Journal articles on the topic 'Alpha Transu'
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1
Pasenkiewicz-Gierula, M., and T. Róg. "Conformations, orientations and time scales characterising dimyristoylphosphatidylcholine bilayer membrane. Molecular dynamics simulation studies." Acta Biochimica Polonica 44, no. 3 (September 30, 1997): 607–24. http://dx.doi.org/10.18388/abp.1997_4409.
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The results of molecular dynamics simulation of fully hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase are presented. They show that the probability of a gauche conformation varies periodically along the chain with only a slight increase towards the end of the chain. However, the frequency of transition between conformations increases, due to a decrease in the lifetime of the trans conformation, along the chain. The average lifetimes for trans conformations are in the range of 1-2 x 10(-10) s and for gauche conformations in the range of 4-7 x 10(-11) s. The alpha-chain of the DMPC head group has mainly an extended conformation, due to predominantly trans conformation of alpha5 torsion. The rotational correlation time for the P-N vector is 3.7 ns. The C2-C1-O11-P fragment of the DMPC head group (theta1, alpha1, alpha2 torsions) is rigid while the P-O12-C11-C12 fragment (alpha3, alpha4, alpha5 torsions) is flexible. The lateral diffusion coefficient for DMPC self-diffusion in the membrane is 2 x 10(-7) cm2/s; the rate of transverse diffusion is the same. Large differences in the calculated rotational correlation times for the alpha-, beta-, gamma-chains and for the O21-C1 vector indicate that in the liquid-crystalline bilayer each segment of the DMPC molecule exhibits its own rotational freedom, in addition to its internal flexibility resulting from rotational isomerism. The results obtained in these calculations, although in general agreement with some experimental data, shed new light on the dynamical behaviour of phosphatidylcholine molecules in the bilayer membrane in the liquid-crystalline phase.
2
Hunter, Aaron. "When is the now in the here and there? Trans-diegetic music in Hal Ashby’s Coming Home." Alphaville: Journal of Film and Screen Media, no. 3 (August 8, 2012): 36–48. http://dx.doi.org/10.33178/alpha.3.03.
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While it would be a stretch to classify Hal Ashby as a postmodernist filmmaker (with that term’s many attendant ambiguities), his films of the 1970s regularly evince post-Classical stylistic and narrative strategies, including non-linear time structures, inter-textual self-references, open endings, and nuanced subversions of the fourth wall. Ashby’s most consistently playful approach to form comes by way of his integration and development of trans-diegetic musical sequences within his body of work. Music in Ashby films creates a lively sense of unpredictability, and each of his seven films of the 1970s employs this strategy at least once. Moreover, trans-diegetic music in Ashby’s films becomes a device that allows the director to elide moments in time. It functions as an editing tool, creating a bridge between often disparate events. However, it is also a narrative device that both compresses and stretches time, allowing for an on-screen confluence of events that at first appear to take place simultaneously or sequentially, but which actually occur over different moments or lengths of time. Yet while Ashby is not alone as a Hollywood director interested in exploring the formal possibilities that trans-diegesis might bring to his movies, film studies has begun only relatively recently to explore and analyse this technique. After briefly discussing the current critical discussion of trans-diegetic music and explicating patterns of its use in Ashby’s career, this paper explores an extended display of the strategy in the film Coming Home (1978). By interrogating its use as both narrative device and formal convention in this instance, the paper attempts both to understand trans-diegesis as a key component of Ashby’s filmmaking style and also to forge ahead in expanding the discussion of trans-diegesis within film studies.
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Igwe, Onyeka, and JD Stokely. "Hiraeth, or queering time in archives otherwise." Alphaville: Journal of Film and Screen Media, no. 16 (January 30, 2019): 9–23. http://dx.doi.org/10.33178/alpha.16.01.
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Archives are the physical manifestations of our collective understanding of history, a way of proving and so legitimising the existence of cultures, practices, and peoples. However, for queer and trans people of colour (QTPOC), entrance into the archive is not easily permitted; the truths of their lives have been, and are presently, excluded, claimed as contingent and/or rendered “folk”—lesser forms of knowledge. “Hiraeth” is a Welsh word that is difficult to translate into English. It speaks of a longing or homesickness for a place that is no longer, or never was. For QTPOC, the archive is this, a hiraeth space. We use “hiraeth” to describe the liminal space in which experiences of home, media practices, and a relationship to the archive can exist. As two Black queer artists who in their work have been exploring ways to implode the archive, in this article we look at how our practices can expand what the archive holds and further provide a space to render the untranslatable, the im/possible, as archive material. It is a strategy of both redefinition and defiance.
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Pos, Robert. "A Developmental Theory of Personality Producing Two Time Orientations." KronoScope 6, no. 1 (2006): 83–104. http://dx.doi.org/10.1163/156852406777505327.
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AbstractThis paper describes a theory of genetically determined personality development, including the development of two mutually exclusive time orientations: alphas preferentially relate to their present, often being inattentive to their past and future; betas preferentially relate to their future and past, and tend to be inattentive to their present. These time orientations or perspectives derive from two types of autobiographic memory materializing around age 6, which cause two types of personality. For example, the theory views alphas as extroverted and betas as introverted although there are significant differences between Jung's view of these traits and the theory's definitions. Beginning at birth, the amalgam of inborn motivational contexts induces a context-specific partitioning of reality. This is left intact when an alpha child's autobiographic memory, which is free of trans-contextual sequencing of memories, becomes active and the child's identity becomes therefore multi-focal (situational), as represented by Jung's personality model. By contrast, a beta child spontaneously produces a trans-contextual sequence of memories. The youngster's partitioned reality fuses, leading to a singular, monofocal identity, as represented by Freud's personality model. Self-reflection in adolescence makes both implicit identities self-conscious. The underlying genes possibly belong to the X-chromosome, the alpha gene being dominant and the beta gene recessive.
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Wu, Q., M. I. Dawson, Y. Zheng, P. D. Hobbs, A. Agadir, L. Jong, Y. Li, R. Liu, B. Lin, and X. K. Zhang. "Inhibition of trans-retinoic acid-resistant human breast cancer cell growth by retinoid X receptor-selective retinoids." Molecular and Cellular Biology 17, no. 11 (November 1997): 6598–608. http://dx.doi.org/10.1128/mcb.17.11.6598.
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All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.
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Atweh, G. F., J. M. Liu, H. E. Brickner, and X. X. Zhu. "A silencer element from the alpha-globin gene inhibits expression of beta-like genes." Molecular and Cellular Biology 8, no. 11 (November 1988): 5047–51. http://dx.doi.org/10.1128/mcb.8.11.5047-5051.1988.
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We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.
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Atweh, G. F., J. M. Liu, H. E. Brickner, and X. X. Zhu. "A silencer element from the alpha-globin gene inhibits expression of beta-like genes." Molecular and Cellular Biology 8, no. 11 (November 1988): 5047–51. http://dx.doi.org/10.1128/mcb.8.11.5047.
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We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.
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Knight, K. L., M. Kingzette, M. A. Crane, and S. K. Zhai. "Transchromosomally derived Ig heavy chains." Journal of Immunology 155, no. 2 (July 15, 1995): 684–91. http://dx.doi.org/10.4049/jimmunol.155.2.684.
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Abstract During an immune response, activated B cells undergo isotype switching and begin to express isotypes other than IgM and IgD. Isotype switching occurs when downstream C gamma, C alpha, or C epsilon genes are rearranged into the S mu chromosomal region, resulting in the deletion of the region in between. These rearrangements usually occur in cis, i.e., intrachromosomally. In previous studies, we analyzed allotypic specificities of rabbit secretory IgA and identified a substantial number of IgA heavy chains with VH and C alpha allotypes that were encoded by VH and C alpha genes in trans. In those studies, however, we could not determine whether the trans association of VH and C alpha occurred during VDJ gene rearrangement or during isotype switching. Here, we cloned rabbit cDNA which encodes these trans IgA heavy chains and determined the chromosomal origin of the VH, JH, and C alpha regions. To determine whether the trans association occurred during VDJ gene rearrangement, we analyzed the nucleotide polymorphism of the JH region and the VH allotype encoded by the cDNA. We found that the VH and JH genes used in the VDJ gene rearrangements were from the same chromosome, indicating that the VH, D, and JH gene rearrangements occurred in cis. Furthermore, we analyzed the DNA polymorphisms of JH and C alpha and showed that the VDJ and C alpha genes encoding the trans IgA molecules were derived from different parental chromosomes. We suggest that the trans association occurred during isotype switching. This study shows that VH and CH can associate transchromosomally as part of a normal immune response.
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Worley, D. S., J. M. Pisano, E. D. Choi, L. Walus, C. A. Hession, R. L. Cate, M. Sanicola, and S. J. Birren. "Developmental regulation of GDNF response and receptor expression in the enteric nervous system." Development 127, no. 20 (October 15, 2000): 4383–93. http://dx.doi.org/10.1242/dev.127.20.4383.
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The development of the enteric nervous system is dependent upon the actions of glial cell line-derived neurotrophic factor (GDNF) on neural crest-derived precursor cells in the embryonic gut. GDNF treatment of cultured enteric precursor cells leads to an increase in the number of neurons that develop and/or survive. Here we demonstrate that, although GDNF promoted an increase in neuron number at all embryonic ages examined, there was a developmental shift from a mitogenic to a trophic response by the developing enteric neurons. The timing of this shift corresponded to developmental changes in gut expression of GFR alpha-1, a co-receptor in the GDNF-Ret signaling complex. GFR alpha-1 was broadly expressed in the gut at early developmental stages, at which times soluble GFR alpha-1 was released into the medium by cultured gut cells. At later times, GFR alpha-1 became restricted to neural crest-derived cells. GFR alpha-1 could participate in GDNF signaling when expressed in cis on the surface of enteric precursor cells, or as a soluble protein. The GDNF-mediated response was greater when cell surface, compared with soluble, GFR alpha-1 was present, with the maximal response seen the presence of both cis and trans forms of GFR alpha-1. In addition to contributing to GDNF signaling, cell-surface GFR alpha-1 modulated the specificity of interactions between GDNF and soluble GFR alphas. These experiments demonstrate that complex, developmentally regulated, signaling interactions contribute to the GDNF-dependent development of enteric neurons.
10
Jameson, J. L., P. J. Deutsch, G. D. Gallagher, R. C. Jaffe, and J. F. Habener. "trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene." Molecular and Cellular Biology 7, no. 9 (September 1987): 3032–40. http://dx.doi.org/10.1128/mcb.7.9.3032-3040.1987.
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The alpha subunit of the placental hormone chorionic gonadotropin is regulated by cyclic AMP (cAMP) at the transcriptional level. A cAMP-responsive fusion gene (alpha-CAT) containing 1.5 kilobases of the alpha gene 5'-flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene was used as a transcriptional reporter in competition assays in transfected JEG-3 choriocarcinoma cells. Expression of the alpha-CAT fusion gene increased linearly with increasing amounts of transfected plasmid and was maximal at the same amount of alpha-CAT DNA (2 micrograms) with or without cAMP treatment. Various amounts of different competitor DNA sequences were cotransfected with the alpha-CAT reporter plasmid to examine the interactions of intracellular trans-acting factors with the regulatory elements of the alpha gene promoter. An 800-base-pair fragment of alpha gene 5'-flanking sequence inhibited both basal and cAMP-stimulated transcription of the alpha-CAT reporter plasmid in a dose-dependent manner, indicative of interactions with one or more trans-acting factors that activate alpha gene expression. The alpha gene sequences that interact with intracellular regulatory factors were defined by using several discrete regions of the 5'-flanking sequence as competitors for alpha-CAT expression. A proximal promoter sequence (-99 to +44) containing the CCAAT box, TATA box, and transcriptional initiation site was a relatively ineffective competitor of alpha-CAT transcription. In contrast, an upstream sequence between -236 and -100 was an effective competitor for transcriptional activators of alpha-CAT expression. Competition for alpha-CAT expression by this regulatory sequence did not require cis interactions with downstream promoter elements and was equally effective with or without cAMP treatment. An 18-base-pair repeated sequence within this region of the alpha gene (-146 to -111) greatly enhanced both basal gene expression and cAMP responsivity and also competed for limiting cellular transcription factors. These findings suggest that JEG-3 cells contain trans-acting factors that interact with a cAMP response element to activate alpha gene transcription. The chorionic gonadotropin beta gene 5'-flanking sequence also competed for alpha-CAT expression, suggesting that a common trans-acting factor is shared by the regulatory sequences of the alpha and beta genes.
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Jameson, J. L., P. J. Deutsch, G. D. Gallagher, R. C. Jaffe, and J. F. Habener. "trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene." Molecular and Cellular Biology 7, no. 9 (September 1987): 3032–40. http://dx.doi.org/10.1128/mcb.7.9.3032.
Full textAbstract:
The alpha subunit of the placental hormone chorionic gonadotropin is regulated by cyclic AMP (cAMP) at the transcriptional level. A cAMP-responsive fusion gene (alpha-CAT) containing 1.5 kilobases of the alpha gene 5'-flanking sequence linked to the chloramphenicol acetyltransferase (CAT) gene was used as a transcriptional reporter in competition assays in transfected JEG-3 choriocarcinoma cells. Expression of the alpha-CAT fusion gene increased linearly with increasing amounts of transfected plasmid and was maximal at the same amount of alpha-CAT DNA (2 micrograms) with or without cAMP treatment. Various amounts of different competitor DNA sequences were cotransfected with the alpha-CAT reporter plasmid to examine the interactions of intracellular trans-acting factors with the regulatory elements of the alpha gene promoter. An 800-base-pair fragment of alpha gene 5'-flanking sequence inhibited both basal and cAMP-stimulated transcription of the alpha-CAT reporter plasmid in a dose-dependent manner, indicative of interactions with one or more trans-acting factors that activate alpha gene expression. The alpha gene sequences that interact with intracellular regulatory factors were defined by using several discrete regions of the 5'-flanking sequence as competitors for alpha-CAT expression. A proximal promoter sequence (-99 to +44) containing the CCAAT box, TATA box, and transcriptional initiation site was a relatively ineffective competitor of alpha-CAT transcription. In contrast, an upstream sequence between -236 and -100 was an effective competitor for transcriptional activators of alpha-CAT expression. Competition for alpha-CAT expression by this regulatory sequence did not require cis interactions with downstream promoter elements and was equally effective with or without cAMP treatment. An 18-base-pair repeated sequence within this region of the alpha gene (-146 to -111) greatly enhanced both basal gene expression and cAMP responsivity and also competed for limiting cellular transcription factors. These findings suggest that JEG-3 cells contain trans-acting factors that interact with a cAMP response element to activate alpha gene transcription. The chorionic gonadotropin beta gene 5'-flanking sequence also competed for alpha-CAT expression, suggesting that a common trans-acting factor is shared by the regulatory sequences of the alpha and beta genes.
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Vilms Pedersen, Simon, Ester Scotto di Perta, Sasha D. Hafner, Andreas S. Pacholski, and Sven G. Sommer. "Evaluation of a Simple, Small-Plot Meteorological Technique for Measurement of Ammonia Emission: Feasibility, Costs, and Recommendations." Transactions of the ASABE 61, no. 1 (2018): 103–15. http://dx.doi.org/10.13031/trans.12445.
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Abstract. Ammonia emission reduces the reliability and nitrogen (N) fertilizer efficiency of animal manure and mineral fertilizers applied to fields. The loss of ammonia to the atmosphere is frequently compensated for by costly over-application of N fertilizers. New technologies to reduce ammonia emission are regularly developed, and their efficacy needs to be tested using accurate methods. To date, a major obstacle to many available emission measurement techniques is the requirement of large plot sizes of homogeneous surface characteristics, which particularly is a challenge to the number of plot-level replicates that can be carried out on a field providing uniform surface characteristics throughout. The objectives of this research were to test three different methods for measuring NH3 flux when applied to small plots (<315 m2) by comparison with conventional micrometeorological methods and to determine the labor intensity and expenses related to the respective methods in their entirety. The integrated horizontal flux (IHF) method and the ZINST method were used with passive flux Leuning samplers as micrometeorological reference methods. As examples of conventional small-plot emission measurement techniques, wind tunnels measuring gas-phase ammonia using ALPHA passive diffusion samplers and a flux chamber method using Dräger tubes for measurements of ammonia concentration (DTM) were used. As an inexpensive alternative small-plot method, we studied the feasibility of applying ALPHA passive diffusion samplers and battery-driven cup anemometers at ZINST height on small source areas (<315 m2), coupled with a backward Lagrangian stochastic (bLS) dispersion model to calculate emission fluxes (referred to as the AbLS method). When exposure duration was appropriate and weather conditions were not extreme, tests showed no significant difference in NH3 emission fluxes measured with AbLS, compared to those obtained with IHF and ZINST using Leuning samplers. However, the AbLS method did not give reliable emission measurements in periods with high wind speeds and heavy rain. It was also shown that the AbLS method provided valid results when reducing the plot radius from the standard 20 m to 10 m, or even 5 m, provided that the ALPHA samplers were exposed for at least 5 or 6 h. Emission from 200 kg urea-N ha-1 was between 20 and 30 kg N ha-1 in the two trials. The cost for one study running for one week using the ZINST or bLS methodology, including equipment for four plots and eight measurement intervals, was $2785 if horizontal fluxes were measured using the ALPHA samplers, compared to $12,301 using the Leuning samplers and $13,928 using gas washing bottles. Using the DTM flux chamber method once is a little more expensive than using the AbLS method, but less expensive if the cost of purchasing the equipment is distributed over five studies in five years. Using wind tunnels is as costly as measuring emissions with the Leuning samplers or gas washing bottles using the bLS or ZINST method. Keywords: ALPHA samplers, Ammonia emission, AbLS, bLS method, DTM method, IHF method, Labor cost, Passive ammonia samplers, Wind tunnels.
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Sowell, A. L., D. L. Huff, P. R. Yeager, S. P. Caudill, and E. W. Gunter. "Retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and four retinyl esters in serum determined simultaneously by reversed-phase HPLC with multiwavelength detection." Clinical Chemistry 40, no. 3 (March 1, 1994): 411–16. http://dx.doi.org/10.1093/clinchem/40.3.411.
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Abstract We describe the use of HPLC with multiwavelength detection to measure retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, beta-carotene, and the linoleate, oleate, palmitate, and stearate esters of retinol in a single 200-microL serum sample. The method is sensitive enough to detect individual retinyl esters in fasting serum from a nonhyperlipidemic population and requires only 12 min for each sample. Serum concentration ranges and means are reported for retinol, alpha-tocopherol, lutein/zeaxanthin, beta-cryptoxanthin, lycopene, alpha-carotene, trans-beta-carotene, and the sum of the retinyl esters from serum analyses of 3480 participants from several different studies.
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Díaz-González, F., J. Forsyth, B. Steiner, and M. H. Ginsberg. "Trans-dominant inhibition of integrin function." Molecular Biology of the Cell 7, no. 12 (December 1996): 1939–51. http://dx.doi.org/10.1091/mbc.7.12.1939.
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Occupancy of integrin adhesion receptors can alter the functions of other integrins and cause partition of the ligand-occupied integrin into focal adhesions. Ligand binding also changes the conformation of integrin extracellular domains. To explore the relationship between ligand-induced conformational change and integrin signaling, we examined the effect of ligands specific for integrin alpha IIb beta 3 on the functions of target integrins alpha 5 beta 1 and alpha 2 beta 1. We report that binding of integrin-specific ligands to a suppressive integrin can inhibit the function of other target integrins (trans-dominant inhibition). Trans-dominant inhibition is due to a blockade of integrin signaling. Furthermore, this inhibition involves both a conformational change in the extracellular domain and the presence of the beta cytoplasmic tail in the suppressive integrin. Similarly, ligand-induced recruitment of alpha IIb beta 3 to focal adhesions also involves a conformational rearrangement of its extracellular domain. These findings imply that the ligand-induced conformational changes can propagate from an integrin's extracellular to its intracellular face. Trans-dominant inhibition by integrin ligands may coordinate integrin signaling and can lead to unexpected biological effects of integrin-specific inhibitors.
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Hiscott, J., A. Wong, D. Alper, and S. Xanthoudakis. "trans activation of type 1 interferon promoters by simian virus 40 T antigen." Molecular and Cellular Biology 8, no. 8 (August 1988): 3397–405. http://dx.doi.org/10.1128/mcb.8.8.3397-3405.1988.
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A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.
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Hiscott, J., A. Wong, D. Alper, and S. Xanthoudakis. "trans activation of type 1 interferon promoters by simian virus 40 T antigen." Molecular and Cellular Biology 8, no. 8 (August 1988): 3397–405. http://dx.doi.org/10.1128/mcb.8.8.3397.
Full textAbstract:
A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.
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Miller, WH Jr, K. Levine, A. DeBlasio, SR Frankel, E. Dmitrovsky, and RP Jr Warrell. "Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RAR-alpha fusion mRNA." Blood 82, no. 6 (September 15, 1993): 1689–94. http://dx.doi.org/10.1182/blood.v82.6.1689.1689.
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Abstract The characteristic reciprocal translocation t(15;17) of acute promyelocytic leukemia (APL) disrupts the PML gene on chromosome 15 and the retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17. PML/RAR-alpha fusion mRNAs are then transcribed and can be detected by a newly described reverse transcription polymerase chain reaction (RT- PCR) assay. Using RT followed by nested PCR amplification for PML/RAR- alpha, we serially evaluated bone marrow aspirates from patients with APL who were treated with all-trans retinoic acid (RA) for induction, followed by all-trans RA as maintenance or cytotoxic drugs as consolidation. At diagnosis, PML/RAR-alpha mRNA was detected in all patients. After initial therapy with all-trans RA, the RT-PCR assay remained positive after induction of complete remission in 31 of 32 evaluable patients. Maintenance treatment by all-trans RA alone was associated with persistent assay positivity and subsequent clinical relapse in 13 of 13 patients. By contrast, the test became negative in 19 of 20 newly diagnosed patients who received consolidation chemotherapy; the 1 patient who remained positive relapsed at 12 months. Three of the 19 assay-negative patients later converted to positive and subsequently relapsed; the remaining 16 patients have remained RT-PCR negative in sustained first remission, with a median follow-up duration that exceeds 24 months (range, 12+ to 34+ months). Despite induction of complete remission in a high proportion of patients, all-trans RA rarely eradicates molecular evidence of disease in patients with APL; however, subsequent treatment with cytotoxic chemotherapy frequently converts the RT-PCR assay for PML/RAR-alpha to negative. Serial negative tests are associated with prolonged disease- free survival, whereas persistence of a positive test after treatment is highly correlated with subsequent relapse. This test identifies patients in remission at high risk for relapse who may benefit from additional antileukemic therapy.
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Miller, WH Jr, K. Levine, A. DeBlasio, SR Frankel, E. Dmitrovsky, and RP Jr Warrell. "Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RAR-alpha fusion mRNA." Blood 82, no. 6 (September 15, 1993): 1689–94. http://dx.doi.org/10.1182/blood.v82.6.1689.bloodjournal8261689.
Full textAbstract:
The characteristic reciprocal translocation t(15;17) of acute promyelocytic leukemia (APL) disrupts the PML gene on chromosome 15 and the retinoic acid receptor-alpha (RAR-alpha) gene on chromosome 17. PML/RAR-alpha fusion mRNAs are then transcribed and can be detected by a newly described reverse transcription polymerase chain reaction (RT- PCR) assay. Using RT followed by nested PCR amplification for PML/RAR- alpha, we serially evaluated bone marrow aspirates from patients with APL who were treated with all-trans retinoic acid (RA) for induction, followed by all-trans RA as maintenance or cytotoxic drugs as consolidation. At diagnosis, PML/RAR-alpha mRNA was detected in all patients. After initial therapy with all-trans RA, the RT-PCR assay remained positive after induction of complete remission in 31 of 32 evaluable patients. Maintenance treatment by all-trans RA alone was associated with persistent assay positivity and subsequent clinical relapse in 13 of 13 patients. By contrast, the test became negative in 19 of 20 newly diagnosed patients who received consolidation chemotherapy; the 1 patient who remained positive relapsed at 12 months. Three of the 19 assay-negative patients later converted to positive and subsequently relapsed; the remaining 16 patients have remained RT-PCR negative in sustained first remission, with a median follow-up duration that exceeds 24 months (range, 12+ to 34+ months). Despite induction of complete remission in a high proportion of patients, all-trans RA rarely eradicates molecular evidence of disease in patients with APL; however, subsequent treatment with cytotoxic chemotherapy frequently converts the RT-PCR assay for PML/RAR-alpha to negative. Serial negative tests are associated with prolonged disease- free survival, whereas persistence of a positive test after treatment is highly correlated with subsequent relapse. This test identifies patients in remission at high risk for relapse who may benefit from additional antileukemic therapy.
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Martín, M., J. M. Sanz, M. Ros, and A. Cubero. "Metabotropic glutamate receptor analogues inhibit p[NH]ppG-stimulated phospholipase C activity in bovine brain coated vesicles: involvement of a pertussis toxin-sensitive G-protein." Biochemical Journal 307, no. 3 (May 1, 1995): 851–57. http://dx.doi.org/10.1042/bj3070851.
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Guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG)-stimulated phospholipase C (PLC) activity in bovine brain coated vesicles is inhibited by glutamate agonists. In the present study we show that quisqualic acid (QA), (+/-)-trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD), glutamic acid and ibotenic acid inhibited p[NH]ppG-stimulated PLC by 44, 41, 36 and 25% respectively. Carbachol also produced an inhibition of p[NH]ppG-stimulated PLC by 45%. The inhibition caused by trans-ACPD and QA was dose-dependent. DL-2-Amino-3-phosphonopropionic acid and (RS)-alpha-methyl-4-carboxyphenylglycine, specific antagonists of metabotropic glutamate receptors (mGluRs), abolished these inhibitory effects. trans-ACPD inhibition of p[NH]ppG-stimulated PLC was also observed in the presence of ionotropic glutamate receptor antagonists. When carbachol and QA or trans-ACPD were combined, additive inhibitory effects were observed. Preincubation of bovine brain coated vesicles with pertussis toxin abolished the inhibitory effects of mGluR analogues and carbachol on p[NH]ppG-stimulated PLC activity. The presence of Gs alpha and pertussis toxin substrates, Gi alpha and Go alpha subunits as well as PLC beta 1 in bovine brain coated vesicles has been confirmed by immunoblot. These results support the coupling of mGluRs to a PLC in an inhibitory manner through a pertussis toxin-sensitive G-protein in bovine brain coated vesicles.
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JURKOVÁ, Marie, Tomáš HORÁK, Jiří ČULÍK, Pavel ČEJKA, and Vladimír KELLNER. "Simultaneous determination of iso-alpha acids in their cis- and transforms and tetrahydroiso-alpha acids." Kvasny Prumysl 56, no. 3 (March 1, 2010): 163–66. http://dx.doi.org/10.18832/kp2010023.
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Kurimoto, I., and J. W. Streilein. "cis-urocanic acid suppression of contact hypersensitivity induction is mediated via tumor necrosis factor-alpha." Journal of Immunology 148, no. 10 (May 15, 1992): 3072–78. http://dx.doi.org/10.4049/jimmunol.148.10.3072.
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Abstract Ultraviolet B (UVB) light impairs the induction of contact hypersensitivity to epicutaneously applied haptens in certain strains of mice by a genetically determined mechanism that depends upon the participation of TNF-alpha. Because the superficial epidermis contains large amounts of trans-urocanic acid (trans-UCA), because exposure to UVB radiation converts this compound to cis-UCA, and because cis-UCA has been reported to be immunosuppressive, we have examined the possibility that the TNF-alpha-dependent effects of UVB on contact hypersensitivity induction in mice are mediated via conversion of trans- to cis-UCA. By injecting cis-UCA intradermally before application of dinitrofluorobenzene, by treating cis-UCA-injected mice systemically with neutralizing anti-TNF-alpha antibodies, and by comparing the consequences of these maneuvers in UVB-susceptible and UVB-resistant strains of mice, we have determined a) that cis-UCA can impair the induction of contact hypersensitivity in a manner similar to UVB radiation, and that the impairment is dependent upon TNF-alpha; b) that cis-UCA altered the morphology of epidermal Langerhans cells in a manner similar to UVB radiation, and that the alteration was dependent, in part, upon TNF-alpha; and c) that the inhibitory effects of cis-UCA on induction of contact hypersensitivity and the histologic effects of this compound on epidermal Langerhans cells appear to be influenced by alleles at the Tnf alpha and Lps loci. Based on these findings we propose that UVB radiation impairs the induction of contact hypersensitivity in mice by converting trans-urocanic acid to cis-UCA within the epidermis; cis-UCA in turn causes the local release of TNF-alpha, which thwarts sensitization by its ability to trap epidermal Langerhans cells transiently within the epidermis, and thereby prevents the immunogenic signal from reaching the draining lymph node where activation of unprimed, Ag-specific T cells must occur.
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Stancheva, Mona, Diana A. Dobreva, and Bistra Galunska. "Retinol, cholecalciferol and alpha-tocopherol contents of Bulgarian Black Sea fish species." Analele Universitatii "Ovidius" Constanta - Seria Chimie 23, no. 1 (June 1, 2012): 31–34. http://dx.doi.org/10.2478/v10310-012-0004-7.
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AbstractThe aim of the present study is to determine and to compare the content of retinol, cholecalciferol and alpha-tocopherol in edible tissue of two Black sea fishes - Garfish (Belone belone) and Turbot (Psetta maxima). All-trans-retinol (vitamin A), cholecalciferol (vitamin D3) and alpha-tocopherol (vitamin E) were analyzed simultaneously using HPLC/UV/FL system (Thermo Scientific Spectra SYSTEM) equipped with RP analytical column. The mobile phase was composed of 97:3 = MeOH:H2O. Retinol and cholecalciferol were monitored by UV detection at lmax = 325 nm and lmax = 265 nm, respectively. Alpha-tocopherol was detected by fluorescence at lex=288 nm and lem=332 nm. The sample preparation procedure includes alkaline saponification, followed by liquid-liquid extraction. Quantities of all-trans-retinol and cholecalciferol were higher in garfish tissues while alpha-tocopherol content in turbot showed seven times higher values.
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Cleanthous, Galatia, and Athanasios G. Georgiadis. "Mixed-norm $\alpha $-modulation spaces." Transactions of the American Mathematical Society 373, no. 5 (February 19, 2020): 3323–56. http://dx.doi.org/10.1090/tran/8023.
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ZHOU, Kaijie, Huali WANG, Peipei CAO, and Zhangkai LUO. "Performance Evaluation for Chirp-BOK Modulation Scheme under Alpha-Stable Noise." IEICE Transactions on Fundamentals of Electronics, Communications and Computer Sciences E103.A, no. 4 (April 1, 2020): 723–27. http://dx.doi.org/10.1587/transfun.2019eal2134.
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Rabouille, C., N. Hui, F. Hunte, R. Kieckbusch, E. G. Berger, G. Warren, and T. Nilsson. "Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides." Journal of Cell Science 108, no. 4 (April 1, 1995): 1617–27. http://dx.doi.org/10.1242/jcs.108.4.1617.
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The distribution of beta 1,2 N-acetylglucosaminyltransferase I (NAGT I), alpha 1,3-1,6 mannosidase II (Mann II), beta 1,4 galactosyltransferase (GalT), alpha 2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.
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Davodeau, F., M. A. Peyrat, J. Gaschet, M. M. Hallet, F. Triebel, H. Vié, D. Kabelitz, and M. Bonneville. "Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes." Journal of Experimental Medicine 180, no. 5 (November 1, 1994): 1685–91. http://dx.doi.org/10.1084/jem.180.5.1685.
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Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire.
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MacCrehan, W. A., and E. Schönberger. "Determination of retinol, alpha-tocopherol, and beta-carotene in serum by liquid chromatography with absorbance and electrochemical detection." Clinical Chemistry 33, no. 9 (September 1, 1987): 1585–92. http://dx.doi.org/10.1093/clinchem/33.9.1585.
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Abstract We describe a method for the determination of retinol, alpha-tocopherol, and beta-carotene in serum, using a liquid-chromatographic separation with wavelength-programmed ultraviolet/visible absorbance and amperometric electrochemical detection with a glassy carbon electrode. After protein denaturation and addition of an internal standard, tocol, 250-microL samples are twice extracted with hexane. The reversed-phase, gradient-elution chromatographic separation provides baseline resolution of: the all-trans isomer of retinol from the cis isomers, alpha- from gamma-tocopherol, and all-trans-beta-carotene from alpha-carotene and from cis-beta-carotene isomers. The linearity of response and the detection limits for the two detectors for the three analytes are measured. A comparison of the values obtained for serum extracts shows good agreement between the absorbance and electrochemical detectors.
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Lundin, K. E., L. M. Sollid, E. Qvigstad, G. Markussen, H. A. Gjertsen, J. Ek, and E. Thorsby. "T lymphocyte recognition of a celiac disease-associated cis- or trans-encoded HLA-DQ alpha/beta-heterodimer." Journal of Immunology 145, no. 1 (July 1, 1990): 136–39. http://dx.doi.org/10.4049/jimmunol.145.1.136.
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Abstract The HLA-associated susceptibility to develop celiac disease may to a large extent be attributed to the combination of particular DQA1 and DQB1 genes, i.e., the DQA1*0501 and DBQB1*0201 alleles, located either in cis position (on the DR3DQw2 haplotype) or in trans position (DR5DQw7/DR7DQw2 heterozygous individuals). We report three alloreactive T lymphocyte clones that recognize an HLA-DQ alpha/beta heterodimer both when the DQA1*0501 and DQB1*0201 alleles are located in cis or in trans position. Thus, the celiac disease associated DQA1 and DQB1 genes encode a functionally expressed DQ alpha/beta heterodimer.
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Perrotta, S., E. Miraglia del Giudice, N. Alloisio, G. Sciarratta, L. Pinto, J. Delaunay, S. Cutillo, and A. Iolascon. "Mild elliptocytosis associated with the alpha 34 Arg-->Trp mutation in spectrin Genova (alpha I/74)." Blood 83, no. 11 (June 1, 1994): 3346–49. http://dx.doi.org/10.1182/blood.v83.11.3346.3346.
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Abstract We report a new mutation responsible for nonhemolytic hereditary elliptocytosis (HE). The proband displayed an impaired spectrin self- association and an increase of the alpha I 74-kD fragment (alpha I/74 abnormality). The responsible mutation occurred in exon 2 of spectrin alpha-gene: alpha 34 Arg-->Trp (CGG-->TGG), defining spectrin Genova. In Trans to allele alpha Genova, the proband disclosed allele alpha LELY, a common low-expression allele of spectrin alpha-gene. It was recognized through particular peptide maps as well as characteristic mutations in exon 40 and intron 45, respectively. The father, who carried allele alpha Genova, but not allele alpha LELY, had a milder presentation. The sensitization of allele alpha Genova by allele alpha LELY was noticeable in the proband as compared with his father. Nevertheless, it was not as sharp as that observed with many other alpha I/74 HE alleles. Therefore, each alpha I/74 HE allele has a distinct intrinsic severity.
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Perrotta, S., E. Miraglia del Giudice, N. Alloisio, G. Sciarratta, L. Pinto, J. Delaunay, S. Cutillo, and A. Iolascon. "Mild elliptocytosis associated with the alpha 34 Arg-->Trp mutation in spectrin Genova (alpha I/74)." Blood 83, no. 11 (June 1, 1994): 3346–49. http://dx.doi.org/10.1182/blood.v83.11.3346.bloodjournal83113346.
Full textAbstract:
We report a new mutation responsible for nonhemolytic hereditary elliptocytosis (HE). The proband displayed an impaired spectrin self- association and an increase of the alpha I 74-kD fragment (alpha I/74 abnormality). The responsible mutation occurred in exon 2 of spectrin alpha-gene: alpha 34 Arg-->Trp (CGG-->TGG), defining spectrin Genova. In Trans to allele alpha Genova, the proband disclosed allele alpha LELY, a common low-expression allele of spectrin alpha-gene. It was recognized through particular peptide maps as well as characteristic mutations in exon 40 and intron 45, respectively. The father, who carried allele alpha Genova, but not allele alpha LELY, had a milder presentation. The sensitization of allele alpha Genova by allele alpha LELY was noticeable in the proband as compared with his father. Nevertheless, it was not as sharp as that observed with many other alpha I/74 HE alleles. Therefore, each alpha I/74 HE allele has a distinct intrinsic severity.
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Kwok, W. W., P. Thurtle, and G. T. Nepom. "A genetically controlled pairing anomaly between HLA-DQ alpha and HLA-DQ beta chains." Journal of Immunology 143, no. 11 (December 1, 1989): 3598–601. http://dx.doi.org/10.4049/jimmunol.143.11.3598.
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Abstract The HLA-DQ region contains highly polymorphic alpha and beta loci, so that a diverse set of cis- and trans-associated class II alpha/beta dimers are potentially generated in heterozygous individuals. To evaluate the extent of this predicted diversity, DQ2 beta or DQ3.2 beta cDNA were introduced into a panel of homozygous B cell lines that expressed different DQ alpha alleles. Restricted patterns of alpha/beta pairing were observed in which DQ2 beta and DQ3 beta molecules were unable to pair efficiently with DQ1 alpha chains. This pairing anomaly may contribute to altered class II phenotypes in heterozygous individuals, and is reflected in the absence of either DQ1 alpha, DQ2 beta or DQ1 alpha, DQ3 beta haplotypes in the known human gene pool.
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Lezon-Geyda, Kimberly, Yelena Maksimova, Vincent P. Schulz, Bernard G. Forget, and Patrick G. Gallagher. "Severe Alpha-Spectrin Linked Recessive Hereditary Spherocytosis." Blood 124, no. 21 (December 6, 2014): 4011. http://dx.doi.org/10.1182/blood.v124.21.4011.4011.
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Abstract Approximately one quarter of cases of hereditary spherocytosis exhibit autosomal recessive inheritance. Patients with recessive hereditary spherocytosis (rHS) are more severely affected than patients with typical, dominant HS. Many rHS patients present in infancy with life-threatening hemolytic anemia; some are transfusion-dependent (TD). Erythrocytes from most rHS patients are spectrin deficient, leading to destabilization of the lipid bilayer. In most cases, the genetic defects in the SPTA1 gene leading to rHS are unknown. We studied 19 families with rHS suspected by clinical and laboratory data including 5 of the original rHS kindreds described by Agre et al. Probands from 9 of the 19 families were severely affected, demonstrating life-long transfusion dependence. After obtaining a genetic diagnosis, two probands were splenectomized and became transfusion independent. In the remaining 10 families, most patients had a history of receiving blood transfusions, particularly during infancy. Most underwent early splenectomy that ameliorated but did not cure their anemia. Hematocrits of affected patients postsplenectomy ranged from 21-51%. In untransfused patients, biochemical analyses included SDS-PAGE of erythrocyte membrane proteins. Quantitative analyses of spectrin content, measured by spectrin/band 3 ratios, demonstrated spectrin deficiency from 43-78% in rHS erythrocyte membranes. Genetic studies included Sanger sequencing (6 families) and/or whole exome analyses (WES, 13 families). Genomic DNA was amplified using primers flanking the 52 coding exons and promoter region of the SPTA1 gene, followed by Sanger sequencing analysis of the amplicons. Alternatively, genomic DNA was subjected to WES via targeted exon capture followed by ultrahigh throughput sequencing of captured DNA. Sequencing data were mapped and variants called by the GATK algorithm. Variants were filtered then further assessed via conservation and mutation prediction programs. Numerous unique null SPTA1 alleles were found. Two TD patients had deleterious mutations in both SPTA1 alleles; one with nonsense mutations in trans died of liver failure associated with transfusion-related iron overload, the other with nonsense and splicing mutations in trans remains transfusion dependent. Additional null alleles included 6 nonsense, 4 splicing, and 3 frameshift mutations. These null alleles were frequently in trans to novel missense mutations. Eight unique missense mutations (in spectrin repeats 2, 4 and 14) were identified in conserved residues and all were predicted to be deleterious by mutation prediction algorithms. Two missense mutations were found in multiple families (repeat 14 in 4 families, repeat 2 in 2 families). Two TD patients were homozygous for missense mutations located in critical residues of the alpha-beta spectrin self-association domain. One of these patients had a sibling homozygous for the same mutation die in the perinatal period due to complications of anemia. The repeat 2 mutation was found in the heterozygous state in 3 other rHS kindreds in trans to 3 different null alleles. Probands from two kindreds suffered from homozygous missense mutations in conserved residues that were predicted to be deleterious by mutation prediction algorithms (repeat 2 and repeat 14). In 7 alpha-spectrin linked rHS probands, only a single deleterious allele was identified. Erythrocyte membranes from 6 of these patients (the seventh was TD) exhibited severe spectrin deficiency, indicating that another mutation associated with a production-defective SPTA1 allele was present in trans. In summary, 27 novel mutations were identified on 38 SPTA1 alleles. The data demonstrate how the synthesis of complementary lines of investigation, i.e. clinical, laboratory, biochemical and genetic data, can be leveraged to define and advance our understanding of inherited hematologic disease. They also demonstrate the significant genetic heterogeneity in hereditary spherocytosis and support the use of genomic strategies for diagnosis, particularly in severe cases, allowing design of appropriate therapy. Disclosures No relevant conflicts of interest to declare.
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Watanabe, H., I. Nakanishi, K. Yamashita, T. Hayakawa, and Y. Okada. "Matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase) from U937 monoblastoid cells: correlation with cellular invasion." Journal of Cell Science 104, no. 4 (April 1, 1993): 991–99. http://dx.doi.org/10.1242/jcs.104.4.991.
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The role of matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase/type IV collagenase) in invasion of mononuclear phagocytes was studied with U937 monoblastoid cells. 12-o-tetradecanoyl 13-phorbol acetate (TPA) differentiated them to macrophage-like cells with induction of MMP-9, and tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) stimulated the production of MMP-9 by TPA-treated cells. TNF alpha also induced the production of MMP-9 by TPA-untreated U937 cells without morphological differentiation. Other agents including dimethyl sulfoxide (DMSO), all-trans-retinoic acid (all-trans-RA), platelet-derived growth factor and 3′;5′-cyclic monophosphate had no effects on MMP-9 production by TPA-treated or -untreated cells, but all-trans-RA and DMSO did have a morphological effect on the differentiation of the cells. These data suggest that MMP-9 production by U937 cells is regulated by a mechanism independent of the differentiation to macrophage-like cells. MMP-9 was purified to homogeneity as an inactive zymogen with M(r) 92,000 (proMMP-9) from TPA-differentiated U937 cells treated with TNF alpha. ProMMP-9 was activated by p-aminophenylmercuric acetate (APMA) generating an active species of M(r) 67,000. Trypsin and cathepsin G also attained activation of the zymogen to its full activity obtained by APMA activation, but plasmin, leukocyte elastase, thrombin and plasma kallikrein had no ability to activate it. APMA-activated MMP-9 degraded type I gelatin readily and cleaved native collagen types III, IV and V. Invasion assays using reconstituted basement membrane coupled with a type IV collagenolysis assay showed good correlations between invasiveness, type IV collagenolysis and proMMP-9 production. Invasion was significantly inhibited by EDTA, alpha 2-macroglobulin and tissue inhibitor of metalloproteinases-1, but not by inhibitors of cathepsin G and leukocyte elastase. These data suggest that MMP-9 plays an important role in the invasion of mononuclear phagocytes through basement membranes.
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Kizaki, M., Y. Ikeda, R. Tanosaki, H. Nakajima, M. Morikawa, A. Sakashita, and HP Koeffler. "Effects of novel retinoic acid compound, 9-cis-retinoic acid, on proliferation, differentiation, and expression of retinoic acid receptor-alpha and retinoid X receptor-alpha RNA by HL-60 cells." Blood 82, no. 12 (December 15, 1993): 3592–99. http://dx.doi.org/10.1182/blood.v82.12.3592.3592.
Full textAbstract:
Abstract Retinoic acid modulates proliferation and differentiation of a wide variety of normal and leukemic cells through two distinct families of transcriptional factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A stereoisomer of retinoic acid, 9-cis- retinoic acid, is a high-affinity ligand for RXR and binds efficiently to RAR. In contrast, all-trans-retinoic acid interacts 40-fold less efficiently with RXR as compared with RAR. To clarify the biologic role of retinoic acid compounds (all-trans,- 9-cis-, and 13-cis-retinoic acid) in hematopoietic cells, we studied their effects on clonal growth, differentiation, and expression of RAR-alpha and RXR-alpha genes in HL-60 cells. At very low concentrations (10(-15) to 10(-12) mmol/L), each retinoid enhanced clonal growth of HL-60 cells. These concentrations of the retinoids had no capacity to induce differentiation of leukemic cells as measured by ability either to reduce nitroblue tetrazolium and to express CD11b antigens, suggesting that retinoids at very low concentrations may stimulate proliferation of leukemic cells rather than induce their differentiation. These findings may help explain why patients with acute promyelocytic leukemia may relapse while receiving retinoic acids. With continuous therapy, retinoids are metabolized rapidly with increased urinary excretion, lowering their plasma levels to a range that may stimulate proliferation without inducing differentiation of leukemic cells. In contrast, we found that at higher concentrations (> or = 10(-11) mmol/L) each retinoid inhibited clonal growth, reduced c-myc RNA levels, and induced differentiation of HL-60 cells. 9-cis-retinoic acid was a slightly more potent inducer of differentiation than all-trans- retinoic acid; the mechanism for this increased potency and its clinical potential requires additional studies. Steady-state levels of RAR-alpha mRNA in HL-60 cells were not affected by either 9-cis- and all-trans-retinoic acid. In contrast, 9-cis-retinoic acid, but not all- trans-retinoic acid, reduced RXR-alpha mRNA accumulation in a dose- dependent manner.
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Kizaki, M., Y. Ikeda, R. Tanosaki, H. Nakajima, M. Morikawa, A. Sakashita, and HP Koeffler. "Effects of novel retinoic acid compound, 9-cis-retinoic acid, on proliferation, differentiation, and expression of retinoic acid receptor-alpha and retinoid X receptor-alpha RNA by HL-60 cells." Blood 82, no. 12 (December 15, 1993): 3592–99. http://dx.doi.org/10.1182/blood.v82.12.3592.bloodjournal82123592.
Full textAbstract:
Retinoic acid modulates proliferation and differentiation of a wide variety of normal and leukemic cells through two distinct families of transcriptional factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A stereoisomer of retinoic acid, 9-cis- retinoic acid, is a high-affinity ligand for RXR and binds efficiently to RAR. In contrast, all-trans-retinoic acid interacts 40-fold less efficiently with RXR as compared with RAR. To clarify the biologic role of retinoic acid compounds (all-trans,- 9-cis-, and 13-cis-retinoic acid) in hematopoietic cells, we studied their effects on clonal growth, differentiation, and expression of RAR-alpha and RXR-alpha genes in HL-60 cells. At very low concentrations (10(-15) to 10(-12) mmol/L), each retinoid enhanced clonal growth of HL-60 cells. These concentrations of the retinoids had no capacity to induce differentiation of leukemic cells as measured by ability either to reduce nitroblue tetrazolium and to express CD11b antigens, suggesting that retinoids at very low concentrations may stimulate proliferation of leukemic cells rather than induce their differentiation. These findings may help explain why patients with acute promyelocytic leukemia may relapse while receiving retinoic acids. With continuous therapy, retinoids are metabolized rapidly with increased urinary excretion, lowering their plasma levels to a range that may stimulate proliferation without inducing differentiation of leukemic cells. In contrast, we found that at higher concentrations (> or = 10(-11) mmol/L) each retinoid inhibited clonal growth, reduced c-myc RNA levels, and induced differentiation of HL-60 cells. 9-cis-retinoic acid was a slightly more potent inducer of differentiation than all-trans- retinoic acid; the mechanism for this increased potency and its clinical potential requires additional studies. Steady-state levels of RAR-alpha mRNA in HL-60 cells were not affected by either 9-cis- and all-trans-retinoic acid. In contrast, 9-cis-retinoic acid, but not all- trans-retinoic acid, reduced RXR-alpha mRNA accumulation in a dose- dependent manner.
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Muscat, G. E., T. A. Gustafson, and L. Kedes. "A common factor regulates skeletal and cardiac alpha-actin gene transcription in muscle." Molecular and Cellular Biology 8, no. 10 (October 1988): 4120–33. http://dx.doi.org/10.1128/mcb.8.10.4120-4133.1988.
Full textAbstract:
The skeletal and cardiac alpha-actin genes are coexpressed in muscle development but exhibit distinctive tissue-specific patterns of expression. We used an in vivo competition assay and an in vitro electrophoretic mobility shift assay to demonstrate that both genes interact with a common trans-acting factor(s). However, there was at least one gene-specific cis-acting sequence in the skeletal alpha-actin gene that interacted with a trans-acting factor which was not rate limiting in the expression of the cardiac alpha-actin gene. The common factor(s) interacted with several cis-acting regions that corresponded to sequences that are required for the transcriptional modulation of these sarcomeric alpha-actin genes in muscle cells. These regulatory regions contained the sequence motif CC(A + T-rich)6GG, which is known as a CArG box. Results of in vivo competition assays demonstrated that the factor(s) bound by the skeletal alpha-actin gene is also essential for the maximal activity of the cardiac alpha-actin, simian virus 40 (SV40), alpha 2(I)-collagen, and the beta-actin promoters in muscle cells. In contrast, fibroblastic cells contained functionally distinct transcription factor(s) that were used by the SV40 enhancer but that did not interact with the sarcomeric alpha-actin cis-acting sequences. The existence of functionally different factors in these cell types may explain the myogenic specificity of these sarcomeric alpha-actin genes. Results of in vitro studies suggested that both the sarcomeric alpha-actin genes interact with the CArG box-binding factor CBF and that the skeletal alpha-actin promoter contains multiple CBF-binding sites. In contrast, CBF did not interact in vitro with a classical CAAT box, the SV40 enhancer, or a linker scanner mutation of an alpha-actin CArG box. Furthermore, methylation interference and DNase I footprinting assays demonstrated the precise sites of interaction of CBF with three CArG motifs at positions -98, -179, and -225 in the human skeletal alpha-actin gene.
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Muscat, G. E., T. A. Gustafson, and L. Kedes. "A common factor regulates skeletal and cardiac alpha-actin gene transcription in muscle." Molecular and Cellular Biology 8, no. 10 (October 1988): 4120–33. http://dx.doi.org/10.1128/mcb.8.10.4120.
Full textAbstract:
The skeletal and cardiac alpha-actin genes are coexpressed in muscle development but exhibit distinctive tissue-specific patterns of expression. We used an in vivo competition assay and an in vitro electrophoretic mobility shift assay to demonstrate that both genes interact with a common trans-acting factor(s). However, there was at least one gene-specific cis-acting sequence in the skeletal alpha-actin gene that interacted with a trans-acting factor which was not rate limiting in the expression of the cardiac alpha-actin gene. The common factor(s) interacted with several cis-acting regions that corresponded to sequences that are required for the transcriptional modulation of these sarcomeric alpha-actin genes in muscle cells. These regulatory regions contained the sequence motif CC(A + T-rich)6GG, which is known as a CArG box. Results of in vivo competition assays demonstrated that the factor(s) bound by the skeletal alpha-actin gene is also essential for the maximal activity of the cardiac alpha-actin, simian virus 40 (SV40), alpha 2(I)-collagen, and the beta-actin promoters in muscle cells. In contrast, fibroblastic cells contained functionally distinct transcription factor(s) that were used by the SV40 enhancer but that did not interact with the sarcomeric alpha-actin cis-acting sequences. The existence of functionally different factors in these cell types may explain the myogenic specificity of these sarcomeric alpha-actin genes. Results of in vitro studies suggested that both the sarcomeric alpha-actin genes interact with the CArG box-binding factor CBF and that the skeletal alpha-actin promoter contains multiple CBF-binding sites. In contrast, CBF did not interact in vitro with a classical CAAT box, the SV40 enhancer, or a linker scanner mutation of an alpha-actin CArG box. Furthermore, methylation interference and DNase I footprinting assays demonstrated the precise sites of interaction of CBF with three CArG motifs at positions -98, -179, and -225 in the human skeletal alpha-actin gene.
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Mastrangelo, M. F., K. G. Weinstock, B. K. Shafer, A. M. Hedge, D. J. Garfinkel, and J. N. Strathern. "Disruption of a silencer domain by a retrotransposon." Genetics 131, no. 3 (July 1, 1992): 519–29. http://dx.doi.org/10.1093/genetics/131.3.519.
Full textAbstract:
Abstract A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.
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Knezetic, J. A., and G. Felsenfeld. "Mechanism of developmental regulation of alpha pi, the chicken embryonic alpha-globin gene." Molecular and Cellular Biology 13, no. 8 (August 1993): 4632–39. http://dx.doi.org/10.1128/mcb.13.8.4632-4639.1993.
Full textAbstract:
The chicken alpha pi-globin gene is expressed during development only in the primitive erythrocyte lineage and not in the definitive lineage. We show that stage-specific expression is maintained when plasmids containing the alpha pi promoter are transfected into primitive and definitive lineage primary erythroid cells and that the information contained in the promoter is sufficient to confer this specificity. Detailed analysis of binding sites in the promoter for trans-acting factors, together with studies of the effects of mutagenesis on expression, reveals that the factors critical to stage-specific expression are all present in both primitive and definitive lineages, but at various concentrations. We identify three proteins, an NF1 family member, a Y-box factor, and an Sp1-like factor, which interact to stimulate or inhibit transcription. We propose that the concentration-dependent action of these factors, together with the general erythroid factor GATA-1, is responsible for the stage-specific expression of the alpha pi-globin gene.
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Knezetic, J. A., and G. Felsenfeld. "Mechanism of developmental regulation of alpha pi, the chicken embryonic alpha-globin gene." Molecular and Cellular Biology 13, no. 8 (August 1993): 4632–39. http://dx.doi.org/10.1128/mcb.13.8.4632.
Full textAbstract:
The chicken alpha pi-globin gene is expressed during development only in the primitive erythrocyte lineage and not in the definitive lineage. We show that stage-specific expression is maintained when plasmids containing the alpha pi promoter are transfected into primitive and definitive lineage primary erythroid cells and that the information contained in the promoter is sufficient to confer this specificity. Detailed analysis of binding sites in the promoter for trans-acting factors, together with studies of the effects of mutagenesis on expression, reveals that the factors critical to stage-specific expression are all present in both primitive and definitive lineages, but at various concentrations. We identify three proteins, an NF1 family member, a Y-box factor, and an Sp1-like factor, which interact to stimulate or inhibit transcription. We propose that the concentration-dependent action of these factors, together with the general erythroid factor GATA-1, is responsible for the stage-specific expression of the alpha pi-globin gene.
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Chan, Kwai S. "Constitutive Relations for Inelastic Deformation and Damage Accumulation in Hard Alpha Titanium." Journal of Engineering Materials and Technology 123, no. 3 (March 11, 2001): 281–86. http://dx.doi.org/10.1115/1.1373653.
Full textAbstract:
The constitutive properties of hard alpha Ti were recently generated by Chan et al., 2000, “Constitutive Properties of Hard Alpha Titanium,” Metall. and Maters. Trans. A, Vol. 31A, pp. 3029–3040, using uniaxial compression, indirect tension, indentation, and plane strain compression tests. Flow stress and fracture strength of hard alpha were measured as a function of stress state, strain rate, temperature, and nitrogen content. Hard alpha exhibited plastic flow at low nitrogen contents (<4 wt. percent N), but elastic fracture at high nitrogen contents (>6 wt. percent N). In this paper, this set of constitutive data is used to develop constitutive equations for describing the inelastic deformation and damage accumulation behavior of hard alpha Ti.
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Weiss, I., FE Cash, MB Coleman, A. Pressley, JG Adams, T. Sanguansermsri, SA Liebhaber, and MH Steinberg. "Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]." Blood 76, no. 12 (December 15, 1990): 2630–36. http://dx.doi.org/10.1182/blood.v76.12.2630.2630.
Full textAbstract:
Abstract Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.
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Weiss, I., FE Cash, MB Coleman, A. Pressley, JG Adams, T. Sanguansermsri, SA Liebhaber, and MH Steinberg. "Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]." Blood 76, no. 12 (December 15, 1990): 2630–36. http://dx.doi.org/10.1182/blood.v76.12.2630.bloodjournal76122630.
Full textAbstract:
Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.
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Pontes de Carvalho, L. C., S. Tomlinson, F. Vandekerckhove, E. J. Bienen, A. B. Clarkson, M. S. Jiang, G. W. Hart, and V. Nussenzweig. "Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor." Journal of Experimental Medicine 177, no. 2 (February 1, 1993): 465–74. http://dx.doi.org/10.1084/jem.177.2.465.
Full textAbstract:
Here we report the presence of a trans-sialidase on the surface of Trypanosoma brucei culture-derived procyclic trypomastigotes. The enzyme is not detected in lysates of bloodstream trypomastigotes enriched for either stumpy or slender forms. The trans-sialidase catalyzes the transfer of alpha(2-3)-linked sialic acid residues to lactose. beta-galactopyranosyl residues are at least 100 times better acceptors for sialic acid than alpha-galactopyranosyl residues. In the absence of efficient acceptors, the purified enzyme transfers sialic acid to water, i.e., it acts as a sialidase. Although the T. cruzi and T. brucei trans-sialidases have very similar donor and acceptor specificities, they are antigenically distinct. Sodium dodecyl sulfate-polyacramide gel electrophoresis under nonreducing conditions and silver staining of the purified trans-sialidase reveals a single band of 63 kD. When the surface membrane of live procyclic trypomastigotes is trans-sialylated, using radioactive sialyllactose as the donor substrate, it appears that the only sialylated surface molecule is procyclin. Pronase treatment of live parasites removes only part of the surface sialic acid, in agreement with recent data showing that the glycosylphosphatidylinositol anchor of procyclin is sialylated (Ferguson, M. A. J., M. Murray, H. Rutherford, and M. J. McConville. 1993. Biochem. J. In press).
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Warmerdam, P. A., E. O. Long, and P. A. Roche. "Isoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments." Journal of Cell Biology 133, no. 2 (April 15, 1996): 281–91. http://dx.doi.org/10.1083/jcb.133.2.281.
Full textAbstract:
Newly synthesized class II molecules of the major histocompatibility complex must be transported to endosomal compartments where antigens are processed for presentation to class II-restricted T cells. The invariant chain (Ii), which assembles with newly synthesized class II alpha- and beta-chains in the endoplasmic reticulum, carries one or more targeting signals for transport to endosomal compartments where Ii dissociates from alpha beta Ii complexes. Here we show that the transport route of alpha beta Ii complexes is regulated selectively by two forms of Ii (p33 and p35) that are generated by the use of alternative translation initiation sites. Using a novel quantitative surface arrival assay based on labeling with [6-3H]-D-galactose combined with biochemical modification at the cell surface with neuraminidase, we demonstrate that newly synthesized alpha beta Ii molecules containing the Ii-p33 isoform can be detected on the cell surface shortly after passage through the Golgi apparatus/trans-Golgi network. A substantial amount of these alpha beta Ii complexes are targeted to early endosomes either directly from the trans-Golgi network or after internalization from the cell surface before their delivery to antigen processing compartments. The fraction of alpha beta Ii complexes containing the p35 isoform of Ii with a longer cytosolic domain was not detected at the cell surface as determined by iodination of intact cells and the lack of susceptibility to neuraminidase trimming on ice. However, treatment with neuraminidase at 37 degrees C did reveal that some of the alpha beta Ii-p35 complexes traversed early endosomes. These results demonstrate that a fraction of newly synthesized class II molecules arrive at the cell surface as alpha beta Ii complexes before delivery to antigen processing compartments and that class II alpha beta Ii complexes associated with the two isoforms of Ii are sorted to these compartments by different transport routes.
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Vilane, V. N., R. D. Knutsen, and J. E. Westraadt. "Contribution of Hydrogen Promoted Aluminium Partitioning to Tempered ά Martensite Embrittlement in Ti-6Al-4V." Materials Science Forum 828-829 (August 2015): 181–87. http://dx.doi.org/10.4028/www.scientific.net/msf.828-829.181.
Full textAbstract:
A thermohydrogen process promoting metastable phase decomposition (THP-MD) treatment was performed on wrought Ti-6Al-4V to determine the effects of microstructure evolution on tensile ductility. Tensile ductility was affected by the nature of phase and morphology evolution in which dissolved hydrogen played a key role. Hydrogen reduced the beta transus and stabilised more beta phase at aging/tempering temperature. A reduced beta transus in a similar heat treatment resulted in a bimodal morphology (in non-hydrogenated samples) or a fully acicular morphology (in hydrogenated samples). It also reduced the volume fraction of alpha at aging/tempering temperature which resulted in the extensive enrichment of reduced alpha with aluminium (Al) during tempering. The increased Al content in the reduced alpha promoted ordering of the HCP lattice to the brittle titanium aluminide (Ti3Al) phase. In addition to Ti3Al embrittlement, the acicular morphology of Ti-6Al-4V tempered hexagonal martensite (ά) offers limited resistance to crack propagation. The highest degree of embrittlement was observed in prior hydrogenated samples because of the combined effect of the acicular morphology and Ti3Al embrittlement.
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Chonat, Satheesh, Mary Risinger, Neha Dagaonkar, Tamara Maghathe, Jennifer Rothman, Jessica Connor, Karen Kalinyak, Amber H. Begtrup, Kejian Zhang, and Theodosia A. Kalfa. "The Spectrum of Alpha-Spectrin Associated Hereditary Spherocytosis." Blood 126, no. 23 (December 3, 2015): 941. http://dx.doi.org/10.1182/blood.v126.23.941.941.
Full textAbstract:
Abstract Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation. Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C>T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B). A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G>TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype. A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth. Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 2. Figure 2. Disclosures Begtrup: GeneDx: Employment.
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Becker, PS, Z. Li, T. Potselueva, JA Madri, PE Newburger, and N. Berliner. "Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid." Blood 88, no. 1 (July 1, 1996): 261–67. http://dx.doi.org/10.1182/blood.v88.1.261.261.
Full textAbstract:
Abstract The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all- trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.
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Becker, PS, Z. Li, T. Potselueva, JA Madri, PE Newburger, and N. Berliner. "Laminin promotes differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid." Blood 88, no. 1 (July 1, 1996): 261–67. http://dx.doi.org/10.1182/blood.v88.1.261.bloodjournal881261.
Full textAbstract:
The promyelocytic leukemia cell line, NB4, carries the t(15; 17) translocation and undergoes limited maturation in response to differentiation agents. Growth on laminin enhanced the ability of all- trans retinoic acid (ATRA) to promote morphologic maturation of these cells. Although exposure to ATRA in suspension yielded minimal maturation beyond the myelocyte stage, after 72 hours of exposure to ATRA on laminin the cells acquired the histologic appearance of metamyelocytes, band forms, and segmented neutrophils. After 96 hours, some cells acquired a spindle shape and became tightly adherent. Growth on collagen types I, III, IV, or fibronectin did not have this effect, although some cells did adhere to fibronectin. NB4 cells treated with ATRA in suspension or on laminin acquired the equivalent ability to reduce nitroblue tetrazolium or cytochrome C. Despite the improved morphologic maturation on laminin, the cells did not express secondary granule proteins such as lactoferrin or neutrophil collagenase. In addition, growth on laminin abolished cell proliferation in the presence of ionomycin. Growth on laminin and/or with ATRA induced new expression of alpha 6 integrin, a laminin receptor, as assessed by reverse transcription-polymerase chain reaction. Different conditions of growth (laminin or differentiation agent) resulted in specific patterns of expression of the alpha 6A and alpha 6B isoforms. Treatment with ATRA also resulted in the acquisition of high-level surface expression of alpha 6 integrin, as assessed by flow cytometry. Thus, treatment of NB4 promyelocytic leukemia cells with ATRA induced expression of alpha 6 integrin (a laminin receptor alpha-chain) and enabled more advanced maturation when the cells were grown on the extracellular matrix component, laminin, compared with tissue culture plastic.
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Tobler, A., R. Munker, D. Heitjan, and HP Koeffler. "In vitro interaction of recombinant tumor necrosis factor alpha and all- trans-retinoic acid with normal and leukemic hematopoietic cells." Blood 70, no. 6 (December 1, 1987): 1940–46. http://dx.doi.org/10.1182/blood.v70.6.1940.bloodjournal7061940.
Full textAbstract:
Both human recombinant tumor necrosis factor alpha (TNF alpha) and all- trans-retinoic acid (RA) inhibit the in vitro clonal growth of human myeloid leukemic cells. We investigated the in vitro interaction of TNF alpha and RA with normal and a variety of leukemic myeloid cells. With the promyelocytic HL-60 cells, TNF alpha (greater than or equal to 2.5 U/mL) in combination with RA synergistically inhibited clonal growth; TNF alpha at lower concentrations (less than or equal to 1 U/mL) plus RA (10(-9) mol/L) were antagonistic in their inhibition of growth. The ability of RA (10(-8) mol/L) plus TNF alpha (2.5, 5 U/mL) to enhance differentiation of HL-60 cells paralleled their ability to inhibit clonal growth of these cells. In addition, RA (10(-9) to 10(-7) mol/L) increased the number of TNF alpha receptors on HL-60 cells 1.3- to 1.7- fold without changing the affinity for the TNF alpha receptor. With the more immature KG-1 myeloblasts, concentrations of TNF alpha greater than 10 U/mL synergistically interacted with RA to inhibit clonal growth; at lower concentrations of TNF alpha (less than 10 U/mL), RA appeared to inhibit the expected effect of TNF alpha. KG-1 cells were not induced to differentiate with either agent alone or in combination. With four of nine leukemic patients, TNF alpha in combination with RA (10(-7) mol/L) inhibited leukemic clonal growth to a greater extent than each agent alone. No marked effect of the combined treatment was seen in two other patients. The RA reversed the inhibitory action of TNF alpha on normal human granulocyte-macrophage colony forming cells (GM-CFC) and on clonal growth of leukemic cells from three patients. Our study suggests that TNF alpha and RA interact in a complex manner with normal and leukemic hematopoietic cells.