well as some forms of cancer. Understanding the mechanisms by which smoking contributes to disease remains a major research focus. Increased levels of carbonylated serum proteins are present in smokers; albumin is the major carbonylated protein in the bronchoalveolar lavage fluid of older smokers. We have investigated the susceptibility of human serum albumin (HSA) to alpha,beta-unsaturated aldehyde-induced carbonylation when exposed to whole-phase cigarette smoke extract (CSE). Fluorescence studies with fluorescent probes showed depletion of HSA Cys34 free thiol and marked decrease of free Lys residues. Spectrophotometric and immunochemical carbonyl assays after carbonyl derivatization with 2,4-dinitrophenylhydrazine revealed the formation of covalent carbonyl adducts. Nanoscale capillary liquid chromatography and electrospray tandem mass spectrometry analysis detected acrolein and crotonaldehyde Michael adducts at Cys34, Lys525, Lys351, and His39 at all the CSE concentrations tested. Lys541 and Lys545 were also found to form a Schiff base with acrolein. The carbonyl scavenger drugs, hydralazine and pyridoxamine, partially prevented CSE-induced HSA carbonylation. Carbonylation of HSA associated with cigarette smoking might result in modifications of its antioxidant properties and transport functions of both endogenous and exogenous compounds.

  7. Cloning of the beta 3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa.

    PubMed

    Pulkkinen, L; Gerecke, D R; Christiano, A M; Wagman, D W; Burgeson, R E; Uitto, J

    1995-01-01

    Laminin 5 consists of three polypeptides, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping lambda phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 bp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. PMID:7774918