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Sample records for modifies neuroprotecting globins

  1. Running, swimming and diving modifies neuroprotecting globins in the mammalian brain

    PubMed Central

    Williams, Terrie M; Zavanelli, Mary; Miller, Melissa A; Goldbeck, Robert A; Morledge, Michael; Casper, Dave; Pabst, D. Ann; McLellan, William; Cantin, Lucas P; Kliger, David S

    2007-01-01

    The vulnerability of the human brain to injury following just a few minutes of oxygen deprivation with submergence contrasts markedly with diving mammals, such as Weddell seals (Leptonychotes weddellii), which can remain underwater for more than 90 min while exhibiting no neurological or behavioural impairment. This response occurs despite exposure to blood oxygen levels concomitant with human unconsciousness. To determine whether such aquatic lifestyles result in unique adaptations for avoiding ischaemic–hypoxic neural damage, we measured the presence of circulating (haemoglobin) and resident (neuroglobin and cytoglobin) oxygen-carrying globins in the cerebral cortex of 16 mammalian species considered terrestrial, swimming or diving specialists. Here we report a striking difference in globin levels depending on activity lifestyle. A nearly 9.5-fold range in haemoglobin concentration (0.17–1.62 g Hb 100 g brain wet wt−1) occurred between terrestrial and deep-diving mammals; a threefold range in resident globins was evident between terrestrial and swimming specialists. Together, these two globin groups provide complementary mechanisms for facilitating oxygen transfer into neural tissues and the potential for protection against reactive oxygen and nitrogen groups. This enables marine mammals to maintain sensory and locomotor neural functions during prolonged submergence, and suggests new avenues for averting oxygen-mediated neural injury in the mammalian brain. PMID:18089537

  2. Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

    PubMed

    Neya, Saburo; Nagai, Masako; Nagatomo, Shigenori; Hoshino, Tyuji; Yoneda, Tomoki; Kawaguchi, Akira T

    2016-05-01

    Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson

  3. The Neuroprotective Disease-Modifying Potential of Psychotropics in Parkinson's Disease

    PubMed Central

    Lauterbach, Edward C.; Fontenelle, Leonardo F.; Teixeira, Antonio L.

    2012-01-01

    Neuroprotective treatments in Parkinson's disease (PD) have remained elusive. Psychotropics are commonly prescribed in PD without regard to their pathobiological effects. The authors investigated the effects of psychotropics on pathobiological proteins, proteasomal activity, mitochondrial functions, apoptosis, neuroinflammation, trophic factors, stem cells, and neurogenesis. Only findings replicated in at least 2 studies were considered for these actions. Additionally, PD-related gene transcription, animal model, and human neuroprotective clinical trial data were reviewed. Results indicate that, from a PD pathobiology perspective, the safest drugs (i.e., drugs least likely to promote cellular neurodegenerative mechanisms balanced against their likelihood of promoting neuroprotective mechanisms) include pramipexole, valproate, lithium, desipramine, escitalopram, and dextromethorphan. Fluoxetine favorably affects transcription of multiple genes (e.g., MAPT, GBA, CCDC62, HIP1R), although it and desipramine reduced MPTP mouse survival. Haloperidol is best avoided. The most promising neuroprotective investigative priorities will involve disease-modifying trials of the safest agents alone or in combination to capture salutary effects on H3 histone deacetylase, gene transcription, glycogen synthase kinase-3, α-synuclein, reactive oxygen species (ROS), reactive nitrogen species (RNS), apoptosis, inflammation, and trophic factors including GDNF and BDNF. PMID:22254151

  4. B6 peptide-modified PEG-PLA nanoparticles for enhanced brain delivery of neuroprotective peptide.

    PubMed

    Liu, Zhongyang; Gao, Xiaoling; Kang, Ting; Jiang, Mengyin; Miao, Deyu; Gu, Guangzhi; Hu, Quanyin; Song, Qingxiang; Yao, Lei; Tu, Yifan; Chen, Hongzhuan; Jiang, Xinguo; Chen, Jun

    2013-06-19

    The blood-brain barrier (BBB), which is formed by the brain capillary wall, greatly hinders the development of new drugs for the brain. Over the past decades, among the various receptor-mediated endogenous BBB transport systems, the strategy of using transferrin or anti-transferrin receptor antibodies to facilitate brain drug delivery system is of particular interest. However, the application of large proteins still suffers from the drawbacks including synthesis procedure, stability, and immunological response. Here, we explored a B6 peptide discovered by phase display as a substitute for transferrin, and conjugated it to PEG-PLA nanoparticles (NP) with the aim of enhancing the delivery of neuroprotective drug across the BBB for the treatment of Alzheimer's disease. B6-modified NP (B6-NP) exhibited significantly higher accumulation in brain capillary endothelial cells via lipid raft-mediated and clathrin-mediated endocytosis. In vivo, fluorescently labeled B6-NP exhibited much higher brain accumulation when compared with NP. Administration of B6-NP encapsulated neuroprotective peptide-NAPVSIPQ (NAP)-to Alzheimer's disease mouse models showed excellent amelioration in learning impairments, cholinergic disruption, and loss of hippocampal neurons even at lower dose. These findings together suggested that B6-NP might serve as a promising DDS for facilitating the brain delivery of neuropeptides. PMID:23718945

  5. A phylogenomic profile of globins

    PubMed Central

    Vinogradov, Serge N; Hoogewijs, David; Bailly, Xavier; Arredondo-Peter, Raúl; Gough, Julian; Dewilde, Sylvia; Moens, Luc; Vanfleteren, Jacques R

    2006-01-01

    Background Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. Results A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. Conclusion Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals. PMID:16600051

  6. Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.

    PubMed

    Wienert, Beeke; Funnell, Alister P W; Norton, Laura J; Pearson, Richard C M; Wilkinson-White, Lorna E; Lester, Krystal; Vadolas, Jim; Porteus, Matthew H; Matthews, Jacqueline M; Quinlan, Kate G R; Crossley, Merlin

    2015-01-01

    Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter. PMID:25971621

  7. Mitochondrial therapy for Parkinson’s disease: Neuroprotective pharmaconutrition may be disease-modifying

    PubMed Central

    Kones, Richard

    2010-01-01

    Progressive destruction of neurons that produce dopamine in the basal ganglia of the brain, particularly the substantia nigra, is a hallmark of Parkinson’s disease. The syndrome of the Parkinsonian phenotype is caused by many etiologies, involving multiple contributing mechanisms. Characteristic findings are pathologic inclusions called Lewy bodies, which are protein aggregates inside nerve cells. Environmental insults are linked with the disease, and a number of associated genes have also been identified. Neuroinflammation, microglia activation, oxidative stress, and mitochondrial dysfunction are central processes producing nerve damage. In addition, protein misfolding, driven by accumulation and condensation of α-synuclein, compounded by inadequate elimination of defective protein through the ubiquitin- proteasome system, promote apoptosis. Current pharmacologic therapy is palliative rather than disease- modifying, and typically becomes unsatisfactory over time. Coenzyme Q10 and creatine, two agents involved in energy production, may be disease-modifying, and able to produce sufficient beneficial pathophysiologic changes in preclinical studies to warrant large studies now in progress. Use of long-chain omega-3 fatty acids and vitamin D in PD are also topics of current interest. PMID:22291504

  8. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  9. Globin gene switching in primates.

    PubMed

    Johnson, Robert M; Gumucio, Deborah; Goodman, Morris

    2002-11-01

    Evolutionary approaches to the identification of DNA sequences required for transcription of the genes of the beta-globin cluster are reviewed. Sequence alignments of non-coding regions from widely divergent species revealed many conserved motifs (phylogenetic footprints) that were putative transcription factor binding sites and candidate binding proteins were identified. The differential timing of the prosimian and simian gamma-globin genes was analyzed by identifying base changes in the vicinity of the phylogenetic footprints. These differential phylogenetic footprints were shown to bind different nuclear factors, and the behavior of constructs with human or galago gamma-promoters in transgenic mice indicated that DNA motifs near the gamma-globin genes are sufficient to determine the developmental stage of expression. Locus control region alignments have identified many conserved sequence differences outside of the hypersensitive sites. Globin protein and mRNA expression profiles during embryological development in a series of catarrhine (Old World monkeys and apes) and platyrrhine (New World monkeys) primates have been determined. While all catarrhines examined to date have globin expression patterns that are highly similar to the well-established human switching behavior, platyrrhines have inactivated their gamma 1 genes by a variety of mechanisms, and have an earlier gamma-beta switch. PMID:12443943

  10. The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and Functions

    PubMed Central

    Schwarze, Kim; Singh, Abhilasha; Burmester, Thorsten

    2015-01-01

    Globins are small heme proteins that play an important role in oxygen supply, but may also have other functions. Globins offer a unique opportunity to study the functional evolution of genes and proteins. We have characterized the globin repertoire of two different turtle species: the Chinese softshell turtle (Pelodiscus sinensis) and the western painted turtle (Chrysemys picta bellii). In the genomes of both species, we have identified eight distinct globin types: hemoglobin (Hb), myoglobin, neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Therefore, along with the coelacanth, turtles are so far the only known vertebrates with a full globin repertoire. This fact allows for the first time a comparative analysis of the expression of all eight globins in a single species. Phylogenetic analysis showed an early divergence of neuroglobin and globin X before the radiation of vertebrates. Among the other globins, cytoglobin diverged first, and there is a close relationship between myoglobin and globin E; the position of globin Y is not resolved. The globin E gene was selectively lost in the green anole, and the genes coding for globin X and globin Y were deleted in chicken. Quantitative real-time reverse transcription polymerase chain reaction experiments revealed that myoglobin, neuroglobin, and globin E are highly expressed with tissue-specific patterns, which are in line with their roles in the oxidative metabolism of the striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment epithelium of the eye. Globin E probably has a myoglobin-like role in transporting O2 across the pigment epithelium to supply in the metabolically highly active retina. PMID:26078264

  11. Neuroprotective effects of a chromatin modifier on ischemia/reperfusion neurons: implication of its regulation of BCL2 transactivation by ERα signaling.

    PubMed

    Guo, Jun; Zhang, Tao; Yu, Jia; Li, Hong-Zeng; Zhao, Cong; Qiu, Jing; Zhao, Bo; Zhao, Jie; Li, Wei; Zhao, Tian-Zhi

    2016-06-01

    An understanding of the molecular mechanisms involved in the regulation of estrogen receptor alpha (ERα)-mediated neuroprotective effects is valuable for the development of therapeutic strategy against neuronal ischemic injury. Here, we report the upregulated expression of metastasis-associated protein 1 (MTA1), a master chromatin modifier and transcriptional regulator, in the murine middle cerebral artery occlusion (MCAO) model. Inhibition of MTA1 expression by in vivo short interfering RNA treatment potentiated neuronal apoptosis in a caspase-3-dependent manner and thereafter aggravated MCAO-induced neuronal damage. Mechanistically, the pro-survival effects of MTA1 required the participation of ERα signaling. We also provide in vitro evidence that MTA1 enhances the binding of ERα with the BCL2 promoter upon ischemic insults via recruitment of HDAC2 together with other unidentified coregulators, thus promoting the ERα-mediated transactivation of the BCL2 gene. Collectively, our results suggest that the augmentation of endogenous MTA1 expression during neuronal ischemic injury acts additionally to an endocrinous cascade orchestrating intimate interactions between ERα and BCL2 pathways and operates as an indispensable defensive mechanism in response to neuronal ischemia/reperfusion stress. Future studies in this field will shed light on the modulation of the complicated neuroprotective effects by estrogen signaling. PMID:26728277

  12. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    SciTech Connect

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  13. Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes.

    PubMed

    Perez-Stable, C; Costantini, F

    1990-03-01

    The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin

  14. Neuroprotection For Huntington’s Disease: Ready, Set, Slow

    PubMed Central

    Hersch, Steven M.; Rosas, H. Diana

    2008-01-01

    Summary The ultimate goal for Huntington’s disease (HD) is to develop disease-modifying neuroprotective therapies able to delay or prevent illness in those who are at genetic risk and able to slow progression in those who are affected clinically. Neuroprotection is the preservation of neuronal structure, function, and viability and neuroprotective therapy is thus targeted at the underlying pathology of HD, rather than at its specific symptoms. Preclinical target discovery research in HD is identifying numerous distinct targets and options for modulating them with some proceeding into large-scale efficacy studies in early symptomatic HD subjects. The first pilot studies of neuroprotective compounds in premanifest HD are also soon to begin. This review discusses the opportunities for neuroprotection in HD, clinical methodology in premanifest and manifest HD, the clinical assessment of neuroprotection, molecular targets and therapeutic leads, and the current state of clinical development. PMID:18394565

  15. A Phylogenetic Analysis of the Globins in Fungi

    PubMed Central

    Hoogewijs, David; Dewilde, Sylvia; Vierstraete, Andy; Moens, Luc; Vinogradov, Serge N.

    2012-01-01

    Background All globins belong to one of three families: the F (flavohemoglobin) and S (sensor) families that exhibit the canonical 3/3 α-helical fold, and the T (truncated 3/3 fold) globins characterized by a shortened 2/2 α-helical fold. All eukaryote 3/3 hemoglobins are related to the bacterial single domain F globins. It is known that Fungi contain flavohemoglobins and single domain S globins. Our aims are to provide a census of fungal globins and to examine their relationships to bacterial globins. Results Examination of 165 genomes revealed that globins are present in >90% of Ascomycota and ∼60% of Basidiomycota genomes. The S globins occur in Blastocladiomycota and Chytridiomycota in addition to the phyla that have FHbs. Unexpectedly, group 1 T globins were found in one Blastocladiomycota and one Chytridiomycota genome. Phylogenetic analyses were carried out on the fungal globins, alone and aligned with representative bacterial globins. The Saccharomycetes and Sordariomycetes with two FHbs form two widely divergent clusters separated by the remaining fungal sequences. One of the Saccharomycete groups represents a new subfamily of FHbs, comprising a previously unknown N-terminal and a FHb missing the C-terminal moiety of its reductase domain. The two Saccharomycete groups also form two clusters in the presence of bacterial FHbs; the surrounding bacterial sequences are dominated by Proteobacteria and Bacilli (Firmicutes). The remaining fungal FHbs cluster with Proteobacteria and Actinobacteria. The Sgbs cluster separately from their bacterial counterparts, except for the intercalation of two Planctomycetes and a Proteobacterium between the Fungi incertae sedis and the Blastocladiomycota and Chytridiomycota. Conclusion Our results are compatible with a model of globin evolution put forward earlier, which proposed that eukaryote F, S and T globins originated via horizontal gene transfer of their bacterial counterparts to the eukaryote ancestor, resulting from

  16. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals

    PubMed Central

    Patel, Vidushi S; Cooper, Steven JB; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer AM

    2008-01-01

    Background Vertebrate alpha (α)- and beta (β)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the α- and β-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil β-globin gene (ω) in the marsupial α-cluster, however, suggested that duplication of the α-β cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous α- and β-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. Results The platypus α-globin cluster (chromosome 21) contains embryonic and adult α- globin genes, a β-like ω-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-ζ-ζ'-αD-α3-α2-α1-ω-GBY-3'. The platypus β-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-ε-β-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate α-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal β-globin clusters are embedded in olfactory genes. Thus, the mammalian α- and β-globin clusters are orthologous to the bird α- and β-globin clusters respectively. Conclusion We propose that α- and β-globin clusters evolved from an ancient MPG-C16orf35-α-β-GBY-LUC7L arrangement 410 million years ago. A copy of the original β (represented by ω in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of β-globin genes with different expression profiles in different lineages. PMID:18657265

  17. Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

    PubMed Central

    2010-01-01

    Background The vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions. Results The Atlantic cod genome was shown to contain 14 globin genes, including nine hemoglobin genes organized in two unlinked clusters designated β5-α1-β1-α4 and β3-β4-α2-α3-β2. The diverged cod hemoglobin genes displayed different expression levels in adult fish, and tetrameric hemoglobins with or without a Root effect were predicted. The novel finding of maternally inherited hemoglobin mRNAs is consistent with a potential role played by fish hemoglobins in the non-specific immune response. In silico analysis of the six teleost genomes available showed that the two α-β globin clusters are flanked by paralogs of five duplicated genes, in agreement with the proposed teleost-specific duplication of the ancestral vertebrate globin cluster. Screening the genome of extant urochordate and cephalochordate species for conserved globin-flanking genes revealed linkage of RHBDF1, MPG and ARHGAP17 to globin genes in the tunicate Ciona intestinalis, while these genes together with LCMT are closely positioned in amphioxus (Branchiostoma floridae), but seem to be unlinked to the multiple globin genes identified in this species. Conclusion The plasticity of Atlantic cod to variable environmental conditions probably involves the expression of multiple globins with potentially different properties. The

  18. Globin gene structure in a reptile supports the transpositional model for amniote α- and β-globin gene evolution.

    PubMed

    Patel, Vidushi S; Ezaz, Tariq; Deakin, Janine E; Graves, Jennifer A Marshall

    2010-12-01

    The haemoglobin protein, required for oxygen transportation in the body, is encoded by α- and β-globin genes that are arranged in clusters. The transpositional model for the evolution of distinct α-globin and β-globin clusters in amniotes is much simpler than the previously proposed whole genome duplication model. According to this model, all jawed vertebrates share one ancient region containing α- and β-globin genes and several flanking genes in the order MPG-C16orf35-(α-β)-GBY-LUC7L that has been conserved for more than 410 million years, whereas amniotes evolved a distinct β-globin cluster by insertion of a transposed β-globin gene from this ancient region into a cluster of olfactory receptors flanked by CCKBR and RRM1. It could not be determined whether this organisation is conserved in all amniotes because of the paucity of information from non-avian reptiles. To fill in this gap, we examined globin gene organisation in a squamate reptile, the Australian bearded dragon lizard, Pogona vitticeps (Agamidae). We report here that the α-globin cluster (HBK, HBA) is flanked by C16orf35 and GBY and is located on a pair of microchromosomes, whereas the β-globin cluster is flanked by RRM1 on the 3' end and is located on the long arm of chromosome 3. However, the CCKBR gene that flanks the β-globin cluster on the 5' end in other amniotes is located on the short arm of chromosome 5 in P. vitticeps, indicating that a chromosomal break between the β-globin cluster and CCKBR occurred at least in the agamid lineage. Our data from a reptile species provide further evidence to support the transpositional model for the evolution of β-globin gene cluster in amniotes. PMID:21116705

  19. O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter.

    PubMed

    Zhang, Zhen; Costa, Flávia C; Tan, Ee Phie; Bushue, Nathan; DiTacchio, Luciano; Costello, Catherine E; McComb, Mark E; Whelan, Stephen A; Peterson, Kenneth R; Slawson, Chad

    2016-07-22

    One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter. PMID:27231347

  20. Widespread occurrence of N-terminal acylation in animal globins and possible origin of respiratory globins from a membrane-bound ancestor.

    PubMed

    Blank, Miriam; Burmester, Thorsten

    2012-11-01

    Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or

  1. Comparison of ligand migration and binding in heme proteins of the globin family

    NASA Astrophysics Data System (ADS)

    Karin, Nienhaus; Ulrich Nienhaus, G.

    2015-12-01

    The binding of small diatomic ligands such as carbon monoxide or dioxygen to heme proteins is among the simplest biological processes known. Still, it has taken many decades to understand the mechanistic aspects of this process in full detail. Here, we compare ligand binding in three heme proteins of the globin family, myoglobin, a dimeric hemoglobin, and neuroglobin. The combination of structural, spectroscopic, and kinetic experiments over many years by many laboratories has revealed common properties of globins and a clear mechanistic picture of ligand binding at the molecular level. In addition to the ligand binding site at the heme iron, a primary ligand docking site exists that ensures efficient ligand binding to and release from the heme iron. Additional, secondary docking sites can greatly facilitate ligand escape after its dissociation from the heme. Although there is only indirect evidence at present, a preformed histidine gate appears to exist that allows ligand entry to and exit from the active site. The importance of these features can be assessed by studies involving modified proteins (via site-directed mutagenesis) and comparison with heme proteins not belonging to the globin family.

  2. α-Globin as a molecular target in the treatment of β-thalassemia

    PubMed Central

    Mettananda, Sachith; Gibbons, Richard J.

    2015-01-01

    The thalassemias, together with sickle cell anemia and its variants, are the world’s most common form of inherited anemia, and in economically undeveloped countries, they still account for tens of thousands of premature deaths every year. In developed countries, treatment of thalassemia is also still far from ideal, requiring lifelong transfusion or allogeneic bone marrow transplantation. Clinical and molecular genetic studies over the course of the last 50 years have demonstrated how coinheritance of modifier genes, which alter the balance of α-like and β-like globin gene expression, may transform severe, transfusion-dependent thalassemia into relatively mild forms of anemia. Most attention has been paid to pathways that increase γ-globin expression, and hence the production of fetal hemoglobin. Here we review the evidence that reduction of α-globin expression may provide an equally plausible approach to ameliorating clinically severe forms of β-thalassemia, and in particular, the very common subgroup of patients with hemoglobin E β-thalassemia that makes up approximately half of all patients born each year with severe β-thalassemia. PMID:25869286

  3. Determination of Ligand Pathways in Globins

    PubMed Central

    Salter, Mallory D.; Blouin, George C.; Soman, Jayashree; Singleton, Eileen W.; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S.

    2012-01-01

    Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  4. Duplication and Divergence: The Evolution of Nematode Globins

    PubMed Central

    McNally, J.; Barris, W.; Blaxter, M. L.

    2009-01-01

    In common with many other groups, nematodes express globins with unknown functions. Nematode globin-like genes can be divided into class 1 globins, similar to vertebrate myoglobins, and a wide range of additional classes. Here we show that class 1 nematode globins possess a huge amount of diversity in gene sequence and structure. There is evidence for multiple events of gene duplication, intron insertion and loss between species, and for allelic variation effecting both synonymous and non-synonymous sites within species. We have also examined gene expression patterns in class I globins from a variety of species. The results show variation in the degree of gene expression, but the tissue specificity and temporal specificity of expression may be more conserved in the phylum. Because the structure-function relationships for the binding and transport of oxygen by globins are well understood, the consequences of genetic variation causing amino acid changes are explored. The gene family shows great promise for discovering unique insights into both structure-function relationships of globins and their physiologial roles. PMID:22661776

  5. Phylogenetic Diversification of the Globin Gene Superfamily in Chordates

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    Summary Phylogenetic reconstructions provide a means of inferring the branching relationships among members of multigene families that have diversified via successive rounds of gene duplication and divergence. Such reconstructions can illuminate the pathways by which particular expression patterns and protein functions evolved. For example, phylogenetic analyses can reveal cases in which similar expression patterns or functional properties evolved independently in different lineages, either through convergence, parallelism, or evolutionary reversals. The purpose of this paper is to provide a robust phylogenetic framework for interpreting experimental data and for generating hypotheses about the functional evolution of globin proteins in chordate animals. To do this we present a consensus phylogeny of the chordate globin gene superfamily. We document the relative roles of gene duplication and whole-genome duplication in fueling the functional diversification of vertebrate globins, and we unravel patterns of shared ancestry among globin genes from representatives of the three chordate subphyla (Craniata, Urochordata, and Cephalochordata). Our results demonstrate the value of integrating phylogenetic analyses with genomic analyses of conserved synteny to infer the duplicative origins and evolutionary histories of globin genes. We also discuss a number of case studies that illustrate the importance of phylogenetic information when making inferences about the evolution of globin gene expression and protein function. Finally, we discuss why the globin gene superfamily presents special challenges for phylogenetic analysis, and we describe methodological approaches that can be used to meet those challenges. PMID:21557448

  6. Neuroprotection in Glaucoma

    PubMed Central

    Doozandeh, Azadeh; Yazdani, Shahin

    2016-01-01

    Glaucoma is a degenerative optic neuropathy characterized by retinal ganglion cell (RGC) loss and visual field defects. It is known that in some glaucoma patients, death of RGCs continues despite intraocular pressure (IOP) reduction. Neuroprotection in the field of glaucoma is defined as any treatment, independent of IOP reduction, which prevents RGC death. Glutamate antagonists, ginkgo biloba extract, neurotrophic factors, antioxidants, calcium channel blockers, brimonidine, glaucoma medications with blood regulatory effect and nitric oxide synthase inhibitors are among compounds with possible neuroprotective activity in preclinical studies. A few agents (such as brimonidine or memantine) with neuroprotective effects in experimental studies have advanced to clinical trials; however the results of clinical trials for these agents have not been conclusive. Nevertheless, lack of compelling clinical evidence has not prevented the off-label use of some of these compounds in glaucoma practice. Stem cell transplantation has been reported to halt experimental neurodegenerative disease processes in the absence of cell replacement. It has been hypothesized that transplantation of some types of stem cells activates multiple neuroprotective pathways via secretion of various factors. The advantage of this approach is a prolonged and targeted effect. Important concerns in this field include the secretion of unwanted harmful mediators, graft survival issues and tumorigenesis. Neuroprotection in glaucoma, pharmacologically or by stem cell transplantation, is an interesting subject waiting for broad and multidisciplinary collaborative studies to better clarify its role in clinical practice. PMID:27413504

  7. Neuroprotection in Glaucoma.

    PubMed

    Doozandeh, Azadeh; Yazdani, Shahin

    2016-01-01

    Glaucoma is a degenerative optic neuropathy characterized by retinal ganglion cell (RGC) loss and visual field defects. It is known that in some glaucoma patients, death of RGCs continues despite intraocular pressure (IOP) reduction. Neuroprotection in the field of glaucoma is defined as any treatment, independent of IOP reduction, which prevents RGC death. Glutamate antagonists, ginkgo biloba extract, neurotrophic factors, antioxidants, calcium channel blockers, brimonidine, glaucoma medications with blood regulatory effect and nitric oxide synthase inhibitors are among compounds with possible neuroprotective activity in preclinical studies. A few agents (such as brimonidine or memantine) with neuroprotective effects in experimental studies have advanced to clinical trials; however the results of clinical trials for these agents have not been conclusive. Nevertheless, lack of compelling clinical evidence has not prevented the off-label use of some of these compounds in glaucoma practice. Stem cell transplantation has been reported to halt experimental neurodegenerative disease processes in the absence of cell replacement. It has been hypothesized that transplantation of some types of stem cells activates multiple neuroprotective pathways via secretion of various factors. The advantage of this approach is a prolonged and targeted effect. Important concerns in this field include the secretion of unwanted harmful mediators, graft survival issues and tumorigenesis. Neuroprotection in glaucoma, pharmacologically or by stem cell transplantation, is an interesting subject waiting for broad and multidisciplinary collaborative studies to better clarify its role in clinical practice. PMID:27413504

  8. Triplex-forming Peptide Nucleic Acids Induce Heritable Elevations in Gamma-globin Expression in Hematopoietic Progenitor Cells

    PubMed Central

    Chin, Joanna Y; Reza, Faisal; Glazer, Peter M

    2013-01-01

    Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes. PMID:23337982

  9. Neurogenic neuroprotection: clinical perspectives

    PubMed Central

    Mandel, Mauricio; Fonoff, Erich Talamoni; Bor-Seng-Shu, Edson; Teixeira, Manoel Jacobsen; Chadi, Gerson

    2012-01-01

    Summary Neurogenic neuroprotection is a promising approach for treating patients with ischemic brain lesions. In rats, stimulation of the deep brain nuclei has been shown to reduce the volume of focal infarction. In this context, protection of neural tissue can be a rapid intervention that has a relatively long-lasting effect, making fastigial nucleus stimulation (FNS) a potentially valuable method for clinical application. Although the mechanisms of neuroprotection induced by FNS remain partially unclear, important data have been presented in the last two decades. A 1-h electrical FNS reduced, by 59%, infarctions triggered by permanent occlusion of the middle cerebral artery in Fisher rats. The acute effect of electrical FNS is likely mediated by a prolonged opening of potassium channels, and the sustained effect appears to be linked to inhibition of the apoptotic cascade. A better understanding of the neuronal circuitry underlying neurogenic neuroprotection may contribute to improving neurological outcomes in ischemic brain insults. PMID:23597434

  10. Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells.

    PubMed

    Corti, Paola; Xue, Jianmin; Tejero, Jesús; Wajih, Nadeem; Sun, Ming; Stolz, Donna B; Tsang, Michael; Kim-Shapiro, Daniel B; Gladwin, Mark T

    2016-07-26

    The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, six-coordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins. PMID:27407144

  11. Molecular cloning and expression of α-globin and β-globin genes from crocodile (Crocodylus siamensis).

    PubMed

    Anwised, Preeyanan; Kabbua, Thai; Temsiripong, Theeranan; Dhiravisit, Apisak; Jitrapakdee, Sarawut; Araki, Tomohiro; Yoneda, Kazunari; Thammasirirak, Sompong

    2013-03-01

    The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co(2+)-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein. PMID:23463382

  12. Genetics Home Reference: methemoglobinemia, beta-globin type

    MedlinePlus

    ... blood cells. Specifically, it alters a molecule called hemoglobin within these cells. Hemoglobin within red blood cells attaches (binds) to oxygen ... in tissues throughout the body. Instead of normal hemoglobin, people with methemoglobinemia, beta-globin type have an ...

  13. Neuroprotective effects of Asiaticoside

    PubMed Central

    Qi, Feng-yan; Yang, Le; Tian, Zhen; Zhao, Ming-gao; Liu, Shui-bing; An, Jia-ze

    2014-01-01

    In the central nervous system, Asiaticoside has been shown to attenuate in vitro neuronal damage caused by exposure to β-amyloid. In vivo studies demonstrated that Asiaticoside could attenuate neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion animals. In addition, Asiaticoside showed anxiolytic effects in acute and chronic stress animals. However, its potential neuroprotective properties in glutamate-induced excitotoxicity have not been fully studied. We investigated the neuroprotective effects of Asiaticoside in primary cultured mouse cortical neurons exposed to glutamate-induced excitotoxicity invoked by N-methyl-D-aspartate. Pretreatment with Asiaticoside decreased neuronal cell loss in a concentration-dependent manner and restored changes in expression of apoptotic-related proteins Bcl-2 and Bax. Asiaticoside pretreatment also attenuated the upregulation of NR2B expression, a subunit of N-methyl-D-aspartate receptors, but did not affect expression of NR2A subunits. Additionally, in cultured neurons, Asiaticoside significantly inhibited Ca2+ influx induced by N-methyl-D-aspartate. These experimental findings provide preliminary evidence that during excitotoxicity induced by N-methyl-D-aspartate exposure in cultured cortical neurons, the neuroprotective effects of Asiaticoside are mediated through inhibition of calcium influx. Aside from its anti-oxidant activity, down-regulation of NR2B-containing N-methyl-D-aspartate receptors may be one of the underlying mechanisms in Asiaticoside neuroprotection. PMID:25221579

  14. Asynchronous DNA replication within the human. beta. -globin gene locus

    SciTech Connect

    Epner, E.; Forrester, W.C.; Groudine, M. )

    1988-11-01

    The timing of DNA replication of the human {beta}-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human {beta}-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-{gamma}-globin gene region and approximately 20 kilobases 5' to the {epsilon}-globin gene and 20 kilobases 3' to the {beta}-globin gene, replicate later and throughout S phase. A similar area is also present in the {alpha}-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks.

  15. Fetal Globin Gene Inducers: Novel Agents & New Potential

    PubMed Central

    Perrine, Susan P.; Castaneda, Serguei A.; Chui, David H.; Faller, Douglas V.; Berenson, Ronald J.; Fucharoen, Suthat

    2013-01-01

    Inducing expression of endogenous fetal globin (γ-globin) gene expression to 60-70% of alpha globin synthesis produces β-thalassemia trait globin synthetic ratios and can reduce anemia to a mild level. Several classes of therapeutics have induced γ-globin expression in beta thalassemia patients and subsequently raised total hemoglobin levels, demonstrating proof-of-concept of the approach. Butyrate treatment eliminated transfusion requirements in formerly transfusion-dependent patients with treatment for as long as 7 years. However, prior generations were not readily applicable for widespread use. Currently, a novel oral dual-action therapeutic sodium 2,2-dimethylbutyrate is in clinical trials, an oral decitabine formulation is under development, and agents with complementary mechanisms of action can be applied in combined regimens. Identification of 3 major genetic trait loci which modulate clinical severity provides avenues for developing tailored regimens. These refinements offer renewed potential to apply fetal globin induction as a treatment approach in patient-friendly regimens that can be used world-wide. PMID:20712788

  16. Neuroprotection in glaucoma

    PubMed Central

    Vasudevan, Sushil K; Gupta, Viney; Crowston, Jonathan G

    2011-01-01

    Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells and their axons. Recent evidence suggests that intraocular pressure (IOP) is only one of the many risk factors for this disease. Current treatment options for this disease have been limited to the reduction of IOP; however, it is clear now that the disease progression continues in many patients despite effective lowering of IOP. In the search for newer modalities in treating this disease, much data have emerged from experimental research the world over, suggesting various pathological processes involved in this disease and newer possible strategies to treat it. This review article looks into the current understanding of the pathophysiology of glaucoma, the importance of neuroprotection, the various possible pharmacological approaches for neuroprotection and evidence of current available medications. PMID:21150020

  17. Transcriptional promiscuity of the human /alpha/-globin gene

    SciTech Connect

    Whitelaw, E.; Hogben, P.; Hanscombe, O.; Proudfoot, N.J.

    1989-01-01

    The human /alpha/-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. The authors have investigated whether there is a relationship between these two observations. First, they investigated the mouse /alpha/-globin gene since it does not lie in an HTF island. They have demonstrated that it was not transcriptionally promiscuous. Second, they studied the transcriptional activity of the human /alpha/-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human /alpha/-globin gene. They found that in a nonreplicating system, the human //a/-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since they only observed enhancer independence of the human /alpha/-globin gene in a high-copy-number replicating system, they suggest that competition for trans-acting factors could explain these results. Finally, the authors' experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human /alpha/-globin cap site or within the gene itself.

  18. Human fetal globin gene expression is regulated by LYAR

    PubMed Central

    Ju, Junyi; Wang, Ying; Liu, Ronghua; Zhang, Yichong; Xu, Zhen; Wang, Yadong; Wu, Yupeng; Liu, Ming; Cerruti, Loretta; Zou, Fengwei; Ma, Chi; Fang, Ming; Tan, Renxiang; Jane, Stephen M.; Zhao, Quan

    2014-01-01

    Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene. PMID:25092918

  19. Human fetal globin gene expression is regulated by LYAR.

    PubMed

    Ju, Junyi; Wang, Ying; Liu, Ronghua; Zhang, Yichong; Xu, Zhen; Wang, Yadong; Wu, Yupeng; Liu, Ming; Cerruti, Loretta; Zou, Fengwei; Ma, Chi; Fang, Ming; Tan, Renxiang; Jane, Stephen M; Zhao, Quan

    2014-09-01

    Human globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5'-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene. PMID:25092918

  20. Alpha-globin loci in homozygous beta-thalassemia intermedia.

    PubMed

    Triadou, P; Lapoumeroulie, C; Girot, R; Labie, D

    1983-01-01

    Homozygous beta-thalassemia intermediate (TI) differs from thalassemia major (TM) in being less severe clinically. Associated alpha-thalassemia could account for the TI phenotype by reducing the alpha/non-alpha chain imbalance. We have analyzed the alpha loci of 9 TI and 11 TM patients by restriction endonuclease mapping. All the TM and 7 of the TI patients have the normal complement of four alpha-globin genes (alpha alpha/alpha alpha). One TI patient has three alpha-globin genes (alpha alpha/-alpha), and another TI patient has five alpha genes (alpha alpha/alpha alpha alpha). PMID:6305827

  1. Neuroprotection in Preterm Infants

    PubMed Central

    Berger, R.; Söder, S.

    2015-01-01

    Preterm infants born before the 30th week of pregnancy are especially at risk of perinatal brain damage which is usually a result of cerebral ischemia or an ascending intrauterine infection. Prevention of preterm birth and early intervention given signs of imminent intrauterine infection can reduce the incidence of perinatal cerebral injury. It has been shown that administering magnesium intravenously to women at imminent risk of a preterm birth leads to a significant reduction in the likelihood of the infant developing cerebral palsy and motor skill dysfunction. It has also been demonstrated that delayed clamping of the umbilical cord after birth reduces the rate of brain hemorrhage among preterm infants by up to 50%. In addition, mesenchymal stem cells seem to have significant neuroprotective potential in animal experiments, as they increase the rate of regeneration of the damaged cerebral area. Clinical tests of these types of therapeutic intervention measures appear to be imminent. In the last trimester of pregnancy, the serum concentrations of estradiol and progesterone increase significantly. Preterm infants are removed abruptly from this estradiol and progesterone rich environment. It has been demonstrated in animal experiments that estradiol and progesterone protect the immature brain from hypoxic-ischemic lesions. However, this neuroprotective strategy has unfortunately not yet been subject to sufficient clinical investigation. PMID:25650134

  2. Neuroprotective Actions of Neurosteroids

    PubMed Central

    Borowicz, Kinga K.; Piskorska, Barbara; Banach, Monika; Czuczwar, Stanislaw J.

    2011-01-01

    Neurosteroids were initially defined as steroid hormones locally synthesized within the nervous tissue. Subsequently, they were described as steroid hormone derivatives that devoid hormonal action but still affect neuronal excitability through modulation of ionotropic receptors. Neurosteroids are further subdivided into natural (produced in the brain) and synthetic. Some authors distinguish between hormonal and regular neurosteroids in the group of natural ones. The latter group, including hormone metabolites like allopregnanolone or tetrahydrodeoxycorticosterone, is devoid of hormonal activity. Both hormones and their derivatives share, however, most of the physiological functions. It is usually very difficult to distinguish the effects of hormones and their metabolites. All these substances may influence seizure phenomena and exhibit neuroprotective effects. Neuroprotection offered by steroid hormones may be realized in both genomic and non-genomic mechanisms and involve regulation of the pro- and anti-apoptotic factors expression, intracellular signaling pathways, neurotransmission, oxidative, and inflammatory processes. Since regular neurosteroids show no affinity for steroid receptors, they may act only in a non-genomic mode. Multiple studies have been conducted so far to show efficacy of neurosteroids in the treatment of the central and peripheral nervous system injury, ischemia, neurodegenerative diseases, or seizures. In this review we focused primarily on neurosteroid mechanisms of action and their role in the process of neurodegeneration. Most of the data refers to results obtained in experimental studies. However, it should be realized that knowledge about neuroactive steroids remains still incomplete and requires confirmation in clinical conditions. PMID:22649375

  3. Transcriptional activity of the human pseudogene psi alpha globin compared with alpha globin, its functional gene counterpart.

    PubMed Central

    Whitelaw, E; Proudfoot, N J

    1983-01-01

    Transcriptional analysis of the human pseudogene psi alpha globin has revealed the following features: (1) The promoter with a 23 bp deletion between the CCAAT and ATA boxes is functional both in vitro and in vivo, 3 fold and 10 fold less efficient, respectively, than alpha. (2) Both the psi alpha and alpha globin gene promoters are active in the absence of transcriptional enhancers, either a gene-encoded or viral enhancer. (3) The mutated poly(A) addition signal in psi alpha (AATGAA) appears to be completely nonfunctional. This result provides an explanation for the absence of psi alpha transcripts in human erythroid cells. Images PMID:6316269

  4. Transcriptional interference among the murine β-like globin genes

    PubMed Central

    Hu, Xiao; Eszterhas, Susan; Pallazzi, Nicolas; Bouhassira, Eric E.; Fields, Jennifer; Tanabe, Osamu; Gerber, Scott A.; Bulger, Michael; Engel, James Douglas; Groudine, Mark

    2007-01-01

    Mammalian β-globin loci contain multiple genes that are activated at different developmental stages. Studies have suggested that the transcription of one gene in a locus can influence the expression of the other locus genes. The prevalent model to explain this transcriptional interference is that all potentially active genes compete for locus control region (LCR) activity. To investigate the influence of transcription by the murine embryonic genes on transcription of the other β-like genes, we generated mice with deletions of the promoter regions of Ey and βh1 and measured transcription of the remaining genes. Deletion of the Ey and βh1 promoters increased transcription of βmajor and βminor 2-fold to 3-fold during primitive erythropoiesis. Deletion of Ey did not affect βh1 nor did deletion of βh1 affect Ey, but Ey deletion uniquely activated transcription from βh0, a β-like globin gene immediately downstream of Ey. Protein analysis showed that βh0 encodes a translatable β-like globin protein that can pair with alpha globin. The lack of transcriptional interference between Ey and βh1 and the gene-specific repression of βh0 did not support LCR competition among the embryonic genes and suggested that direct transcriptional interference from Ey suppressed βh0. PMID:17077320

  5. Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia.

    PubMed

    Bean, Christopher J; Boulet, Sheree L; Yang, Genyan; Payne, Amanda B; Ghaji, Nafisa; Pyle, Meredith E; Hooper, W Craig; Bhatnagar, Pallav; Keefer, Jeffrey; Barron-Casella, Emily A; Casella, James F; Debaun, Michael R

    2013-10-01

    Genetic diversity at the human β-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the β-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the β-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (β-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the β(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with β(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined β(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA. PMID:23952145

  6. Functional Identification of Neuroprotective Molecules

    PubMed Central

    Dai, Cheng; Liang, Dong; Li, Huiwu; Sasaki, Masayuki; Dawson, Ted M.; Dawson, Valina L.

    2010-01-01

    The central nervous system has the capacity to activate profound neuroprotection following sub-lethal stress in a process termed preconditioning. To gain insight into this potent survival response we developed a functional cloning strategy that identified 31 putative neuroprotective genes of which 28 were confirmed to provide protection against oxygen-glucose deprivation (OGD) or excitotoxic exposure to N-methyl-D-aspartate (NMDA) in primary rat cortical neurons. These results reveal that the brain possesses a wide and diverse repertoire of neuroprotective genes. Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease. PMID:21124846

  7. Therapeutic Hypothermia for Neuroprotection

    PubMed Central

    Karnatovskaia, Lioudmila V.; Wartenberg, Katja E.

    2014-01-01

    The earliest recorded application of therapeutic hypothermia in medicine spans about 5000 years; however, its use has become widespread since 2002, following the demonstration of both safety and efficacy of regimens requiring only a mild (32°C-35°C) degree of cooling after cardiac arrest. We review the mechanisms by which hypothermia confers neuroprotection as well as its physiological effects by body system and its associated risks. With regard to clinical applications, we present evidence on the role of hypothermia in traumatic brain injury, intracranial pressure elevation, stroke, subarachnoid hemorrhage, spinal cord injury, hepatic encephalopathy, and neonatal peripartum encephalopathy. Based on the current knowledge and areas undergoing or in need of further exploration, we feel that therapeutic hypothermia holds promise in the treatment of patients with various forms of neurologic injury; however, additional quality studies are needed before its true role is fully known. PMID:24982721

  8. Neuroprotection and antioxidants

    PubMed Central

    Lalkovičová, Maria; Danielisová, Viera

    2016-01-01

    Ischemia as a serious neurodegenerative disorder causes together with reperfusion injury many changes in nervous tissue. Most of the neuronal damage is caused by complex of biochemical reactions and substantial processes, such as protein agregation, reactions of free radicals, insufficient blood supply, glutamate excitotoxicity, and oxidative stress. The result of these processes can be apoptotic or necrotic cell death and it can lead to an irreversible damage. Therefore, neuroprotection and prevention of the neurodegeneration are highly important topics to study. There are several approaches to prevent the ischemic damage. Use of many modern therapeutical methods and the incorporation of several substances into the diet of patients is possible to stimulate the endogenous protective mechanisms and improve the life quality. PMID:27482198

  9. Neuroprotection and antioxidants.

    PubMed

    Lalkovičová, Maria; Danielisová, Viera

    2016-06-01

    Ischemia as a serious neurodegenerative disorder causes together with reperfusion injury many changes in nervous tissue. Most of the neuronal damage is caused by complex of biochemical reactions and substantial processes, such as protein agregation, reactions of free radicals, insufficient blood supply, glutamate excitotoxicity, and oxidative stress. The result of these processes can be apoptotic or necrotic cell death and it can lead to an irreversible damage. Therefore, neuroprotection and prevention of the neurodegeneration are highly important topics to study. There are several approaches to prevent the ischemic damage. Use of many modern therapeutical methods and the incorporation of several substances into the diet of patients is possible to stimulate the endogenous protective mechanisms and improve the life quality. PMID:27482198

  10. Neuroprotective potential of phytochemicals

    PubMed Central

    Kumar, G. Phani; Khanum, Farhath

    2012-01-01

    Cognitive dysfunction is a major health problem in the 21st century, and many neuropsychiatric disorders and neurodegenerative disorders, such as schizophrenia, depression, Alzheimer's Disease dementia, cerebrovascular impairment, seizure disorders, head injury and Parkinsonism, can be severly functionally debilitating in nature. In course of time, a number of neurotransmitters and signaling molecules have been identified which have been considered as therapeutic targets. Conventional as well newer molecules have been tried against these targets. Phytochemicals from medicinal plants play a vital role in maintaining the brain's chemical balance by influencing the function of receptors for the major inhibitory neurotransmitters. In traditional practice of medicine, several plants have been reported to treat cognitive disorders. In this review paper, we attempt to throw some light on the use of medicinal herbs to treat cognitive disorders. In this review, we briefly deal with some medicinal herbs focusing on their neuroprotective active phytochemical substances like fatty acids, phenols, alkaloids, flavonoids, saponins, terpenes etc. The resistance of neurons to various stressors by activating specific signal transduction pathways and transcription factors are also discussed. It was observed in the review that a number of herbal medicines used in Ayurvedic practices as well Chinese medicines contain multiple compounds and phytochemicals that may have a neuroprotective effect which may prove beneficial in different neuropsychiatric and neurodegenerative disorders. Though the presence of receptors or transporters for polyphenols or other phytochemicals of the herbal preparations, in brain tissues remains to be ascertained, compounds with multiple targets appear as a potential and promising class of therapeutics for the treatment of diseases with a multifactorial etiology. PMID:23055633

  11. Neuroprotective potential of phytochemicals.

    PubMed

    Kumar, G Phani; Khanum, Farhath

    2012-07-01

    Cognitive dysfunction is a major health problem in the 21st century, and many neuropsychiatric disorders and neurodegenerative disorders, such as schizophrenia, depression, Alzheimer's Disease dementia, cerebrovascular impairment, seizure disorders, head injury and Parkinsonism, can be severly functionally debilitating in nature. In course of time, a number of neurotransmitters and signaling molecules have been identified which have been considered as therapeutic targets. Conventional as well newer molecules have been tried against these targets. Phytochemicals from medicinal plants play a vital role in maintaining the brain's chemical balance by influencing the function of receptors for the major inhibitory neurotransmitters. In traditional practice of medicine, several plants have been reported to treat cognitive disorders. In this review paper, we attempt to throw some light on the use of medicinal herbs to treat cognitive disorders. In this review, we briefly deal with some medicinal herbs focusing on their neuroprotective active phytochemical substances like fatty acids, phenols, alkaloids, flavonoids, saponins, terpenes etc. The resistance of neurons to various stressors by activating specific signal transduction pathways and transcription factors are also discussed. It was observed in the review that a number of herbal medicines used in Ayurvedic practices as well Chinese medicines contain multiple compounds and phytochemicals that may have a neuroprotective effect which may prove beneficial in different neuropsychiatric and neurodegenerative disorders. Though the presence of receptors or transporters for polyphenols or other phytochemicals of the herbal preparations, in brain tissues remains to be ascertained, compounds with multiple targets appear as a potential and promising class of therapeutics for the treatment of diseases with a multifactorial etiology. PMID:23055633

  12. Zebrafish globin Switching Occurs in Two Developmental Stages and is Controlled by the LCR

    PubMed Central

    Ganis, Jared J.; Hsia, Nelson; Trompouki, Eirini; de Jong, Jill L.O.; DiBiase, Anthony; Lambert, Janelle S.; Jia, Zhiying; Sabo, Peter J.; Weaver, Molly; Sandstrom, Richard; Stamatoyannopoulos, John A.; Zhou, Yi; Zon, Leonard I.

    2012-01-01

    Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5′-3′ to 3′-5′ orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching. PMID:22537494

  13. A physiological delay in human fetal hemoglobin switching is associated with specific globin DNA hypomethylation.

    PubMed

    Perrine, S P; Greene, M F; Cohen, R A; Faller, D V

    1988-02-01

    The human fetal-to-adult globin switch normally occurs on a fixed schedule, beginning at 32-34 weeks gestation, and recent studies have suggested an association between this developmental inactivation of the fetal (gamma) globin genes and the appearance of methylation within and around these genes. We have studied a population of infants in whom this switch does not occur before birth (infants of diabetic mothers, IDM) and examined the patterns of methylation surrounding their active gamma-globin genes, in comparison to the gamma-globin genes of age-matched controls who have switched their pattern of globin gene expression on schedule. All genomic DNA samples from infants with delays in the globin switch demonstrated extensive hypomethylation in the region of the gamma-globin genes, comparable to that found in the genomes of fetuses of less than 21 weeks gestation. DNA from the erythroid cells of infants of 32-40 weeks gestation had no detectable hypomethylation in the gamma-globin region. These findings support the concept that hypomethylation is an accurate developmental marker of globin gene switching, and suggest that globin gene expression in IDM may be arrested at an early preswitch stage. PMID:2449361

  14. Insulin Neuroprotection and the Mechanisms

    PubMed Central

    Yu, Li-Yun; Pei, Yu

    2015-01-01

    Objective: To analyze the mechanism of neuroprotection of insulin and which blood glucose range was benefit for insulin exerting neuroprotective action. Data Sources: The study is based on the data from PubMed. Study Selection: Articles were selected with the search terms “insulin”, “blood glucose”, “neuroprotection”, “brain”, “glycogen”, “cerebral ischemia”, “neuronal necrosis”, “glutamate”, “γ-aminobutyric acid”. Results: Insulin has neuroprotection. The mechanisms include the regulation of neurotransmitter, promoting glycogen synthesis, and inhibition of neuronal necrosis and apoptosis. Insulin could play its role in neuroprotection by avoiding hypoglycemia and hyperglycemia. Conclusions: Intermittent and long-term infusion insulin may be a benefit for patients with ischemic brain damage at blood glucose 6–9 mmol/L. PMID:25836621

  15. Alternative sites of transcription initiation upstream of the canonical cap site in human gamma-globin and beta-globin genes.

    PubMed Central

    Grindlay, G J; Lanyon, W G; Allan, M; Paul, J

    1984-01-01

    Using S1 mapping and primer extension analysis, we have identified a number of human kappa-globin and beta-globin 5' RNA termini originating in the 200 bp upstream of the canonical mRNA cap sites. Upstream initiation sites have previously been reported for the human epsilon-globin gene (4,5) and the present work indicates that this is a general feature of the human beta-type globin genes. We have attempted to identify features common to such sites between the three genes. One site 170 bp upstream of the major beta-globin cap site and a site 1400 bp upstream of the major epsilon-globin cap site are located near putative PolIII promoter sequences and may therefore be transcribed by this enzyme. Alternative initiation sites located 200 bp and 50-100 bp upstream of the epsilon-globin and kappa-globin cap sites respectively are located within S1 hypersensitive regions of chromatin. Images PMID:6701091

  16. Butyrate Infusions in the Ovine Fetus Delay the Biologic Clock for Globin Gene Switching

    NASA Astrophysics Data System (ADS)

    Perrine, Susan P.; Rudolph, Abraham; Faller, Douglas V.; Roman, Christine; Cohen, Ruth A.; Chen, Shao-Jing; Kan, Yuet Wai

    1988-11-01

    The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal β -globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switching.

  17. Microglia and neuroprotection.

    PubMed

    Chen, Zhihong; Trapp, Bruce D

    2016-01-01

    Microglia were first identified over a century ago, but our knowledge about their ontogeny and functions has significantly expanded only recently. Microglia colonize the central nervous system (CNS) in utero and play essential roles in brain development. Once neural development is completed, microglia function as the resident innate immune cells of the CNS by surveying their microenvironment and becoming activated when the CNS is challenged by infection, injury, or disease. Despite the traditional view of microglia as being destructive in neurological diseases, recent studies have shown that microglia maintain CNS homeostasis and protect the CNS under various pathological conditions. Microglia can be prophylactically activated by modeling infection with systemic lipopolysaccharide injections and these activated microglia can protect the brain from traumatic injury through modulation of neuronal synapses. Microglia can also protect the CNS by promoting neurogenesis, clearing debris, and suppressing inflammation in diseases such as stroke, autism, and Alzheimer's. Microglia are the resident innate immune cells of the CNS. Despite the traditional view of microglia as being destructive in neurological diseases, recent studies have shown that they maintain tissue homeostasis and protect the CNS under various pathological conditions. They achieve so by clearing debris, promoting neurogenesis, suppressing inflammation and stripping inhibitory synapses. This review summarizes recent advances of our understanding on the multi-dimensional neuroprotective roles of microglia. PMID:25693054

  18. Genomic evidence for independent origins of β-like globin genes in monotremes and therian mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    Phylogenetic reconstructions of the β-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult β-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic β-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the “ε-globin” gene in birds is not orthologous to the ε-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto β-globin gene. Here, we report evidence that the early and late expressed β-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto β-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the ε- and β-globin genes of therian mammals arose via duplication of a proto β-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of β-like globin genes that originated via duplication of a proto β-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto β-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development. PMID:18216242

  19. The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification

    PubMed Central

    2008-01-01

    Background Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles. Results In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection. Conclusion Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying

  20. The Evolution of the Globins: We Thought We Understood It

    NASA Astrophysics Data System (ADS)

    Lesk, Arthur M.

    Protein crystallography achieved its first results in the late 1950s with the structure determinations of sperm whale myoglobin and human haemoglobin. These gave us our first glimpse of the structural changes that take place during protein evolution. Many other structures of proteins in the globin family have continued to reveal interesting and important details of the coordinated divergence during evolution of amino acid sequences and protein structures and functions.

  1. Decitabine Increases Fetal Hemoglobin in P. Anubis by Increasing γ-globin Gene Transcription

    PubMed Central

    Akpan, Imo; Banzon, Virryan; Ibanez, Vinzon; Vaitkus, Kestis; DeSimone, Joseph; Lavelle, Donald

    2014-01-01

    1) Objective The mechanism responsible for increased fetal hemoglobin (HbF) levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional versus translational mechanisms in the ability of decitabine to increase HbF levels in vivo. 2) Methods Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin mRNA levels was measured in pre- and post-treatment bone marrow (BM) aspirates by biosynthetic radiolabelling with [3H] leucine followed by separation of globin chains by HPLC, and real time PCR, respectively. The effect on DNA methylation of the ε- and γ-globin gene promoters was determined by bisulfite sequence analysis. 3) Results Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+β chain synthetic ratio and the γ/total β-like globin RNA ratio and also increased expression of ε-globin transcripts. Increased expression of ε- and γ-globin was associated with decreased DNA methylation of the ε- and γ-globin gene promoters. 4) Conclusion Decitabine increases HbF in vivo by transcriptional activation of the γ-globin gene. PMID:20713129

  2. Globin-coupled sensors, protoglobins, and the last universal common ancestor.

    PubMed

    Freitas, Tracey Allen K; Saito, Jennifer A; Hou, Shaobin; Alam, Maqsudul

    2005-01-01

    The strategy for detecting oxygen, carbon monoxide, nitric oxide, and sulfides is predominantly through heme-based sensors utilizing either a globin domain or a PAS domain. Whereas PAS domains bind various cofactors, globins bind only heme. Globin-coupled sensors (GCSs) were first described as regulators of the aerotactic responses in Bacillus subtilis and Halobacterium salinarum. GCSs were also identified in diverse microorganisms that appear to have roles in regulating gene expression. Functional and evolutionary analyses of the GCSs, their protoglobin ancestor, and their relationship to the last universal common ancestor (LUCA) are discussed in the context of globin-based signal transduction. PMID:15598488

  3. Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH.

    PubMed

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D; Peterson, Kenneth R

    2016-04-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the (A)γ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the (A)γ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of (A)γ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to

  4. Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: −175 Black HPFH and −195 Brazilian HPFH

    PubMed Central

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D; Peterson, Kenneth R

    2016-01-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the Aγ-globin −175 T >C or −195 C >G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult −175 and −195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In −175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the Aγ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of Aγ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study

  5. Clinical trials for neuroprotection in ALS.

    PubMed

    Siciliano, G; Carlesi, C; Pasquali, L; Piazza, S; Pietracupa, S; Fornai, F; Ruggieri, S; Murri, L

    2010-07-01

    Owing to uncertainty on the pathogenic mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) riluzole remains the only available therapy, with only marginal effects on disease survival. Here we review some of the recent advances in the search for disease-modifying drugs for ALS based on their putative neuroprotective effetcs. A number of more or less established agents have recently been investigated also in ALS for their potential role in neuroprotection and relying on antiglutamatergic, antioxidant or antiapoptotic strategies. Among them Talampanel, beta-lactam antibiotics, Coenzyme Q10, and minocycline have been investigated. Progress has also been made in exploiting growth factors for the treatment of ALS, partly due to advances in developing effective delivery systems to the central nervous system. A number of new therapies have also been identified, including a novel class of compounds, such as heat-shock protein co-inducers, which upregulate cell stress responses, and agents promoting autophagy and mitochondriogenesis, such as lithium and rapamycin. More recently, alterations of mRNA processing were described as a pathogenic mechanism in genetically defined forms of ALS, as those related to TDP-43 and FUS-TLS gene mutations. This knowledge is expected to improve our understanding of the pathogenetic mechanism in ALS and developing more effective therapies. PMID:20406180

  6. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

    PubMed

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  7. Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation

    PubMed Central

    Ruiz, Maria Armila; Rivers, Angela; Ibanez, Vinzon; Vaitkus, Kestis; Mahmud, Nadim; DeSimone, Joseph; Lavelle, Donald

    2015-01-01

    The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5mC) and 5hmC at a CCGG site within the 5′ γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5mC and 5hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5hmC and negatively correlated with 5mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter. PMID:25932923

  8. Hypothermia as a clinical neuroprotectant.

    PubMed

    Sherman, Andrew L; Wang, Michael Y

    2014-08-01

    Applying therapeutic hypothermia (TH) for the purposes of neuroprotection, originally termed "hibernation," started nearly 100 years ago. Because TH cooling systems have improved to the point where it is practical and safe for general application, interest in providing such treatment in conditions such as spinal cord injury, traumatic brain injury, stroke, and cardiac arrest has increased. This article reviews the mechanisms by which TH mitigates secondary neurologic injury, the clinical scenarios where TH is being applied, and reviews selected published studies using TH for central nervous system neuroprotection. PMID:25064786

  9. The 3' noncoding region of beta-globin mRNA is not essential for in vitro translation.

    PubMed Central

    Kronenberg, M N; Roberts, B E; Efstratiadis, A

    1979-01-01

    Rabbit beta globin DNA sequence, excised from plasmid pbetaG1, directs in vitro synthesis of beta-globin in a transcription-translation cell-free system, even after specific elimination of the entire 3'-noncoding region. A DNA restriction fragment carrying this 3' noncoding region and hybridized to globin mRNA cannot arrest the cell-free translation of beta-globin mRNA. Images PMID:424286

  10. Neuroprotective therapies in glaucoma: II. Genetic nanotechnology tools

    PubMed Central

    Nafissi, Nafiseh; Foldvari, Marianna

    2015-01-01

    Neurotrophic factor genome engineering could have many potential applications not only in the deeper understanding of neurodegenerative disorders but also in improved therapeutics. The fields of nanomedicine, regenerative medicine, and gene/cell-based therapy have been revolutionized by the development of safer and efficient non-viral technologies for gene delivery and genome editing with modern techniques for insertion of the neurotrophic factors into clinically relevant cells for a more sustained pharmaceutical effect. It has been suggested that the long-term expression of neurotrophic factors is the ultimate approach to prevent and/or treat neurodegenerative disorders such as glaucoma in patients who do not respond to available treatments or are at the progressive stage of the disease. Recent preclinical research suggests that novel neuroprotective gene and cell therapeutics could be promising approaches for both non-invasive neuroprotection and regenerative functions in the eye. Several progenitor and retinal cell types have been investigated as potential candidates for glaucoma neurotrophin therapy either as targets for gene therapy, options for cell replacement therapy, or as vehicles for gene delivery. Therefore, in parallel with deeper understanding of the specific protective effects of different neurotrophic factors and the potential therapeutic cell candidates for glaucoma neuroprotection, the development of non-invasive and highly specific gene delivery methods with safe and effective technologies to modify cell candidates for life-long neuroprotection in the eye is essential before investing in this field. PMID:26528114

  11. Molecular nature of alpha-globin genes in the Saudi population

    PubMed Central

    Borgio, J. Francis

    2015-01-01

    Alpha-thalassemia (α-thal) is a disorder caused by the deletion of single or double α-globin genes, and/or point mutations in the α-globin genes. There are 2 common types of α-globin genes; HBA2 and HBA1. Recently, it has been discovered that the HBA2 gene is replaced by a unique HBA12 gene convert in 5.7% of the Saudi population. The α-globin genes have been emerging as a molecular target for the treatment of β-thalassemia (β-thal). Hence, it is essential to understand the molecular nature of α-globin genes to treat the most prevalent hemoglobin disorders, such as sickle cell disease, α-thal, and β-thal prevalent in the Kingdom of Saudi Arabia. Thirty-two different α-globin genotypes have been observed in the Saudi population. This review outlines the classification of the α-globin genes on the basis of their molecular nature and complex combinations of α-globin genes, and their variants predominant in Saudis. PMID:26593158

  12. Genomic remnants of alpha-globin genes in the hemoglobinless antarctic icefishes.

    PubMed Central

    Cocca, E; Ratnayake-Lecamwasam, M; Parker, S K; Camardella, L; Ciaramella, M; di Prisco, G; Detrich, H W

    1995-01-01

    Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade. Images Fig. 2 Fig. 3 Fig. 4 PMID:7892183

  13. Identification and characterization of globin genes from two lepidopteran insects, Bombyx mori and Samia cynthia ricini.

    PubMed

    Kawaoka, Shinpei; Katsuma, Susumu; Meng, Yan; Hayashi, Nobumitsu; Mita, Kazuei; Shimada, Toru

    2009-02-15

    We describe the characterization of hemoglobin-like genes from two lepidopteran insects, Bombyx mori (Bmglobin) and Samia cynthia ricini (Scglobin). Bmglobin and Scglobin are predicted to be intracellular proteins and contain amino acids required for heme and oxygen binding. Expression profiles of two lepidopteran globins, especially Bmglobin, were different from that of other insect globins. Although other insect globins are mainly associated with the tracheal system, Bmglobin was expressed almost exclusively in the Malpighian tubules, and the strongest signal for Scglobin was detected in the fat body. Furthermore, biochemical fractionation analysis revealed that both Bmglobin and Scglobin were localized in the cytoplasm. These results suggest that each lepidopteran globin has a distinct role in the tissues in which it is expressed and that the functions of insect globins are more divergent than previously thought. PMID:19059317

  14. Locus assignment of alpha-globin structural mutations by hybrid-selected translation.

    PubMed Central

    Liebhaber, S A; Cash, F E

    1985-01-01

    The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants. Images PMID:2981252

  15. Methylation of alpha-type embryonic globin gene alpha pi represses transcription in primary erythroid cells.

    PubMed

    Singal, Rakesh; vanWert, Jane M; Ferdinand, Larry

    2002-12-01

    The inverse relationship between expression and methylation of beta-type globin genes is well established. However, little is known about the relationship between expression and methylation of avian alpha-type globin genes. The embryonic alpha(pi)-globin promoter was unmethylated, and alpha(pi)-globin RNA was easily detected in 5-day chicken erythroid cells. A progressive methylation of the CpG dinucleotides in the alpha(pi) promoter associated with loss of expression of alpha(pi)-globin gene was seen during development in primary erythroid cells. A 315-bp alpha(pi)-globin promoter region was cloned in an expression construct (alpha(pi)pGL3E) containing a luciferase reporter gene and SV40 enhancer. The alpha(pi)pGL3E construct was transfected into primary erythroid cells derived from 5-day-old chicken embryos. Methylation of alpha(pi)pGL3E plasmid and alpha(pi)-globin promoter alone resulted in a 20-fold and 7-fold inhibition of expression, respectively. The fully methylated but not the unmethylated 315-bp alpha(pi)-globin gene promoter fragment formed a methyl cytosine-binding protein complex (MeCPC). Chromatin immunoprecipitation assays were combined with quantitative real-time polymerase chain reaction to assess histone acetylation associated with the alpha(pi)-globin gene promoter. Slight hyperacetylation of histone H3 but a marked hyperacetylation of histone H4 was seen in 5-day when compared with 14-day erythroid cells. These results demonstrate that methylation can silence transcription of an avian alpha-type embryonic globin gene in homologous primary erythroid cells, possibly by interacting with an MeCPC and histone deacetylase complex. PMID:12393573

  16. Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition.

    PubMed

    Hoffmann, Federico G; Opazo, Juan C; Hoogewijs, David; Hankeln, Thomas; Ebner, Bettina; Vinogradov, Serge N; Bailly, Xavier; Storz, Jay F

    2012-07-01

    In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available

  17. Hb Wilde and Hb Patagonia: two novel elongated beta-globin variants causing dominant beta-thalassemia.

    PubMed

    Scheps, Karen G; Hasenahuer, Marcia A; Parisi, Gustavo; Fornasari, María S; Pennesi, Sandra P; Erramouspe, Beatriz; Basack, Felisa N; Veber, Ernesto S; Aversa, Luis; Elena, Graciela; Varela, Viviana

    2015-06-01

    We describe here the molecular and hematological characteristics of novel frameshift mutations in exon 2 of the HBB gene (in heterozygous state) found in two Argentinean pediatric patients with dominant β-thalassemia-like features. In Hb Wilde, HBB:c.270_273delTGAG(p.Glu90Cysfs*67), we detected the deletion of the third base of the codon 89 (T) and the codon 90 (GAG), whereas in Hb Patagonia, HBB:c.296_297dupGT(p.Asp99Trpfs*59), the frameshift mutation was due to a duplication of a 'GT' dinucleotide after the second base of codon 98 (GTG). The Hb Patagonia and Hb Wilde mutations would result in elongated β-globin chains with modified C-terminal sequences and a total of 155 and 157 amino acids residues, respectively. Based on bioinformatics and structural analysis, as well as protein modeling, we predict that the elongated β-globins would affect the formation of the αβ dimers and their stability, which would further support the mechanism for the observed clinical features in both patients. PMID:25284604

  18. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.

    PubMed

    Montagner, Giulia; Gemmo, Chiara; Fabbri, Enrica; Manicardi, Alex; Accardo, Igea; Bianchi, Nicoletta; Finotti, Alessia; Breveglieri, Giulia; Salvatori, Francesca; Borgatti, Monica; Lampronti, Ilaria; Bresciani, Alberto; Altamura, Sergio; Corradini, Roberto; Gambari, Roberto

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  19. Hemoglobin Agenogi--A rare abnormal beta globin chain variant.

    PubMed

    Sharma, Sunita; Sharma, Geetika; Chandra, Jagdish; Colah, Roshan

    2016-01-01

    Haemoglobin (Hb) Agenogi is clinically asymptomatic, rare β-globin chain variant characterized by a substitution of glutamic acid by lysine at position 90 of β-chain. It elutes in the C-window on high-performance liquid chromatography (HPLC). We report a 10-year-old male with easy fatigability, lethargy, pallor, and mild splenomegaly. Hematological parameters revealed microcytic hypochromic anemia and mildly raised red blood cells count, suggestive of thalassemia trait. On HPLC, a predominant peak was observed in the C-window (82.6%) along with raised HbA 2 level (9.3%). Based on these findings, a possibility of HbC disease/β-thalassemia trait doubly heterozygous was considered. Family studies were advised. HPLC findings in father were suggestive of β-thalassemia trait, while both his mother and brother had an abnormal peak in the C-window of 42.7% and 40.8%, respectively, with elevated HbA 2 values of 5% and 4.9%, respectively. Direct DNA sequencing revealed intervening sequences 1-5 (G ; C) in father, confirming β-thalassemia trait. His mother and brother had heterozygous gene mutation at codon 90 of β-globin chain (G ; A) suggestive of Hb Agenogi. The child carried mutations for both β-thalassemia trait as well as Hb Agenogi. PMID:26960650

  20. Intronless β-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements.

    PubMed

    Brown, Jessica A; Steitz, Joan A

    2016-01-01

    The intronless β-globin reporter, whose mRNA is intrinsically unstable due to the lack of introns, is a useful tool to study RNA stability elements in a heterologous transcript. Insertion of a stability element leads to the accumulation of intronless β-globin mRNA that can be visualized by conventional Northern blot analyses. In this chapter, we explain how to perform the β-globin reporter assay using the ENE (expression and nuclear retention element), a triple-helix-forming RNA stability element that protects reporter mRNA from 3'- 5' decay. A list of considerations is included for the use of ENEs as a tool to stabilize other RNAs. In this chapter, we provide a brief description of how to insert an ENE sequence into the 3'-untranslated region of an intronless β-globin reporter plasmid using basic cloning technology. Then, we provide a detailed protocol for quantitative measurements of steady-state levels of β-globin mRNA. This entails the transient transfection of mammalian cells with β-globin reporter plasmids, isolation of total cellular RNA, and detection of reporter mRNA via Northern blot. This methodology can be applied for the study of any nuclear RNA stability element using the intronless β-globin reporter. PMID:27236793

  1. Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG).

    PubMed

    Singh, Neha Atulkumar; Mandal, Abul Kalam Azad; Khan, Zaved Ahmed

    2016-01-01

    Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) enforce an overwhelming social and economic burden on society. They are primarily characterized through the accumulation of modified proteins, which further trigger biological responses such as inflammation, oxidative stress, excitotoxicity and modulation of signalling pathways. In a hope for cure, these diseases have been studied extensively over the last decade to successfully develop symptom-oriented therapies. However, so far no definite cure has been found. Therefore, there is a need to identify a class of drug capable of reversing neural damage and preventing further neural death. This review therefore assesses the reliability of the neuroprotective benefits of epigallocatechin-gallate (EGCG) by shedding light on their biological, pharmacological, antioxidant and metal chelation properties, with emphasis on their ability to invoke a range of cellular mechanisms in the brain. It also discusses the possible use of nanotechnology to enhance the neuroprotective benefits of EGCG. PMID:27268025

  2. Duality of Antidepressants and Neuroprotectants.

    PubMed

    Tizabi, Yousef

    2016-07-01

    The co-morbidity of neuropsychiatric disorders, particularly major depressive disorder (MDD) with neurodegenerative diseases, in particular Parkinson's disease (PD) is now well recognized. Indeed, it is suggested that depressive disorders, especially in late life, may be an indication of latent neurodegeneration. Thus, it is not unreasonable to expect that deterrents of MDD may also deter the onset and/or progression of the neurodegenerative diseases including PD. In this review, examples of neuroprotective efficacy of established as well as prospective antidepressants are provided. Conversely, mood-regulating effects of some neuroprotective drugs are also presented. Thus, in addition to currently used antidepressants, ketamine, nicotine, curcumin, and resveratrol are discussed for their dual efficacy. In addition, potential neurobiological substrates for their actions are presented. It is concluded that pharmacological developments of mood-regulating or neuroprotective drugs can have cross benefit in co-morbid conditions of neuropsychiatric and neurodegenerative disorders and that inflammatory and neurotrophic factors play important roles in both conditions. PMID:26613895

  3. Transcription in vivo of an Alu family member upstream from the human epsilon-globin gene.

    PubMed Central

    Allan, M; Paul, J

    1984-01-01

    The more distal Alu repeat flanking the epsilon-globin gene is transcribed in K562 cells to generate transcripts 350-400 nucleotides in length. Initiation occurs at the start of the repeat, upstream of a putative PolIII control signal. These transcripts originate from the strand which does not code epsilon-globin and are oriented in the opposite direction from the gene. They are non-polyadenylated, nucleus-confined and are only detectable in association with expression of the epsilon-globin gene. Images PMID:6320117

  4. β-Thalassemia Due to Intronic LINE-1 Insertion in the β-Globin Gene (HBB): Molecular Mechanisms Underlying Reduced Transcript Levels of the β-GlobinL1 Allele

    PubMed Central

    Lanikova, Lucie; Kucerova, Jana; Indrak, Karel; Divoka, Martina; Issa, Jean-Pierre; Papayannopoulou, Thalia; Prchal, Josef T.; Divoky, Vladimir

    2016-01-01

    We describe the molecular etiology of β+-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globinL1 allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globinL1 allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globinL1 transcription despite permanent β-globinL1 promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globinL1 allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia. PMID:23878091

  5. Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome

    PubMed Central

    Opazo, Juan C.; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F.

    2015-01-01

    Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about

  6. The 5' flanking region of human epsilon-globin gene.

    PubMed Central

    Baralle, F E; Shoulders, C C; Goodbourn, S; Jeffreys, A; Proudfoot, N J

    1980-01-01

    The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat. Images PMID:6253916

  7. alpha-Thalassemia caused by an unstable alpha-globin mutant.

    PubMed Central

    Liebhaber, S A; Kan, Y W

    1983-01-01

    In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins. Images PMID:6826718

  8. Structure and Properties of a Bis-Histidyl Ligated Globin from Caenorhabditis elegans†

    PubMed Central

    Yoon, Jungjoo; Herzik, Mark A.; Winter, Michael B.; Tran, Rosalie; Olea, Charles; Marletta, Michael A.

    2010-01-01

    Globins are heme-containing proteins that are best known for their roles in oxygen (O2) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes have been recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O2 sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN− do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O2 levels as well as a low redox potential (−193 ± 2 mV). A high resolution (1.40 Å) crystal structure of the ferric form of the heme-domain of GLB-6 confirms both the putative globin-fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O2 in C. elegans via redox signaling and/or electron transfer. PMID:20518498

  9. Characterization of histone H3K27 modifications in the {beta}-globin locus

    SciTech Connect

    Kim, Yea Woon; Kim, AeRi

    2011-02-11

    Research highlights: {yields} The {beta}-globin locus control region is hyperacetylated and monomethylated at histone H3K27. {yields} Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. {yields} Association of PRC2 subunits is comparable with H3K27me3 pattern. {yields} Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human {beta}-globin locus using the ChIP assay. The LCR of the human {beta}-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the {beta}-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  10. Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation.

    PubMed

    Mabaera, Rodwell; Richardson, Christine A; Johnson, Kristin; Hsu, Mei; Fiering, Steven; Lowrey, Christopher H

    2007-08-15

    The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching. PMID:17456718

  11. Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA.

    PubMed Central

    Wilson, J T; deRiel, J K; Forget, B G; Marotta, C A; Weissman, S M

    1977-01-01

    We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A). Images PMID:909779

  12. KLF1 stabilizes GATA-1 and TAL1 occupancy in the human β-globin locus.

    PubMed

    Kang, Yujin; Kim, Yea Woon; Yun, Jangmi; Shin, Jongo; Kim, AeRi

    2015-03-01

    KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the β-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the β-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the β-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the β-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping. PMID:25528728

  13. Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

    PubMed

    Dai, Yan; Sangerman, Jose; Luo, Hong Yuan; Fucharoen, Suthat; Chui, David H K; Faller, Douglas V; Perrine, Susan P

    2016-01-01

    Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy. PMID:26603726

  14. Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

    PubMed Central

    Lee, Tze Yan; Muniandy, Logeswaran; Teh, Lai Kuan; Abdullah, Maha; George, Elizabeth; Sathar, Jameela; Lai, Mei I

    2016-01-01

    Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia. PMID:26377036

  15. [γ-Globin Inductive Therapy of β-thalassemia and Its Relationship with MicroRNA].

    PubMed

    Li, Yao-Yao; Gu, Jian; Yu, Duo-Nan

    2016-04-01

    β-thalassemia is a chronic hemolytic anemia characterized by the reduction or absence of synthesis of β-globin chains because of the β-globin gene mutations. β-thalassemia belongs to the inherited hemoglobin disease, and occurs in some provinces of China, such as in Guangdong, Guangxi, Fujian, its prevalence is about 2%. The treatment of this disease include transfusion, iron chelating agent, hematopoietic stem cell transplantation, splenectomy, induced expression of Fetal Hemoglobin (HbF) and gene therapies. However, the mortality rate of this disease is still higher, thus some new treatments are urgently needed. In recent years, the study was mainly concentrated in 2 aspects: the normal β-globin gene transfer and endogenous γ-globin re-activation. Some studies showed that the expression of miRNAs was dysregulated in β-thalassemia. Some miRNAs could regulate γ-globin at posttranscriptional level, thus, the clarification of relationship between miRNAs and β-thalassemia is expected to provide experimental bases to β-thalassemia therapy. In this review, the induced therapy of γ-globin for β-thalassemia and its relationship with the miRNA are summarized. PMID:27151042

  16. Pharmacological Induction of Human Fetal Globin Gene in Hydroxyurea-Resistant Primary Adult Erythroid Cells.

    PubMed

    Chou, Yu-Chi; Chen, Ruei-Lin; Lai, Zheng-Sheng; Song, Jen-Shin; Chao, Yu-Sheng; Shen, Che-Kun James

    2015-07-01

    Pharmacological induction of the fetal γ globin gene and the consequent formation of HbF (α2/γ2) in adult erythroid cells are one feasible therapeutic strategy for sickle cell disease (SCD) and severe β-thalassemias. Hydroxyurea (HU) is the current drug of choice for SCD, but serious side effects limit its clinical use. Moreover, 30 to 50% of patients are irresponsive to HU treatment. We have used high-throughput screening to identify benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one and its derivatives (compounds I to VI) as potent γ globin inducers. Of the compounds, I to V exert superior γ globin induction and have better therapeutic potential than HU, likely because of their activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and modulation of expression levels and/or chromosome binding of γ globin gene regulators, including BCL11A, and chromatin structure over the γ globin promoter. Unlike sodium butyrate (NaB), the global levels of acetylated histones H3 and H4 are not changed by compound II treatment. Remarkably, compound II induces the γ globin gene in HU-resistant primary human adult erythroid cells, the p38 signaling pathway of which appears to be irresponsive to HU and NaB as well as compound II. This study provides a new framework for the development of new and superior compounds for treating SCD and severe β-thalassemias. PMID:25986606

  17. Understanding α-globin gene regulation and implications for the treatment of β-thalassemia.

    PubMed

    Mettananda, Sachith; Gibbons, Richard J; Higgs, Douglas R

    2016-03-01

    Over the past three decades, a vast amount of new information has been uncovered describing how the globin genes are regulated. This knowledge has provided significant insights into the general understanding of the regulation of human genes. It is now known that molecular defects within and around the α- and β-globin genes, as well as in the distant regulatory elements, can cause thalassemia. Unbalanced production of globin chains owing to defective synthesis of one, and the continued unopposed synthesis of another, is the central causative factor in the cellular pathology and pathophysiology of thalassemia. A large body of clinical, genetic, and experimental evidence suggests that altering globin chain imbalance by reducing the production of α-globin synthesis ameliorates the disease severity in patients with β-thalassemia. With the development of new genetic-based therapeutic tools that have a potential to decrease the expression of a selected gene in a tissue-specific manner, the possibility of decreasing expression of the α-globin gene to improve the clinical severity of β-thalassemia could become a reality. PMID:26695885

  18. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    PubMed

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  19. Neuroprotective compounds of Tilia amurensis

    PubMed Central

    Lee, Bohyung; Weon, Jin Bae; Eom, Min Rye; Jung, Youn Sik; Ma, Choong Je

    2015-01-01

    Background: Tilia amurensis (Tiliacese) has been used for anti-tumor and anti-inflammatory in Korea, China, and Japan. Objective: In this study, we isolated five compounds from T. amurensis and determined whether protected neuronal cells against glutamate-induced oxidative stress in HT22 cells. Materials and Methods: Compounds were isolated using chromatographic techniques including silica gel, Sephadex LH-20 open column and high performance liquid chromatography analysis, and evaluated neuroprotective effect in HT22 cells by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Results: β-D-fructofuranosyl α-D-glucopyranoside (1), (-)-epicatechin (2), nudiposide (3), lyoniside (4), and scopoletin (5) were isolated by bioactivity-guided fractionation from the ethyl acetate fraction of T. amurensis. Among them, (-)-epicatechin, nudiposide, lyoniside, and scopoletin had significant neuroprotective activities against glutamate-injured neurotoxicity in HT22 cells. Conclusion: These results demonstrated that compound two, three, four, and five have a pronounced protective effect against glutamate-induced neurotoxicity in HT22 cells. PMID:26664019

  20. Neuroprotection in vascular dementia: a future path.

    PubMed

    Skoog, Ingmar; Korczyn, Amos D; Guekht, Alla

    2012-11-15

    The burden of cognitive disorders is likely to increase over the coming years due to both increased longevity and altered risk factor patterns, arising from changes in lifestyle, healthcare and society. Vascular dementia with its underlying heterogeneous pathology, is a challenge for clinicians, and is frequently further aggravated by overlap with other neurodegenerative processes. Current Alzheimer's disease drugs have had limited clinical efficacy in treating vascular dementia and none have been approved by major regulatory authorities specifically for this disease. Moving forward, a valid choice may be a multimodal therapy, as has already been successfully proven in Alzheimer's disease. Actovegin, a hemodialysate derived from calf blood, has been shown to have effects on a variety of cellular processes and a recent experimental study has revealed its neuroprotective mechanisms of action. These data, coupled with positive results from clinical trials in mixed dementia populations, have served as a foundation for the design of a new trial investigating the efficacy and disease-modifying effects of Actovegin in post-stroke cognitive impairment. PMID:22472726

  1. Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

    PubMed Central

    2012-01-01

    Background The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation. Results We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified. The same approach was used to generate profile-2

  2. Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

    PubMed Central

    Bruno, J; Reich, N; Lucas, J J

    1981-01-01

    The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed. Images PMID:7346715

  3. Globin mRNAs are primers for the transcription of influenza viral RNA in vitro

    PubMed Central

    Bouloy, Michele; Plotch, Stephen J.; Krug, Robert M.

    1978-01-01

    Because influenza viral RNA transcription in vitro is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by the host cell, specifically by RNA polymerase II, thereby explaining the α-amanitin sensitivity of viral RNA transcription in vivo. Here, we identify such primer RNAs, initially in reticulocyte extracts, where they are shown to be globin mRNAs. Purified globin mRNAs very effectively stimulated viral RNA transcription in vitro, and the resulting transcripts directed the synthesis of all the nonglycosylated virus-specific proteins in micrococcal nuclease-treated L cell extracts. The viral RNA transcripts synthesized in vitro primed by ApG also directed the synthesis of the nonglycosylated virus-specific proteins, but the globin mRNA-primed transcripts were translated about 3 times more efficiently. The translation of the globin mRNA-primed, but not the ApG-primed, viral RNA transcripts was inhibited by 7-methylguanosine 5′-phosphate in the presence of S-adenosylhomocysteine, suggesting that the globin mRNA-primed transcripts contained a 5′-terminal methylated cap structure. We propose that this cap was transferred from the globin mRNA primer to the newly synthesized viral RNA transcripts, because no detectable de novo synthesis of a methylated cap occurred during globin mRNA-primed viral RNA transcription. Preliminary experiments indicate that other purified eukaryotic mRNAs also stimulate influenza viral RNA transcription in vitro. Images PMID:283399

  4. Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

    PubMed Central

    Villeponteau, B; Martinson, H

    1981-01-01

    A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts. Images PMID:6269092

  5. Neuroprotective effects of Lepidium meyenii (Maca).

    PubMed

    Pino-Figueroa, Alejandro; Nguyen, Diane; Maher, Timothy J

    2010-06-01

    The neuroprotective activity of the plant Lepidium meyenii (Maca) was studied in two experimental models: in vitro and in vivo. Crayfish neurons were pretreated with vehicle or the pentane extract from Maca, subjected to H(2)O(2), and their viability determined microscopically and chemically. A significant concentration-neuroprotective effect relationship was demonstrated. The pentane extract was then administered intravenously to rats prior to and following middle cerebral artery occlusion. While infarct volumes were decreased for the lower dose, higher doses increased infarct volumes compared to controls. These results suggest a potential application of Maca as a neuroprotectant. PMID:20633111

  6. Neurodegeneration and Neuroprotection in Glaucoma.

    PubMed

    Gauthier, Angela C; Liu, Ji

    2016-03-01

    Glaucoma is the principal cause of irreversible blindness in the world. The disease leads to progressive optic nerve degeneration with a gradual loss of retinal ganglion cells. Neurodegeneration in glaucoma extends beyond the eye into the lateral geniculate nucleus and visual cortex, and the disease even shares some characteristics with other central nervous system degenerative disorders. Glaucoma destroys neurons through oxidative stress, impairment in axonal transport, neuroinflammation, and excitotoxicity. Autophagy may promote or inhibit disease progression. Currently, lowering intraocular pressure is the only way proven to delay glaucoma advancement. However, many new therapies are being developed, including antioxidants, adenosine receptor antagonists, Rho-pathway inhibitors, stem cell therapy, and neurotrophic factors. These therapies focus on neuroprotection, and they may eventually halt glaucoma progression or reverse the process of the disease itself. PMID:27505018

  7. Neuroprotective flavonoids from Flemingia macrophylla.

    PubMed

    Shiao, Young-Ji; Wang, Chuen-Neu; Wang, Wan-Yu; Lin, Yun-Lian

    2005-09-01

    Using an Abeta-induced neurotoxicity blocking assay to direct fractionation, three new flavonoids, fleminginin (1), flemingichromone (2), and flemingichalcone (3), and twenty known compounds were isolated from the active fractions of the aerial parts of Flemingia macrophylla. The structures of 1 - 3 were elucidated on the basis of spectroscopic data. When tested for neuroprotective activity, compound 2, osajin ( 4), 5,7,4'-trihydroxy-6,8-diprenylisoflavone (5), 5,7,4'-trihydroxy-6,3'-diprenylisoflavone (6), and aureole (7) protected neuronal cells from Abeta-induced damage with EC50 values of 31.43 +/- 3.16, 5.01 +/- 1.28, 11.25 +/-1.51, 4.47 +/- 0.65, 12.09 +/- 2.55 microM, respectively. PMID:16206038

  8. Neurodegeneration and Neuroprotection in Glaucoma

    PubMed Central

    Gauthier, Angela C.; Liu, Ji

    2016-01-01

    Glaucoma is the principal cause of irreversible blindness in the world. The disease leads to progressive optic nerve degeneration with a gradual loss of retinal ganglion cells. Neurodegeneration in glaucoma extends beyond the eye into the lateral geniculate nucleus and visual cortex, and the disease even shares some characteristics with other central nervous system degenerative disorders. Glaucoma destroys neurons through oxidative stress, impairment in axonal transport, neuroinflammation, and excitotoxicity. Autophagy may promote or inhibit disease progression. Currently, lowering intraocular pressure is the only way proven to delay glaucoma advancement. However, many new therapies are being developed, including antioxidants, adenosine receptor antagonists, Rho-pathway inhibitors, stem cell therapy, and neurotrophic factors. These therapies focus on neuroprotection, and they may eventually halt glaucoma progression or reverse the process of the disease itself.

  9. [Evolution of the neuroprotection concept].

    PubMed

    Ostrovskaia, R U

    2003-01-01

    Although the modern concept of neuroprotection has been formulated quite recently, the basis of this approach was laid about four decades ago when Zakusov initiated the study of mechanisms involved in the neuroprotector action of GABA shunt metabolites (in particular, alpha-hydroxybutyric acid and succinic semialdehyde) during hypoxia. It was suggested to consider these agents as a system of endogenous neuroprotectors. The interest of Zakusov in endogenous regulators (including oligopeptides) had stimulated research in this direction and gave impact to the investigations of A. P. Skoldinov and T. A. Gudasheva initiated in the early 1980s. Proceeding from the original concept of the possibility of imitation of the action of neurotropic agents by their structural-conformational oligopeptide analogs, a number of biologically active stable dipeptides were obtained, based on pyroglutamate and proline, and high specific bioaccessibility of these dipeptides for the brain was established. Our investigations showed that these compounds not only possess nootropic activity (in a dose 1000 times lower than that of piracetam), but produce a pronounced neuroprotector action as well. Most thoroughly studied in this respect were substituted acyl-prolyl dipeptides, in particular, the drug noopept exhibiting a combined neuroprotector effect both in vitro and in vivo. Noopept decreases the extent of necrotic damage caused by photoinduced thrombosis of cortical blood vessels. It was established that the neuroprotector effect of noopept is related to its action upon the well-known "triad", whereby the drug reduces neurotoxic effects of excess extracellular calcium, glutamate, and free radicals. Two additional components of the neuroprotector action of noopept are related to the antiinflammatory and antithrombotic activity. The prospects of using direct and indirect action upon neurotrophin system for neuroprotection purposes are considered. Taking into account common secondary

  10. Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes

    PubMed Central

    Navas, Patrick A.; Swank, Richard A.; Yu, Man; Peterson, Kenneth R.; Stamatoyannopoulos, George

    2010-01-01

    High-level β-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1–HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb β-globin locus yeast artificial chromosome and measuring the activity of β-globin genes in GT6m β-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the β-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of ε- and γ-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, γ-globin gene expression was significantly reduced while β-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal ε- and γ-globin gene expression during embryonic erythropoiesis and for γ-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression. PMID:14506128

  11. Secondary neuroprotective effects of hypotensive drugs and potential mechanisms of action

    PubMed Central

    Shih, Grace C; Calkins, David J

    2012-01-01

    Primary open-angle glaucoma, a long-term degenerative ocular neuropathy, remains a significant cause of vision impairment worldwide. While many risk factors have been correlated with increased risk for primary open-angle glaucoma, intraocular pressure (IOP) remains the only modifiable risk factor and primary therapeutic target. Pharmacologic therapies are administered topically; these include α2-agonists, β-antagonists, prostaglandin analogs and carbonic anhydrase inhibitors. Some of these topical medications exhibit secondary neuroprotective effects independent of their effect on IOP. This review covers the possible mechanisms of neuroprotection stimulated by drugs currently marketed for the lowering of IOP, based on known literature. While the neuroprotective properties of many glaucoma pharmaceuticals are promising from an experimental standpoint, key challenges for the development of new clinical practices include unknown systemic side effects, limited methods of drug delivery to the retina and optic nerve, and development of extended-release formulations. PMID:22737176

  12. Translation of globin messenger RNA by the mouse ovum

    PubMed Central

    Brinster, R. L.; Chen, H. Y.; Trumbauer, M. E.; Avarbock, M. R.

    2016-01-01

    It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell1. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species2–4. This system has proved useful in understanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte5,6. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell7,8. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system. PMID:7352032

  13. Globin gene expression in correlation with G protein-related genes during erythroid differentiation

    PubMed Central

    2013-01-01

    Background The guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) regulate cell growth, proliferation and differentiation. G proteins are also implicated in erythroid differentiation, and some of them are expressed principally in hematopoietic cells. GPCRs-linked NO/cGMP and p38 MAPK signaling pathways already demonstrated potency for globin gene stimulation. By analyzing erythroid progenitors, derived from hematopoietic cells through in vitro ontogeny, our study intends to determine early markers and signaling pathways of globin gene regulation and their relation to GPCR expression. Results Human hematopoietic CD34+ progenitors are isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and G-CSF stimulated mobilized PB (mPB), and then differentiated in vitro into erythroid progenitors. We find that growth capacity is most abundant in FL- and CB-derived erythroid cells. The erythroid progenitor cells are sorted as 100% CD71+, but we did not find statistical significance in the variations of CD34, CD36 and GlyA antigens and that confirms similarity in maturation of studied ontogenic periods. During ontogeny, beta-globin gene expression reaches maximum levels in cells of adult blood origin (176 fmol/μg), while gamma-globin gene expression is consistently up-regulated in CB-derived cells (60 fmol/μg). During gamma-globin induction by hydroxycarbamide, we identify stimulated GPCRs (PTGDR, PTGER1) and GPCRs-coupled genes known to be activated via the cAMP/PKA (ADIPOQ), MAPK pathway (JUN) and NO/cGMP (PRPF18) signaling pathways. During ontogeny, GPR45 and ARRDC1 genes have the most prominent expression in FL-derived erythroid progenitor cells, GNL3 and GRP65 genes in CB-derived cells (high gamma-globin gene expression), GPR110 and GNG10 in BM-derived cells, GPR89C and GPR172A in PB-derived cells, and GPR44 and GNAQ genes in mPB-derived cells (high beta-globin gene expression). Conclusions These results

  14. Membrane-bound globin X protects the cell from reactive oxygen species.

    PubMed

    Koch, Jonas; Burmester, Thorsten

    2016-01-01

    Globin X (GbX) is a member of the globin family that emerged early in the evolution of Metazoa. In vertebrates, GbX is restricted to lampreys, fish, amphibians and some reptiles, and is expressed in neurons. Unlike any other metazoan globin, GbX is N-terminally acylated and anchored in the cell membrane via myristoyl and palmitoyl groups, suggesting a unique function. Here, we compared the capacity of GbX to protect a mouse neuronal cell line from hypoxia and reactive oxygen species (ROS) with that of myoglobin. To evaluate the contribution of membrane-binding, we generated a mutated version of GbX without acyl groups. All three globins enhanced cell viability under hypoxia, with myoglobin having the most pronounced effect. GbX but not myoglobin protected the cells from hydrogen peroxide (H2O2)-induced stress. Membrane-bound GbX was significantly more efficient than its mutated, soluble form. Furthermore, myoglobin and mutated GbX increased production of ROS upon H2O2-treatment, while membrane-bound GbX did not. The results indicate that myoglobin enhances O2 supply while GbX protects the cell membrane from ROS-stress. The ancient origin of GbX suggests that ROS-protection reflects the function of the early globins before they acquired a respiratory role. PMID:26631962

  15. Antioxidant activity, cellular bioavailability, and iron and calcium management of neuroprotective and nonneuroprotective flavones.

    PubMed

    Echeverry, Carolina; Arredondo, Florencia; Martínez, Marcela; Abin-Carriquiry, Juan Andrés; Midiwo, Jacob; Dajas, Federico

    2015-01-01

    Few studies have been undertaken on the relationship of the structure of flavones and neuroprotection. Previously, we described the structural determinants of the neuroprotective activity of some natural flavones in cerebellar granule neurons in culture against an oxidative insult (H2O2). In the present work, we analyzed anti-oxidant activity, cellular iron, and Ca(2+) levels and cellular bioavailability of neuroprotective and nonneuroprotective flavones in the same experimental paradigm. Oxidative cellular damage produced by H2O2 was prevented by all of the studied flavones with rather similar potency for all of them. Labile Iron Pool was neither affected by protective nor nonprotective flavones. Intracellular Ca(2+) homeostasis was not affected by protective flavones either. Nonetheless, fisetin, the nonprotective flavone, decreased Ca(2+) levels modifying Ca(2+) homeostasis. Methylation of the catechol group, although weakens anti-oxidant capacity, keeps the neuroprotective capacity with less degradation and lower toxicity, constituting promising structural alternatives as leads for the design of neuroprotective molecules. PMID:24972590

  16. Autoinduction, purification, and characterization of soluble α-globin chains of crocodile (Crocodylus siamensis) hemoglobin in Escherichia coli.

    PubMed

    Kabbua, Thai; Anwised, Preeyanan; Boonmee, Atcha; Subedi, Bishnu P; Pierce, Brad S; Thammasirirak, Sompong

    2014-11-01

    We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) α-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile α-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant α-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) α-globin. Both the monomeric and the dimeric protein configuration formed by intermolecular disulfide bond could be purified as soluble protein. Spectroscopic characterization [UV-visible, circular dichroism (CD), and electron paramagnetic resonance (EPR)] of purified recombinant α-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified α-globin was also investigated. These results suggested that C. siamensis α-globin expressed in E. coli was folded correctly with proper incorporation of the heme cofactor. The expression method we now describe can facilitate production and isolation of individual globin chains in order to further study the mechanism and assembly of crocodile hemoglobin. PMID:25175288

  17. Regulation of Gγ-Globin Gene by ATF2 and Its Associated Proteins through the cAMP-Response Element

    PubMed Central

    Dhar, Ruby; Mahajan, Milind; Choudhury, Alina; Weissman, Sherman; Pace, Betty S.

    2013-01-01

    The upstream Gγ-globin cAMP-response element (G-CRE) plays an important role in regulating Gγ-globin expression through binding of ATF2 and its DNA-binding partners defined in this study. ATF2 knockdown resulted in a significant reduction of γ-globin expression accompanied by decreased ATF2 binding to the G-CRE. By contrast, stable ATF2 expression in K562 cells increased γ-globin transcription which was reduced by ATF2 knockdown. Moreover, a similar effect of ATF2 on γ-globin expression was observed in primary erythroid progenitors. To understand the role of ATF2 in γ-globin expression, chromatographically purified G-CRE/ATF2-interacting proteins were subjected to mass spectrometry analysis; major binding partners included CREB1, cJun, Brg1, and histone deacetylases among others. Immunoprecipitation assays demonstrated interaction of these proteins with ATF2 and in vivo GCRE binding in CD34+ cells undergoing erythroid differentiation which was correlated with γ-globin expression during development. These results suggest synergism between developmental stage-specific recruitments of the ATF2 protein complex and expression of γ-globin during erythropoiesis. Microarray studies in K562 cells support ATF2 plays diverse roles in hematopoiesis and chromatin remodeling. PMID:24223142

  18. α:Non-α and Gγ:Aγ globin chain ratios in thalassemia intermedia patients treated with hydroxyurea

    PubMed Central

    Najjari, Abbas; Asouri, Mohsen; Gouhari, Ladan Hosseini; Niaki, Haleh Akhavan; Nejad, Amir Sasan Mozaffari; Eslami, Seyyedeh Masoumeh; Abolghasemi, Hassan; Ataee, Ramin; Ebrahimi, Abdol Ali; Moshaei, Masoumeh Rezaei; Ahmadi, Ali Asghar

    2014-01-01

    Objectives To elucidate the possible ways by which hydroxyurea molecules affect globin chain (α or β-like) synthesis. Methods A total of 23 thalassemia intermedia patients (13 male and 10 female) aged between 5 and 26 years were treated for five months with 15 mg/(kg·day) of hydroxyurea. Hemoglobins electrophoresis and globin chain electrophoresis was performed on each sample at different time points before and during the treatment. Results Fetal hemoglobin increased significantly in most patients and average episodes of transfusion decreased. Both Gγ and Aγ-globin chains increased significantly and α-globin:Nonα-globin chain as well as Gγ-globin:Aγ globin chains ratios decreased. Conclusions Improvement in α:non-α ratio and consequent decrease of free α-globin chain might be the cause of beneficial effects of hydroxyurea therapy. Two patients who felt better didn't show significant increase in their fetal hemoglobin level, and this is in contradiction with the hypothesis claiming that the HbF level increase is the cause of such therapeutic effect. In spite of the unclear mechanism of action of this drug, hydroxyurea therapy had noticeable impacts on thalassemia intermedia and also sickle cell disease and even patients suffering from thalassemia major. PMID:25183077

  19. Complete amino acid sequence of globin chains and biological activity of fragmented crocodile hemoglobin (Crocodylus siamensis).

    PubMed

    Srihongthong, Saowaluck; Pakdeesuwan, Anawat; Daduang, Sakda; Araki, Tomohiro; Dhiravisit, Apisak; Thammasirirak, Sompong

    2012-08-01

    Hemoglobin, α-chain, β-chain and fragmented hemoglobin of Crocodylus siamensis demonstrated both antibacterial and antioxidant activities. Antibacterial and antioxidant properties of the hemoglobin did not depend on the heme structure but could result from the compositions of amino acid residues and structures present in their primary structure. Furthermore, thirteen purified active peptides were obtained by RP-HPLC analyses, corresponding to fragments in the α-globin chain and the β-globin chain which are mostly located at the N-terminal and C-terminal parts. These active peptides operate on the bacterial cell membrane. The globin chains of Crocodylus siamensis showed similar amino acids to the sequences of Crocodylus niloticus. The novel amino acid substitutions of α-chain and β-chain are not associated with the heme binding site or the bicarbonate ion binding site, but could be important through their interactions with membranes of bacteria. PMID:22648692

  20. A redox signalling globin is essential for reproduction in Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    de Henau, Sasha; Tilleman, Lesley; Vangheel, Matthew; Luyckx, Evi; Trashin, Stanislav; Pauwels, Martje; Germani, Francesca; Vlaeminck, Caroline; Vanfleteren, Jacques R.; Bert, Wim; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; de Wael, Karolien; Moens, Luc; Dewilde, Sylvia; Braeckman, Bart P.

    2015-12-01

    Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers.

  1. A redox signalling globin is essential for reproduction in Caenorhabditis elegans

    PubMed Central

    De Henau, Sasha; Tilleman, Lesley; Vangheel, Matthew; Luyckx, Evi; Trashin, Stanislav; Pauwels, Martje; Germani, Francesca; Vlaeminck, Caroline; Vanfleteren, Jacques R.; Bert, Wim; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; De Wael, Karolien; Moens, Luc; Dewilde, Sylvia; Braeckman, Bart P.

    2015-01-01

    Moderate levels of reactive oxygen species (ROS) are now recognized as redox signalling molecules. However, thus far, only mitochondria and NADPH oxidases have been identified as cellular sources of ROS in signalling. Here we identify a globin (GLB-12) that produces superoxide, a type of ROS, which serves as an essential signal for reproduction in C. elegans. We find that GLB-12 has an important role in the regulation of multiple aspects in germline development, including germ cell apoptosis. We further describe how GLB-12 displays specific molecular, biochemical and structural properties that allow this globin to act as a superoxide generator. In addition, both an intra- and extracellular superoxide dismutase act as key partners of GLB-12 to create a transmembrane redox signal. Our results show that a globin can function as a driving factor in redox signalling, and how this signal is regulated at the subcellular level by multiple control layers. PMID:26621324

  2. Alpha globin gene analysis in a Sardinian family with interacting alpha and beta thalassaemia genes.

    PubMed

    Melis, M A; Galanello, R; Cao, A

    1983-04-01

    This paper reports the results of alpha globin gene analysis in a Sardinian family with interacting alpha and beta thalassaemia genes. The propositus, who was identified in a newborn survey as he had 26.0% Hb Bart's and 74.0% Hb F, successively developed the clinical and haematological picture of a transfusion-dependent thalassaemia major. According to the haemoglobin pattern, restriction endonuclease analysis of the DNA from this patient showed the deletion of three of the four alpha-globin structural genes. Thus beta 0-thalassaemia homozygotes with the delection of three alpha-structural genes seem to have a severe clinical phenotype similar to that of patients with a full complement of four alpha-globin structural genes. PMID:6299325

  3. Evolutionary pathway of pseudogenization of globin genes, α5 and β5, in genus Oryzias.

    PubMed

    Maruyama, Kouichi; Wang, Bing; Ishikawa, Yuji; Yasumasu, Shigeki; Iuchi, Ichiro

    2015-09-01

    Hemoglobin transports oxygen in many organisms and consists of α- and β-globin chains. Previously, using molecular phylogenetic analysis, we proposed that both α- and β-globins of teleost could be classified into four groups. We also showed that the Hd-rR strain of medaka (Oryzias latipes) inhabiting southern Japan had all four groups of globin genes but that the α- and β-globin genes of group III were pseudogenized (α5(ψα), β5(ψβ)). Based on the small degree of nucleotide variations, the pseudogenization of β5 was assumed to have occurred at a relatively late stage of evolution. Here, we compared the α5(ψα)-β5(ψβ) of two other strains of O. latipes and found that both α5(ψα) and β5(ψβ) of the northern Japanese and Korean strains were pseudogenized similar to those of Hd-rR. In a Philippine population (Oryzias luzonensis), α5(ψα) was also pseudogenized, but the structure was different from that of O. latipes, and β5(ψβ) was almost deleted. Interestingly, an Indonesian population (Oryzias celebensis) had α5 and β5 genes that were deduced to be functional. Indeed, they were expressed from the young to adult development stages, and this expression pattern was consistent with the expression of α2 and ad.α1 in Hd-rR. Because α2 and ad.α1 in Hd-rR were assigned to groups I and II, respectively, we speculate that their expression patterns might be altered by pseudogenization of group III genes. These results provide a basis for further investigations of recruiting and changing expression patterns of one globin gene after pseudogenization of other globin genes during evolution. PMID:26199047

  4. Evidence on primate phylogeny from epsilon-globin gene sequences and flanking regions.

    PubMed

    Porter, C A; Sampaio, I; Schneider, H; Schneider, M P; Czelusniak, J; Goodman, M

    1995-01-01

    Phylogenetic relationships among various primate groups were examined based on sequences of epsilon-globin genes. epsilon-globin genes were sequenced from five species of strepsirhine primates. These sequences were aligned and compared with other known primate epsilon-globin sequences, including data from two additional strepsirhine species, one species of tarsier, 19 species of New World monkeys (representing all extant genera), and five species of catarrhines. In addition, a 2-kb segment upstream of the epsilon-globin gene was sequenced in two of the five strepsirhines examined. This upstream sequence was aligned with five other species of primates for which data are available in this segment. Domestic rabbit and goat were used as outgroups. This analysis supports the monophyly of order Primates but does not support the traditional prosimian grouping of tarsiers, lorisoids, and lemuroids; rather it supports the sister grouping of tarsiers and anthropoids into Haplorhini and the sister grouping of lorisoids and lemuroids into Strepsirhini. The mouse lemur (Microcebus murinus) and dwarf lemur (Cheirogaleus medius) appear to be most closely related to each other, forming a clade with the lemuroids, and are probably not closely related to the lorisoids, as suggested by some morphological studies. Analysis of the epsilon-globin data supports the hypothesis that the aye-aye (Daubentonia madagascariensis) shares a sister-group relationship with other Malagasy strepsirhines (all being classified as lemuroids). Relationships among ceboids agree with findings from a previous epsilon-globin study in which fewer outgroup taxa were employed. Rates of molecular evolution were higher in lorisoids than in lemuroids. PMID:7714911

  5. Specific repression of β-globin promoter activity by nuclear ferritin

    PubMed Central

    Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

    2001-01-01

    Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

  6. A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum

    PubMed Central

    Tilleman, Lesley; Germani, Francesca; De Henau, Sasha; Helbo, Signe; Desmet, Filip; Berghmans, Herald; Van Doorslaer, Sabine; Hoogewijs, David; Schoofs, Liliane; Braeckman, Bart P.; Moens, Luc; Fago, Angela; Dewilde, Sylvia

    2015-01-01

    We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor. PMID:25666609

  7. Gene duplication, genome duplication, and the functional diversification of vertebrate globins

    PubMed Central

    Storz, Jay F.; Opazo, Juan C.; Hoffmann, Federico G.

    2015-01-01

    The functional diversification of the vertebrate globin gene superfamily provides an especially vivid illustration of the role of gene duplication and whole-genome duplication in promoting evolutionary innovation. For example, key globin proteins that evolved specialized functions in various aspects of oxidative metabolism and oxygen signaling pathways (hemoglobin [Hb], myoglobin [Mb], and cytoglobin [Cygb]) trace their origins to two whole-genome duplication events in the stem lineage of vertebrates. The retention of the proto-Hb and Mb genes in the ancestor of jawed vertebrates permitted a physiological division of labor between the oxygen-carrier function of Hb and the oxygen-storage function of Mb. In the Hb gene lineage, a subsequent tandem gene duplication gave rise to the proto α- and β-globin genes, which permitted the formation of multimeric Hbs composed of unlike subunits (α2β2). The evolution of this heteromeric quaternary structure was central to the emergence of Hb as a specialized oxygen-transport protein because it provided a mechanism for cooperative oxygen-binding and allosteric regulatory control. Subsequent rounds of duplication and divergence have produced diverse repertoires of α- and β-like globin genes that are ontogenetically regulated such that functionally distinct Hb isoforms are expressed during different stages of prenatal development and postnatal life. In the ancestor of jawless fishes, the proto Mb and Hb genes appear to have been secondarily lost, and the Cygb homolog evolved a specialized respiratory function in blood-oxygen transport. Phylogenetic and comparative genomic analyses of the vertebrate globin gene superfamily have revealed numerous instances in which paralogous globins have convergently evolved similar expression patterns and/or similar functional specializations in different organismal lineages. PMID:22846683

  8. Decreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization

    PubMed Central

    Kacian, D. L.; Gambino, R.; Dow, L. W.; Grossbard, E.; Natta, C.; Ramirez, F.; Spiegelman, S.; Marks, P. A.; Bank, A.

    1973-01-01

    In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient β-chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; α and β mRNAs isolated from heavy (β-producing) and light (α-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α cDNA labeled with [32P]dCTP and the β cDNA labeled with [3H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β-thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA. PMID:4124307

  9. Nucleotides in neuroregeneration and neuroprotection.

    PubMed

    Miras-Portugal, M Teresa; Gomez-Villafuertes, Rosa; Gualix, Javier; Diaz-Hernandez, Juan Ignacio; Artalejo, Antonio R; Ortega, Felipe; Delicado, Esmerilda G; Perez-Sen, Raquel

    2016-05-01

    Brain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-associated genes expression. Finally, general signaling pathways triggered by nucleotide receptors in neuronal populations converge on several intracellular kinases, such as PI3K/Akt, GSK3 and ERK1,2, as well as the Nrf-2/heme oxigenase-1 axis, which specifically link them to neuroprotection. In this regard, regulation of dual specificity protein phosphatases can become novel mechanism of actions for nucleotide receptors that associate them to cell homeostasis regulation. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26359530

  10. Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

    PubMed Central

    Moleirinho, Ana; Lopes, Alexandra M.; Seixas, Susana; Morales-Hojas, Ramiro; Prata, Maria J.; Amorim, António

    2015-01-01

    In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a

  11. Coinheritance of a Rare Nucleotide Substitution on the β-Globin Gene and Other Known Mutations in the Globin Clusters: Management in Genetic Counseling.

    PubMed

    Vinciguerra, Margherita; Passarello, Cristina; Leto, Filippo; Crivello, Anna; Fustaneo, Maria; Cassarà, Filippo; Cannata, Monica; Maggio, Aurelio; Giambona, Antonino

    2016-08-01

    A large number of methods for DNA analysis are available to identify defects in globin genes associated with hemoglobin (Hb) disorders. In this study, we report a rare nucleotide (nt) substitution on the β-globin gene, nt 781 in the second intron [IVS-II-781 (C > G); HBB: c.316-70C > G], identified in four patients. This nt substitution was previously described only as a personal communication to the HbVar database and indicated as a β(0) or β(+) mutation. The purpose of this study was to evaluate the clinical implication of this nt change, particularly when coinherited with severe β-thalassemia (β-thal), in order to be able to conduct appropriate genetic counseling. Genetic studies were performed on two subjects, one carried Hb S [β6(A3)Glu→Val; HBB: c.20A > T], and the other carried IVS-I-110 (G > A) (HBB: c.93-21G > A). All these subjects showed this new β nt substitution in association with Hb A2' (or Hb B2) [δ16(A13)Gly→Arg; HBD: c.49G > C]. Another 16 samples, carrying the same δ variant as the probands, were processed by β-globin gene sequencing in order to better understand the correlation between this Hb variant and the rare nt substitution reported in this study. The present investigation emphasizes the importance of sharing the observed nt changes in the globin gene cluster, especially in the case of new or rare undefined mutations, in order to facilitate the determination of their phenotypic expression, the possible interactions with known molecular defects and to formulate appropriate genetic counseling for at-risk couples. PMID:27258795

  12. Neuroprotective Effects of Psychotropic Drugs in Huntington’s Disease

    PubMed Central

    Lauterbach, Edward C.

    2013-01-01

    Psychotropics (antipsychotics, mood stabilizers, antidepressants, anxiolytics, etc.) are commonly prescribed to treat Huntington’s disease (HD). In HD preclinical models, while no psychotropic has convincingly affected huntingtin gene, HD modifying gene, or huntingtin protein expression, psychotropic neuroprotective effects include upregulated huntingtin autophagy (lithium), histone acetylation (lithium, valproate, lamotrigine), miR-222 (lithium-plus-valproate), mitochondrial protection (haloperidol, trifluoperazine, imipramine, desipramine, nortriptyline, maprotiline, trazodone, sertraline, venlafaxine, melatonin), neurogenesis (lithium, valproate, fluoxetine, sertraline), and BDNF (lithium, valproate, sertraline) and downregulated AP-1 DNA binding (lithium), p53 (lithium), huntingtin aggregation (antipsychotics, lithium), and apoptosis (trifluoperazine, loxapine, lithium, desipramine, nortriptyline, maprotiline, cyproheptadine, melatonin). In HD live mouse models, delayed disease onset (nortriptyline, melatonin), striatal preservation (haloperidol, tetrabenazine, lithium, sertraline), memory preservation (imipramine, trazodone, fluoxetine, sertraline, venlafaxine), motor improvement (tetrabenazine, lithium, valproate, imipramine, nortriptyline, trazodone, sertraline, venlafaxine), and extended survival (lithium, valproate, sertraline, melatonin) have been documented. Upregulated CREB binding protein (CBP; valproate, dextromethorphan) and downregulated histone deacetylase (HDAC; valproate) await demonstration in HD models. Most preclinical findings await replication and their limitations are reviewed. The most promising findings involve replicated striatal neuroprotection and phenotypic disease modification in transgenic mice for tetrabenazine and for sertraline. Clinical data consist of an uncontrolled lithium case series (n = 3) suggesting non-progression and a primarily negative double-blind, placebo-controlled clinical trial of lamotrigine. PMID:24248060

  13. Association of Xmn I Polymorphism and Hemoglobin E Haplotypes on Postnatal Gamma Globin Gene Expression in Homozygous Hemoglobin E

    PubMed Central

    Ekwattanakit, Supachai; Monteerarat, Yuwarat; Riolueang, Suchada; Tachavanich, Kalaya; Viprakasit, Vip

    2012-01-01

    Background and Objectives. To explore the role of cis-regulatory sequences within the β globin gene cluster at chromosome 11 on human γ globin gene expression related to Hb E allele, we analyze baseline hematological data and Hb F values together with β globin haplotypes in homozygous Hb E. Patients and Methods. 80 individuals with molecularly confirmed homozygous Hb E were analyzed for the β globin haplotypes and Xmn I polymorphism using PCR-RFLPs. 74 individuals with complete laboratory data were further studied for association analyses. Results. Eight different β globin haplotypes were found linked to Hb E alleles; three major haplotypes were (a) (III), (b) (V), and (c) (IV) accounting for 94% of Hb E chromosomes. A new haplotype (Th-1) was identified and most likely converted from the major ones. The majority of individuals had Hb F < 5%; only 10.8% of homozygous Hb E had high Hb F (average 10.5%, range 5.8–14.3%). No association was found on a specific haplotype or Xmn I in these individuals with high Hb F, measured by alkaline denaturation. Conclusion. The cis-regulation of γ globin gene expression might not be apparent under a milder condition with lesser globin imbalance such as homozygous Hb E. PMID:23049556

  14. An embryonic stage–specific enhancer within the murine β-globin locus mediates domain-wide histone hyperacetylation

    PubMed Central

    Fromm, George; Cadiz-Rivera, Brenda; de Vries, Christina; Getman, Michael; McGrath, Kathleen E.; Kingsley, Paul D.; Fields, Jennifer; Fiering, Steven

    2011-01-01

    In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian β-globin gene loci, and determined that within the murine locus, neither the β-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic β-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ϵ-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine β-globin locus results in significant decrease in the expression of the embryonic β-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for β-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure. PMID:21321362

  15. Open and Lys–His Hexacoordinated Closed Structures of a Globin with Swapped Proximal and Distal Sites

    PubMed Central

    Teh, Aik-Hong; Saito, Jennifer A.; Najimudin, Nazalan; Alam, Maqsudul

    2015-01-01

    Globins are haem-binding proteins with a conserved fold made up of α-helices and can possess diverse properties. A putative globin-coupled sensor from Methylacidiphilum infernorum, HGbRL, contains an N-terminal globin domain whose open and closed structures reveal an untypical dimeric architecture. Helices E and F fuse into an elongated helix, resulting in a novel site-swapped globin fold made up of helices A–E, hence the distal site, from one subunit and helices F–H, the proximal site, from another. The open structure possesses a large cavity binding an imidazole molecule, while the closed structure forms a unique Lys–His hexacoordinated species, with the first turn of helix E unravelling to allow Lys52(E10) to bind to the haem. Ligand binding induces reorganization of loop CE, which is stabilized in the closed form, and helix E, triggering a large conformational movement in the open form. These provide a mechanical insight into how a signal may be relayed between the globin domain and the C-terminal domain of HGbRL, a Roadblock/LC7 domain. Comparison with HGbI, a closely related globin, further underlines the high degree of structural versatility that the globin fold is capable of, enabling it to perform a diversity of functions. PMID:26094577

  16. A Novel Mutation in the Promoter Region of the β-Globin Gene: HBB: c.-127G > C.

    PubMed

    Bilgen, Turker; Canatan, Duran; Delibas, Serpil; Keser, Ibrahim

    2016-08-01

    Novel β-globin gene mutations are still occasionally being reported, especially when evaluating milder phenotypes. We report here a novel putative mutation in the promoter region of the β-globin gene and assess its clinical implications. A family, parents and four siblings, with hematological and clinical features suspected of being β-globin gene mutation(s), were involved in this study. In addition to hematological and clinical evaluations of the whole family, molecular analyses of the β-globin gene were performed by direct sequencing. Sequencing of the β-globin gene revealed a novel genomic alteration in the regulatory region of the gene. This novel genomic alteration was defined as HBB: c.-127G > C according to the Human Genome Variation Society (HGVS) nomenclature. Two siblings were found to be carriers of the HBB: c.-127G > C mutation, while the other two siblings were carriers of the codon 8 (-AA) (HBB: c.25_26delAA) deletion of the β-globin gene. The mother was a compound heterozygote for the codon 8 and HBB: c.-127G > C mutations. Based on hematological and clinical evaluations, we conclude that this novel β-globin gene promoter region change would be associated with a mild phenotype of β-thalassemia (β-thal). PMID:27349616

  17. Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site▿ †

    PubMed Central

    Harju-Baker, Susanna; Costa, Flávia C.; Fedosyuk, Halyna; Neades, Renee; Peterson, Kenneth R.

    2008-01-01

    Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ɛ-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the −566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of the proximal γ-globin promoters. PMID:18347053

  18. Epigenetics and therapeutic targets mediating neuroprotection.

    PubMed

    Qureshi, Irfan A; Mehler, Mark F

    2015-12-01

    The rapidly evolving science of epigenetics is transforming our understanding of the nervous system in health and disease and holds great promise for the development of novel diagnostic and therapeutic approaches targeting neurological diseases. Increasing evidence suggests that epigenetic factors and mechanisms serve as important mediators of the pathogenic processes that lead to irrevocable neural injury and of countervailing homeostatic and regenerative responses. Epigenetics is, therefore, of considerable translational significance to the field of neuroprotection. In this brief review, we provide an overview of epigenetic mechanisms and highlight the emerging roles played by epigenetic processes in neural cell dysfunction and death and in resultant neuroprotective responses. This article is part of a Special Issue entitled SI: Neuroprotection. PMID:26236020

  19. Neuroprotective Mechanisms of Taurine against Ischemic Stroke

    PubMed Central

    Menzie, Janet; Prentice, Howard; Wu, Jang-Yen

    2013-01-01

    Ischemic stroke exhibits a multiplicity of pathophysiological mechanisms. To address the diverse pathophysiological mechanisms observed in ischemic stroke investigators seek to find therapeutic strategies that are multifaceted in their action by either investigating multipotential compounds or by using a combination of compounds. Taurine, an endogenous amino acid, exhibits a plethora of physiological functions. It exhibits antioxidative properties, stabilizes membrane, functions as an osmoregulator, modulates ionic movements, reduces the level of pro-inflammators, regulates intracellular calcium concentration; all of which contributes to its neuroprotective effect. Data are accumulating that show the neuroprotective mechanisms of taurine against stroke pathophysiology. In this review, we describe the neuroprotective mechanisms employed by taurine against ischemic stroke and its use in clinical trial for ischemic stroke. PMID:24961429

  20. Neuroprotection in Stroke: Past, Present, and Future

    PubMed Central

    Majid, Arshad

    2014-01-01

    Stroke is a devastating medical condition, killing millions of people each year and causing serious injury to many more. Despite advances in treatment, there is still little that can be done to prevent stroke-related brain damage. The concept of neuroprotection is a source of considerable interest in the search for novel therapies that have the potential to preserve brain tissue and improve overall outcome. Key points of intervention have been identified in many of the processes that are the source of damage to the brain after stroke, and numerous treatment strategies designed to exploit them have been developed. In this review, potential targets of neuroprotection in stroke are discussed, as well as the various treatments that have been targeted against them. In addition, a summary of recent progress in clinical trials of neuroprotective agents in stroke is provided. PMID:24579051

  1. Vascular damage by unstable hemoglobins: the role of heme-depleted globin.

    PubMed

    Tsemakhovich, V A; Bamm, V V; Shaklai, M; Shaklai, N

    2005-04-15

    The study compared the damage inflicted to endothelial cells (ECs) by intact hemoglobin (Hb) and isolated chains. To compare optional in vivo contact of acellular Hb with the endothelium, oxy-forms of Hb and its isolated alpha- and beta-chains existing in the thalassemias were added to primary confluent cultures of bovine aorta EC. Cell damage was followed by morphological changes or leakage of lactic dehydrogenase and pre-inserted 51Cr from the cells, followed for 27 h. Under these experimental conditions, Hb did not affect the cells but its chains inflicted damage, beta- more than alpha-chains. Based on the literature and our data, we hypothesized that hemin and/or globin should be responsible for the increased endothelial damage by beta-chains. While hemin hardly affected ECs, globin, unlike the plasma protein hemopexin, was harmful. Since hemin release leaves globin with a large hydrophobic surface, the globin-damage appears to result from adsorptive pinocytosis to endothelial membrane. PMID:15797243

  2. Differential loss of embryonic globin genes during the radiation of placental mammals

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Storz, Jay F.

    2008-01-01

    The differential gain and loss of genes from homologous gene families represents an important source of functional variation among the genomes of different species. Differences in gene content between species are primarily attributable to lineage-specific gene gains via duplication and lineage-specific losses via deletion or inactivation. Here, we use a comparative genomic approach to investigate this process of gene turnover in the β-globin gene family of placental mammals. By analyzing genomic sequence data from representatives of each of the main superordinal clades of placental mammals, we were able to reconstruct pathways of gene family evolution during the basal radiation of this physiologically and morphologically diverse vertebrate group. Our analysis revealed that an initial expansion of the nonadult portion of the β-globin gene cluster in the ancestor of placental mammals was followed by the differential loss and retention of ancestral gene lineages, thereby generating variation in the complement of embryonic globin genes among contemporary species. The sorting of ε-, γ-, and η-globin gene lineages among the basal clades of placental mammals has produced species differences in the functional types of hemoglobin isoforms that can be synthesized during the course of embryonic development. PMID:18755893

  3. RNA sequencing for increasing gene discovery and and coverage using globin RNA reduced porcine blood samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Transcriptome analysis in porcine whole blood will provide major insights to decipher genetic mechanisms for host responses to viral infection. The abundance of porcine globin transcripts, however, impedes the ability to detect less abundant transcripts. The objective of our study was to...

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

    SciTech Connect

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

    1988-04-01

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

  5. Beta-globin gene cluster haplotype frequencies in Khalkhs and Buryats of Mongolia.

    PubMed

    Shimizu, Koji; Tokimasa, Kozue; Takeuchi, Yukiko; Gereksaikhan, Tudevdagva; Tanabe, Yuichi; Omoto, Keiichi; Imanishi, Tadashi; Harihara, Shinji; Hao, Luping; Jing, Feng

    2006-12-01

    Beta-globin gene cluster haplotype frequencies of 169 Khalkhs and 145 Buryats were estimated, and their characteristics were compared with those of Evenkis, Oroqens, Koreans, Japanese, and three Colombian Amerindian groups. The present study suggests that Colombian Amerindians diverged first from Asian populations and then Buryats diverged from other Asian populations. PMID:17564253

  6. Wine Polyphenols: Potential Agents in Neuroprotection

    PubMed Central

    Basli, Abdelkader; Soulet, Stéphanie; Chaher, Nassima; Mérillon, Jean-Michel; Chibane, Mohamed; Monti, Jean-Pierre; Richard, Tristan

    2012-01-01

    There are numerous studies indicating that a moderate consumption of red wine provides certain health benefits, such as the protection against neurodegenerative diseases. This protective effect is most likely due to the presence of phenolic compounds in wine. Wine polyphenolic compounds are well known for the antioxidant properties. Oxidative stress is involved in many forms of cellular and molecular deterioration. This damage can lead to cell death and various neurodegenerative disorders, such as Parkinson's or Alzheimer's diseases. Extensive investigations have been undertaken to determine the neuroprotective effects of wine-related polyphenols. In this review we present the neuroprotective abilities of the major classes of wine-related polyphenols. PMID:22829964

  7. Evidence that the recently discovered theta 1-globin gene is functional in higher primates.

    PubMed

    Shaw, J P; Marks, J; Shen, C K

    A new subfamily of the alpha-globin-like family has recently been identified in higher primates, rabbit, galago and possibly the horse. One member of this subfamily, theta 1, is downstream from the adult alpha 1-globin gene. In orang-utan, but not in rabbit or galago, the theta 1-gene appears to be structurally intact, suggesting that it may be functional in this species. The orang-utan theta 1-gene possesses initiation and termination codons, and the predicted polypeptide differs from the orang-utan alpha 1-globin by 55 amino acids. The upstream promoter boxes CCAAT and ATA are present, although approximately 150 base pairs (bp) farther upstream than in the alpha 1-gene. This structural difference in the promoter between the orang-utan theta 1- and alpha 1-genes has led Proudfoot to speculate that the theta 1-gene may be inactive. We have now cloned the theta 1- and alpha 1-globin genes from the olive baboon, and have compared their sequences with those of orang-utan. The unique promoter structure of the orang-utan theta 1-gene is highly conserved in baboon, although the orang-utan and baboon diverged nearly 30 million years ago. The coding sequences of the two theta 1-genes differ by only 6.3% with 22 out of 27 nucleotide substitutions being codon third position silent changes. These data support the view that the theta 1-gene has been functional in the baboon, orang-utan, and by implication, in man. We also estimate that the duplication event generating the theta 1- and alpha-globin-like subfamilies may have occurred as much as 260 million years ago. PMID:3561513

  8. Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Natarajan, Chandrasekhar; Witt, Christopher C.; Berenbrink, Michael; Storz, Jay F.

    2015-01-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa. PMID:25502940

  9. A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds

    PubMed Central

    Durlak, Marta; Fugazza, Cristina; Elangovan, Sudharshan; Marini, Maria Giuseppina; Marongiu, Maria Franca; Moi, Paolo; Fraietta, Ivan; Cappella, Paolo; Barbarani, Gloria; Font-Monclus, Isaura; Mauri, Mario; Ottolenghi, Sergio; Gasparri, Fabio; Ronchi, Antonella

    2015-01-01

    The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies. PMID:26509275

  10. Prehospital Use of Magnesium Sulfate as Neuroprotection in Acute Stroke

    PubMed Central

    Saver, Jeffrey L.; Starkman, Sidney; Eckstein, Marc; Stratton, Samuel J.; Pratt, Franklin D.; Hamilton, Scott; Conwit, Robin; Liebeskind, David S.; Sung, Gene; Kramer, Ian; Moreau, Gary; Goldweber, Robert; Sanossian, Nerses

    2016-01-01

    BACKGROUND Magnesium sulfate is neuroprotective in preclinical models of stroke and has shown signals of potential efficacy with an acceptable safety profile when delivered early after stroke onset in humans. Delayed initiation of neuroprotective agents has hindered earlier phase 3 trials of neuroprotective agents. METHODS We randomly assigned patients with suspected stroke to receive either intravenous magnesium sulfate or placebo, beginning within 2 hours after symptom onset. A loading dose was initiated by paramedics before the patient arrived at the hospital, and a 24-hour maintenance infusion was started on the patient’s arrival at the hospital. The primary outcome was the degree of disability at 90 days, as measured by scores on the modified Rankin scale (range, 0 to 6, with higher scores indicating greater disability). RESULTS Among the 1700 enrolled patients (857 in the magnesium group and 843 in the placebo group), the mean (±SD) age was 69±13 years, 42.6% were women, and the mean pretreatment score on the Los Angeles Motor Scale of stroke severity (range, 0 to 10, with higher scores indicating greater motor deficits) was 3.7±1.3. The final diagnosis of the qualifying event was cerebral ischemia in 73.3% of patients, intracranial hemorrhage in 22.8%, and a stroke-mimicking condition in 3.9%. The median interval between the time the patient was last known to be free of stroke symptoms and the start of the study-drug infusion was 45 minutes (interquartile range, 35 to 62), and 74.3% of patients received the study-drug infusion within the first hour after symptom onset. There was no significant shift in the distribution of 90-day disability outcomes on the global modified Rankin scale between patients in the magnesium group and those in the placebo group (P = 0.28 by the Cochran–Mantel–Haenszel test); mean scores at 90 days did not differ between the magnesium group and the placebo group (2.7 in each group, P = 1.00). No significant between

  11. Erythropoietin: still on the neuroprotection road

    PubMed Central

    del Barco, Diana García; Coro-Antich, Rosa M.

    2012-01-01

    Acute stroke is one of the major causes of death and disabilities. Since the 1980s many clinical studies have been conducted to evaluate neuroprotective approaches to treat this important brain vascular event. However, to date the only drug approved (recombinant tissue plasminogen activator [rtPA]) represents a thrombolytic, nonneuroprotective approach. An important neuroprotective strategy is based on erythropoietin (EPO). Exogenously administered EPO exhibits neuroprotective effects in numerous animal models, through the activation of anti-apoptotic, anti-oxidant and anti-inflammatory pathways as well as through the stimulation of angiogenic and neurogenic events. The capability of EPO to cross the blood–brain barrier after systemic administration and its effective therapeutic window are advantages for human acute stroke therapy. However, a multicenter stroke trial where recombinant human EPO (rhEPO) was combined with rtPA had negative outcomes. The present paper reviews the EPO neuroprotective strategy and its mechanisms in ischemic stroke and in other human nervous system diseases. PMID:22590480

  12. NAP (davunetide) provides functional and structural neuroprotection.

    PubMed

    Gozes, Illana

    2011-01-01

    NAP (davunetide) is an eight amino acid peptide (NAPVSIPQ) that has been shown to provide potent neuroprotection, in vitro and in vivo. In human clinical trials, NAP has been shown to increase memory scores in patients suffering from amnestic mild cognitive impairment, a precursor to Alzheimer's disease and to enhance functional daily behaviors in schizophrenia patients. NAP is derived from activity-dependent neuroprotective protein (ADNP) a molecule that is essential for brain formation, interacting with chromatin associated protein alpha and the chromatin remodeling complex SWI/SNF and regulating >400 genes during embryonic development. Partial loss in ADNP results in cognitive deficits and pathology of the microtubule associated protein tau (tauopathy) that is ameliorated in part by NAP replacement therapy. Recent studies increased the scope of NAP neuroprotection and provided further insights into the NAP mechanisms of action. Thus, it has been hypothesized that the presence of tau on axonal microtubules renders them notably less sensitive to the microtubule-severing protein katanin, and NAP was shown to protect microtubules from katanin disruption in the face of reduced tau expression. Parallel studies showed that NAP reduced the number of apoptotic neurons through activation of PI-3K/Akt pathway in the cortical plate or both PI-3K/Akt and MAPK/MEK1 kinases in the white matter. The interaction of these disparate yet complementary pathways is the subject of future studies toward human brain neuroprotection in the clinical scenario. PMID:21524250

  13. Progress in Neuroprotective Strategies for Preventing Epilepsy

    PubMed Central

    Acharya, Munjal M.; Hattiangady, Bharathi; Shetty, Ashok K.

    2008-01-01

    Neuroprotection is increasingly considered as a promising therapy for preventing and treating temporal lobe epilepsy (TLE). The development of chronic TLE, also termed as epileptogenesis, is a dynamic process. An initial precipitating injury (IPI) such as the status epilepticus (SE) leads to neurodegeneration, abnormal reorganization of the brain circuitry and a significant loss of functional inhibition. All of these changes likely contribute to the development of chronic epilepsy, characterized by spontaneous recurrent motor seizures (SRMS) and learning and memory deficits. The purpose of this review is to discuss the current state of knowledge pertaining to neuroprotection in epileptic conditions, and to highlight the efficacy of distinct neuroprotective strategies for preventing or treating chronic TLE. Although the administration of certain conventional and new generation antiepileptic drugs is effective for primary neuroprotection such as reduced neurodegeneration after acute seizures or the SE, their competence for preventing the development of chronic epilepsy after an IPI is either unknown or not promising. On the other hand, alternative strategies such as the ketogenic diet therapy, administration of distinct neurotrophic factors, hormones or antioxidants seem useful for preventing and treating chronic TLE. However, long term studies on the efficacy of these approaches introduced at different time-points after the SE or an IPI are lacking. Additionally, grafting of fetal hippocampal cells at early time-points after an IPI holds considerable promise for preventing TLE, though issues regarding availability of donor cells, ethical concerns, timing of grafting after SE, and durability of graft-mediated seizure suppression need to be resolved for further advances with this approach. Overall, from the studies performed so far, there is consensus that neuroprotective strategies need to be employed as quickly as possible after the onset of the SE or an IPI for

  14. Molecular cloning and sequencing of mRNAs coding for minor adult globin polypeptides of Xenopus laevis.

    PubMed Central

    Knöchel, W; Meyerhof, W; Hummel, S; Grundmann, U

    1983-01-01

    Globin mRNA was isolated from immature red blood cells of an adult Xenopus laevis female. mRNA/cDNA hybrids were integrated in the Pst I cleavage site of pBR 322 by G/C tailing, and cloned in Escherichia coli strain HB 101. By restriction site analysis as well as hybridization behaviour we identified two clones coding for minor adult alpha and beta globin chains. Nucleotide sequence analysis and derived amino acid sequences are presented. PMID:6298748

  15. Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

    PubMed

    Krivega, Ivan; Byrnes, Colleen; de Vasconcellos, Jaira F; Lee, Y Terry; Kaushal, Megha; Dean, Ann; Miller, Jeffery L

    2015-07-30

    Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult β-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of β-hemoglobinopathies. PMID:25979948

  16. Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions.

    PubMed Central

    Moon, A M; Ley, T J

    1990-01-01

    DNA sequences located in a region 6-18 kilobases (kb) upstream from the human epsilon-globin gene are known as the locus-activating region (LAR) or dominant control region. This region is thought to play a key role in chromatin organization of the beta-like globin gene cluster during erythroid development. The beta-globin LAR activates linked globin genes in transiently or stably transfected erythroleukemia cells and in erythroid cells of transgenic mice. Since the human beta-globin LAR is functional in mice, we reasoned that critical LAR sequence elements might be conserved between mice and humans. We therefore cloned murine genomic sequences homologous to one portion of the human LAR (site II, positions -11,054 to -10,322 with respect to the human epsilon gene). We found that this murine DNA fragment (mouse LAR site II) and sequences homologous to human LAR sites I and III are located upstream from the mouse beta-like globin gene cluster and determined that their locations relative to the cluster are similar to that of their human counterparts. The homologous site II sequences are 70% identical between mice and humans over a stretch of approximately 800 base pairs. Multiple core sequences with greater than 80% identity were present within this region. Transient and stable transfection assays of K562 erythroleukemia cells demonstrated that both human and mouse LAR elements contain enhancer activity and confer hemin inducibility on a linked human gamma-globin promoter. These results suggest that primary structural elements--and the spatial organization of these elements--are important for function of the beta-globin LAR. Images PMID:2217202

  17. Tissue specific transcription of the human epsilon-globin gene following transfection into the embryonic erythroid cell line K562.

    PubMed Central

    Allan, M; Montague, P; Grindlay, G J; Sibbet, G; Donovan-Peluso, M; Bank, A; Paul, J

    1985-01-01

    We have introduced a plasmid containing the human epsilon-globin gene either stably or transiently into a number of erythroid or non-erythroid cell lines, and analysed the accuracy and efficiency of transcription. In non-erythroid cells (or in mouse erythroleukaemia (MEL) cells in which adult but not embryonic globin genes are expressed) transcription of the epsilon-globin gene occurs mainly from a site 200 bp upstream of the major cap site (the -200 cap site). In the human K562 cell line, in which the endogenous epsilon-globin gene is transcribed at high levels, transcription initiation from the introduced gene occurs mainly from the major cap site. Transcriptional activity of the epsilon-globin gene introduced into K562 cell is quantitatively similar to that of the endogenous gene. This suggests the presence (or absence) in K562 cells of factor(s) which activate (or repress) the epsilon-globin gene in a tissue specific manner. Images PMID:2995916

  18. Analysis of β-globin chromatin micro-environment using a novel 3C variant, 4Cv.

    PubMed

    Pink, Ryan C; Eskiw, Christopher H; Caley, Daniel P; Carter, David R F

    2010-01-01

    Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C) have provided us with the tools to probe chromosomal structures. These techniques have been applied to the β-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of β-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv), which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the β-globin locus in mice and show that a significant proportion of the interactions of β-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, β-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the β-globin locus leads to a change in nuclear position relative to the chromosome territory. PMID:20927371

  19. An N-Myristoylated Globin with a Redox-Sensing Function That Regulates the Defecation Cycle in Caenorhabditis elegans

    PubMed Central

    Tilleman, Lesley; De Henau, Sasha; Pauwels, Martje; Nagy, Nora; Pintelon, Isabel; Braeckman, Bart P.; De Wael, Karolien; Van Doorslaer, Sabine; Adriaensen, Dirk; Timmermans, Jean-Pierre; Moens, Luc; Dewilde, Sylvia

    2012-01-01

    Globins occur in all kingdoms of life where they fulfill a wide variety of functions. In the past they used to be primarily characterized as oxygen transport/storage proteins, but since the discovery of new members of the globin family like neuroglobin and cytoglobin, more diverse and complex functions have been assigned to this heterogeneous family. Here we propose a function for a membrane-bound globin of C. elegans, GLB-26. This globin was predicted to be myristoylated at its N-terminus, a post-translational modification only recently described in the globin family. In vivo, this globin is found in the membrane of the head mesodermal cell and in the tail stomato-intestinal and anal depressor muscle cells. Since GLB-26 is almost directly oxidized when exposed to oxygen, we postulate a possible function as electron transfer protein. Phenotypical studies show that GLB-26 takes part in regulating the length of the defecation cycle in C. elegans under oxidative stress conditions. PMID:23251335

  20. Neuroprotective effect of alkyl hydroxytyrosyl ethers in rat brain slices subjected to a hypoxia-reoxygenation model.

    PubMed

    Guerrero, A; De la Cruz, J P; Muñoz-Marín, J; López-Villodres, J A; Madrona, A; Espartero, J L; González-Correa, J A

    2012-10-15

    The aim of the present study was to investigate the antioxidant and possible neuroprotective and antioxidant effects of five alkyl hydroxytyrosyl (HT) ethers (ethyl, butyl, hexyl, octyl and dodecyl) in rat brain slices. None of the compounds modified lipid peroxidation or glutathione concentrations (GSH) in oxygenated samples. The effects of oxidative stress were investigated with ferrous salts to induce lipid peroxidation and diethylmaleate (DEM) to reduce GSH. All compounds inhibited lipid peroxidation with an inhibitory concentration 50% (IC(50)) one tenth that of HT. These compounds, especially the butyl derivative, prevented GSH depletion after incubation with DEM. We also explored the neuroprotective effect of these compounds in an experimental model of hypoxia-reoxygenation in rat brain slices. All compounds showed neuroprotective and antioxidant effects. Our results established a relationship between these effects and the length of the carbon chain (maximum effect in the range of C4-C8). PMID:23442672

  1. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures. PMID:26874070

  2. Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

    PubMed Central

    Marinova, Zoya; Walitza, Susanne; Grünblatt, Edna

    2014-01-01

    Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations – most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential

  3. Globin mRNA reduction for whole-blood transcriptome sequencing

    PubMed Central

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  4. Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry

    PubMed Central

    Silaghi-Dumitrescu, Radu; Svistunenko, Dimitri A.; Cioloboc, Daniela; Bischin, Cristina; Scurtu, Florina; Cooper, Chris E.

    2014-01-01

    The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct. PMID:25172022

  5. Recombination within and between the human insulin and beta-globin gene loci.

    PubMed Central

    Lebo, R V; Chakravarti, A; Buetow, K H; Cheung, M C; Cann, H; Cordell, B; Goodman, H

    1983-01-01

    We detected a large number of polymorphic insulin restriction fragments in black Americans. These different size fragments were probably generated by unequal recombination on both sides of the human insulin gene. Population genetic analysis indicates that recombination occurred 33 times more frequently than expected to generate this large number of polymorphic fragments. Specific properties of the unique repeated 14- to 16-base-pair sequences 5' to the insulin gene suggest that this sequence would promote increased unequal recombination. Additional pedigree analysis showed that the recombination rate between the structural insulin and beta-globin gene loci was 14% with strong evidence for linkage. Since both insulin and beta-globin have been mapped to the short arm of human chromosome 11, this study establishes that the genetic map distance between these genes is 14.2 centimorgans. PMID:6348773

  6. Globin mRNA reduction for whole-blood transcriptome sequencing.

    PubMed

    Krjutškov, Kaarel; Koel, Mariann; Roost, Anne Mari; Katayama, Shintaro; Einarsdottir, Elisabet; Jouhilahti, Eeva-Mari; Söderhäll, Cilla; Jaakma, Ülle; Plaas, Mario; Vesterlund, Liselotte; Lohi, Hannes; Salumets, Andres; Kere, Juha

    2016-01-01

    The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish. PMID:27515369

  7. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

    PubMed

    Kotova, E S; Akopov, S B; Didych, D A; Petrova, N V; Iarovaia, O V; Razin, S V; Nikolaev, L G

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  8. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus

    PubMed Central

    Kotova, E. S.; Akopov, S. B.; Didych, D. A.; Petrova, N. V.; Iarovaia, O. V.; Razin, S. V.; Nikolaev, L. G.

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently – in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  9. Precise nucleosome positioning in the promoter of the chicken beta A globin gene.

    PubMed

    Kefalas, P; Gray, F C; Allan, J

    1988-01-25

    Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer. PMID:3340546

  10. Precise nucleosome positioning in the promoter of the chicken beta A globin gene.

    PubMed Central

    Kefalas, P; Gray, F C; Allan, J

    1988-01-01

    Histone octamers were reconstituted onto 5' end-labelled DNA fragments derived from the promoter region of the chicken beta A globin gene. The location of the reconstituted histone octamer with respect to the DNA sequence of each fragment was assessed by Exonuclease III digestion of purified nucleosome monomers. By this approach we have found a strong preference for histone octamers to be positioned over nucleotides -206 to -62 relative to the gene cap site. This stretch of DNA contains all those 5' beta globin sequences which, by DNase footprinting, bind specific protein factors and incorporates three promoter consensus sequence motifs. The upstream terminal 32 base pairs of this DNA segment contains the binding sites for the erythrocyte specific G-string binding protein and transcription factor Spl and appears to be relatively weakly bound to the histone octamer. Images PMID:3340546

  11. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    SciTech Connect

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. )

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  12. Spectrum of Common α-Globin Deletion Mutations in the Southern Region of Vietnam.

    PubMed

    Bui Thi Kim, Ly; Phu Chi, Dung; Hoang Thanh, Chi

    2016-06-01

    The common deletion mutations of α-globin genes in the Vietnamese population is not well known. Here we report the presence of five deletional mutations of Southeast Asia in the southern region of Vietnam. The - -(SEA) (NG_000006.1: g.26264_45564del19301) mutation is the most common type of deletion (87.35%), followed by the -α(3.7) (rightward) (NG_000006.1: g.34164_37967del3804) deletion (9.64%), -α(4.2) (leftward) (AF221717) deletion (2.41%) and - -(THAI) (NG_000006.1: g.10664_44164del33501) (0.6%) mutation in this region. The - -(FIL) (NG_000006.1: g.11684_43534del31581) mutation was not detected in this study. This result provided a view of the distribution of common α-globin gene mutations in Vietnam and could serve as a baseline for further investigations into these genetic defects. PMID:27117571

  13. Characteristic beta-globin gene cluster haplotypes of Evenkis and Oroqens in north China.

    PubMed

    Shimizu, Koji; Marubayashi, Azusa; Tokimasa, Kozue; Harihara, Shinji; Omoto, Keiichi; Imanishi, Tadashi; Hao, Luping; Jin, Feng

    2004-10-01

    Haplotype frequencies of the beta-globin gene cluster were estimated for 114 Evenkis and 81 Oroqens from northeast China, and their characteristics were compared with those in Japanese, Koreans, and three Colombian Amerindian groups of South America (Wayuu, Kamsa, and Inga tribes). A major 5' subhaplotype (5' to the delta-globin gene) was + - - - - in Evenkis, whereas + - - - -, - + + - +, and - + - + + were the major subhaplotypes in Oroqens. One possible candidate for an ancestral 5' subhaplotype, - - - - -, was found in one Evenki (0.5%) and three Oroqen chromosomes (2.0%). They were observed as heterozygous forms for + ---- and -----. Major haplotypes were +-----+, + -----+-, and + - - - - + + in Evenkis, whereas they were +-----+,-++-+-+, +----+-, and -+-++-+ in Oroqens. The lowest Nei's genetic distance values of Evenkis or Oroqens based on the 5' subhaplotype frequency distributions were observed in relation to the Wayuu or Koreans, respectively, but those of Evenkis and Oroqens based on the haplotype frequency distributions were found in relation to Koreans. PMID:15757246

  14. Investigating alpha-globin structural variants: a retrospective review of 135,000 Brazilian individuals

    PubMed Central

    Kimura, Elza Miyuki; Oliveira, Denise Madureira; Jorge, Susan Elisabeth; Ribeiro, Daniela Maria; Zaccariotto, Tânia Regina; Santos, Magnun Nueldo Nunes; Almeida, Vanessa; Albuquerque, Dulcinéia Martins; Costa, Fernando Ferreira; Sonati, Maria de Fátima

    2015-01-01

    Background Brazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population. Methods A total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC) and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC). Mutations were identified by molecular analyses, and the oxygen affinity, heme–heme cooperativity and Bohr effect of the variants were evaluated by functional tests. Results Four new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia. Conclusion The variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations. PMID:25818820

  15. Characterization of two globin genes from the malaria mosquito Anopheles gambiae: divergent origin of nematoceran haemoglobins.

    PubMed

    Burmester, Thorsten; Klawitter, Sabine; Hankeln, Thomas

    2007-04-01

    The chironomid midges are the only insects that harbour true haemoglobin in their haemolymph. Here we report the identification of haemoglobin genes in two other nematoceran species. Two paralogous haemoglobin genes (glob1 and glob2) from the malaria mosquito Anopheles gambiae were cloned and sequenced. Furthermore, we identified two orthologous haemoglobin genes in the yellow fever mosquito Aedes aegypti. All four haemoglobins were predicted to be intracellular proteins, with the amino acids required for heme- and oxygen-binding being conserved. In situ-hybridization studies showed that glob1 and glob2 expression in An. gambiae is mainly associated with the tracheal system. This pattern resembles that of other insect intracellular globins. We also observed expression of glob2 in visceral muscles. Phylogenetic analyses showed that the globins of the mosquitoes and the Chironomidae are not orthologous. The chironomid haemoglobins share a recent common origin with the brachyceran glob1 proteins. The mosquito glob1 and glob2 proteins, which separated by gene duplication around 170 million years ago, form a distinct clade of more ancient evolutionary origin within the insects. The glob1 genes have introns in the ancestral globin positions B12.2 and G7.0. An additional intron was observed in Ae. aegypti glob1 helix position E18.0, providing evidence for a recent intron gain event. Both mosquito glob2 genes have lost the B12.2 intron. This pattern must be interpreted in terms of dynamic intron gain and loss events in the globin gene lineage. PMID:17298561

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

    SciTech Connect

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

    1991-05-15

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

  17. Analysis of α1 and α2 globin genes among patients with hemoglobin Adana in Malaysia.

    PubMed

    Lee, T Y; Lai, M I; Ismail, P; Ramachandran, V; Tan, J A M A; Teh, L K; Othman, R; Hussein, N H; George, E

    2016-01-01

    Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position. PMID:27173219

  18. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. PMID:493112

  19. Homologous globin cell-free transcription system with comparison of heterologous factors.

    PubMed Central

    Tolunay, H E; Yang, L; Kemper, W M; Safer, B; Anderson, W F

    1984-01-01

    Mouse erythroleukemia (MEL) cells provide a useful model system to examine the regulation of globin gene expression. MEL cells ordinarily do not express globin genes, but in the presence of inducers, such as dimethyl sulfoxide or hexamethylene bisacetamide, they mimic erythroid differentiation. We have developed a cell-free transcription system from uninduced MEL cells to determine the requirements for mRNA synthesis. The MEL system directs accurate transcription of adenovirus type 2 major late DNA and mouse betamaj-globin with an efficiency comparable to those of HeLa and KB cell extracts. Using the procedure of Matsui et al. (T. Matsui, J. Segall, P.A. Weil, and R.G. Roeder, J. Biol. Chem. 255:11992-11996, 1980), we have isolated three active fractions from both MEL and HeLa cell extracts which are required for accurate transcription and have shown that equivalent fractions from MEL and HeLa cell extracts are interchangeable. Our findings suggest that the components required for initiation of transcription are similar in different cell types, at least to the extent that they can be assayed in these in vitro systems. Images PMID:6583493

  20. Oral decitabine reactivates expression of the methylated gamma-globin gene in Papio anubis.

    PubMed

    Lavelle, Donald; Chin, Janet; Vaitkus, Kestis; Redkar, Sanjeev; Phiasivongsa, Pasit; Tang, Chunlin; Will, Roselle; Hankewych, Maria; Roxas, Bryan; Singh, Mahipal; Saunthararajah, Yogen; Desimone, Joseph

    2007-11-01

    The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-2'-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated gamma-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated gamma-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the epsilon- and gamma-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis). PMID:17696208

  1. Quantification of tertiary structural conservation despite primary sequence drift in the globin fold.

    PubMed

    Aronson, H E; Royer, W E; Hendrickson, W A

    1994-10-01

    The globin family of protein structures was the first for which it was recognized that tertiary structure can be highly conserved even when primary sequences have diverged to a virtually undetectable level of similarity. This principle of structural inertia in molecular evolution is now evident for many other protein families. We have performed a systematic comparison of the sequences and structures of 6 representative hemoglobin subunits as diverse in origin as plants, clams, and humans. Our analysis is based on a 97-residue helical core in common to all 6 structures. Amino acid sequence identities range from 12.4% to 42.3% in pairwise comparisons, and, despite these variations, the maximal RMS deviation in alpha-carbon positions is 3.02 A. Overall, sequence similarity and structural deviation are significantly anticorrelated, with a correlation coefficient of -0.71, but for a set of structures having under 20% pairwise identity, this anticorrelation falls to -0.38, which emphasizes the weak connection between a specific sequence and the tertiary fold. There is substantial variability in structure outside the helical core, and functional characteristics of these globins also differ appreciably. Nevertheless, despite variations in detail that the sequence dissimilarities and functional differences imply, the core structures of these globins remain remarkably preserved. PMID:7849587

  2. Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors.

    PubMed

    Dulmovits, Brian M; Appiah-Kubi, Abena O; Papoin, Julien; Hale, John; He, Mingzhu; Al-Abed, Yousef; Didier, Sebastien; Gould, Michael; Husain-Krautter, Sehba; Singh, Sharon A; Chan, Kyle W H; Vlachos, Adrianna; Allen, Steven L; Taylor, Naomi; Marambaud, Philippe; An, Xiuli; Gallagher, Patrick G; Mohandas, Narla; Lipton, Jeffrey M; Liu, Johnson M; Blanc, Lionel

    2016-03-17

    Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies. PMID:26679864

  3. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed Central

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-01-01

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z. Images PMID:493112

  4. GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer.

    PubMed

    Escamilla-Del-Arenal, Martín; Recillas-Targa, Félix

    2008-01-01

    Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development. PMID:17984219

  5. Thalassaemia mutations within the 5'UTR of the human beta-globin gene disrupt transcription.

    PubMed

    Sgourou, Argyro; Routledge, Samantha; Antoniou, Michael; Papachatzopoulou, Adamantia; Psiouri, Lambrini; Athanassiadou, Aglaia

    2004-03-01

    The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment. PMID:15009072

  6. Current perspective of neuroprotection and glaucoma

    PubMed Central

    Tian, Kailin; Shibata-Germanos, Shannon; Pahlitzsch, Milena; Cordeiro, M Francesca

    2015-01-01

    Glaucoma is the second leading cause of blindness worldwide and is most notably characterized by progressive optic nerve atrophy and advancing loss of retinal ganglion cells (RGCs). The main concomitant factor is the elevated intraocular pressure (IOP). Existing treatments are focused generally on lowering IOP. However, both RGC loss and optic nerve atrophy can independently occur with IOP at normal levels. In recent years, there has been substantial progress in the development of neuroprotective therapies for glaucoma in order to restore vital visual function. The present review intends to offer a brief insight into conventional glaucoma treatments and discuss exciting current developments of mostly preclinical data in novel neuroprotective strategies for glaucoma that include recent advances in noninvasive diagnostics going beyond IOP maintenance for an enhanced global view. Such strategies now target RGC loss and optic nerve damage, opening a critical therapeutic window for preventative monitoring and treatment. PMID:26635467

  7. Exosomes: Mediators of Neurodegeneration, Neuroprotection and Therapeutics

    PubMed Central

    Kalani, Anuradha; Tyagi, Alka

    2014-01-01

    Exosomes have emerged as prominent mediators of neurodegenerative diseases where they have been shown to carry disease particles such as beta amyloid and prions from their cells of origin to other cells. Their simple structure and ability to cross the blood-brain barrier allow great opportunity to design a “makeup” with drugs and genetic elements, such as siRNA or miRNA, and use them as delivery vehicles for neurotherapeutics. Their role in neuroprotection is evident by the fact that they are involved in the regeneration of peripheral nerves and repair of neuronal injuries. This review is focused on the role of exosomes in mediating neurodegeneration and neuroprotection. PMID:23999871

  8. Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy

    PubMed Central

    Dixon, Brandon J.; Reis, Cesar; Ho, Wing Mann; Tang, Jiping; Zhang, John H.

    2015-01-01

    Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury and is among the leading cause of death among infants. Currently there are no well-established treatments; thus, it is important to understand the pathophysiology of the disease and elucidate complications that are creating a gap between basic science and clinical translation. In the development of neuroprotective strategies and translation of experimental results in HIE, there are many limitations and challenges to master based on an appropriate study design, drug delivery properties, dosage, and use in neonates. We will identify understudied targets after HIE, as well as neuroprotective molecules that bring hope to future treatments such as melatonin, topiramate, xenon, interferon-beta, stem cell transplantation. This review will also discuss some of the most recent trials being conducted in the clinical setting and evaluate what directions are needed in the future. PMID:26389893

  9. Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy.

    PubMed

    Dixon, Brandon J; Reis, Cesar; Ho, Wing Mann; Tang, Jiping; Zhang, John H

    2015-01-01

    Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury and is among the leading cause of death among infants. Currently there are no well-established treatments; thus, it is important to understand the pathophysiology of the disease and elucidate complications that are creating a gap between basic science and clinical translation. In the development of neuroprotective strategies and translation of experimental results in HIE, there are many limitations and challenges to master based on an appropriate study design, drug delivery properties, dosage, and use in neonates. We will identify understudied targets after HIE, as well as neuroprotective molecules that bring hope to future treatments such as melatonin, topiramate, xenon, interferon-beta, stem cell transplantation. This review will also discuss some of the most recent trials being conducted in the clinical setting and evaluate what directions are needed in the future. PMID:26389893

  10. Neuritogenic and neuroprotective activities of fruit residues.

    PubMed

    Tadtong, Sarin; Kanlayavattanakul, Mayuree; Lourith, Nattaya

    2013-11-01

    Neuritogenic and neuroprotective activities of litchi (Litchi chinensis Sonn., Sapindaceae) and salacca (Salacca edulis Reinw., Arecaceae) pericarp, and sapodilla (Achras sapota L., Sapotaceae) and tamarind Srichompu cultivar (Tamarindus indica L., Caesalpiniaceae) seed coat extracts were evaluated on cultured cholinergic P19-derived neurons. All the extracts, at a very low concentration (1 ng/mL of litchi and salacca pericarp extracts, 10 ng/mL of sapodilla and 100 ng/mL of tamarind seed coat extracts), enhanced the survival of cultured neurons (% viability more than 100%) by XTT reduction assay. The extracts were further evaluated for their neuritogenicity by observing cell morphology by phase-contrast microscopy and neuroprotective activity in serum deprivation and pre- and co-administration of hydrogen peroxide models. The phase-contrast micrographs displayed that all of the extracts possessed neurogenic activity by promoting the neurite outgrowth of the cultured neurons. Moreover, these extracts can protect neurons from oxidative stress-caused cell death in a serum deprivation model, and prevent and protect neuron cells from the toxicity of hydrogen peroxide. In this study we assured that the neuritogenic and neuroprotective activities of these extracts derived from the phenolic components and flavonoids contained in the extracts by acting as signaling molecules to enhance neuron survival and promote neurite outgrowth. These results suggest that all of the extracts are potentially sources of neuritogenic and neuroprotective components which might be used either as pharmaceutical products or dietary supplements for neurodegenerative disorder patients, for example, those suffering from Alzheimer's disease. PMID:24427947

  11. Ginseng: a promising neuroprotective strategy in stroke

    PubMed Central

    Rastogi, Vaibhav; Santiago-Moreno, Juan; Doré, Sylvain

    2015-01-01

    Ginseng is one of the most widely used herbal medicines in the world. It has been used in the treatment of various ailments and to boost immunity for centuries; especially in Asian countries. The most common ginseng variant in traditional herbal medicine is ginseng, which is made from the peeled and dried root of Panax Ginseng. Ginseng has been suggested as an effective treatment for a vast array of neurological disorders, including stroke and other acute and chronic neurodegenerative disorders. Ginseng’s neuroprotective effects are focused on the maintenance of homeostasis. This review involves a comprehensive literature search that highlights aspects of ginseng’s putative neuroprotective effectiveness, focusing on stroke. Attenuation of inflammation through inhibition of various proinflammatory mediators, along with suppression of oxidative stress by various mechanisms, including activation of the cytoprotective transcriptional factor Nrf2, which results in decrease in reactive oxygen species, could account for its neuroprotective efficacy. It can also prevent neuronal death as a result of stroke, thus decreasing anatomical and functional stroke damage. Although there are diverse studies that have investigated the mechanisms involved in the efficacy of ginseng in treating disorders, there is still much that needs to be clarified. Both in vitro and in vivo studies including randomized controlled clinical trials are necessary to develop in-depth knowledge of ginseng and its practical applications. PMID:25653588

  12. Polyphenols and neuroprotection against ischemia and neurodegeneration.

    PubMed

    Lin, B

    2011-12-01

    Neuroprotection of polyphenols in medical plants is getting attention in the world. Scutellaria baicalensis, paeonia veitchii and paeonia suffruticosa have been extensively studied in the last 10 years and show multi-function. They are neuroprotectants, antioxidants, anti-inflammatory and antithrombic agents as well as vasoconstriction inhibitors and amyloid-peptide (Aβ) cleaners by means of their polyphenols: baicalin, baicalein, wogonin (in scutellaria), and paeonol, paeonoside, paeoniflorin (PF) and 1, 2, 3, 4, 6-Penta-O-galloyl-beta-D-glucose (PGG) (in paeonia veitchii and paeonia suffruticosa). Other 4 medical plants: astragali, ligusticum wallichii, angelica sinensis and carthamus tinctorius (saffron) have been the major medicines to treat ischemia for hundreds of years in China, Korea and Japan. Our recent experimental studies demonstrated the neuroprotective efficacy of the combination of these phyotmedicines on mitigating brain infarction and global ischemia as well as preventing the neurodegeneration following ischemia. Owing to their multi-function, including improving cerebral blood circulation, they therefore have the potential to alleviate the symptoms of degenerative diseases, Alzheimer's disease (AD) and Parkinson's disease (PD). Pharmacology of the 7 herbs and their major relative polyphenols is depicted in the article. PMID:22070681

  13. Non-covalent and covalent modifications modulate the reactivity of monomeric mammalian globins.

    PubMed

    Ascenzi, Paolo; Marino, Maria; Polticelli, Fabio; Coletta, Massimo; Gioia, Magda; Marini, Stefano; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Reeder, Brandon J; Wilson, Michael T

    2013-09-01

    Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O2 and facilitates the O2 flux within a myocyte, displays the allosteric modulation of the O2 affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O2 diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue

  14. Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

    PubMed Central

    Gong, Q H; McDowell, J C; Dean, A

    1996-01-01

    Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free

  15. Carbamylated Erythropoietin: A Prospective Drug Candidate for Neuroprotection

    PubMed Central

    Chen, Jianmin; Yang, Zheng; Zhang, Xiao

    2015-01-01

    Carbamylated erythropoietin (cEpo), which is neuroprotective but lacks hematopoietic activity, has been attracting rising concerns. However, the cellular and molecular mechanisms involved in the process of neuroprotection of cEpo are not well known. Based on several recent reports, the neuroprotective effects of cEpo are illustrated, and signaling pathways involved in the different effects of erythropoietin and cEpo are discussed. These newly reported researches may shed new light on the development and application of cEpo, a prospective drug candidate for neuroprotection. PMID:26862298

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

    PubMed Central

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

    1995-01-01

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

  17. Glucocorticoids inhibit coordinated translation of. cap alpha. - and. beta. -globin mRNAs in Friend erythroleukemia cells

    SciTech Connect

    Papaconstantinou, J.; Stewart, J.A.; Rabek, J.P.; McClintock, P.R.; Wong, E.Y.

    1983-12-01

    The dimethylsulfoxide (Me/sub 2/SO)-mediated induction of hemoglobin synthesis in Friend erythroleukemia cells is inhibited by the glucocorticoids hydrocortisone, dexamethasone, and fluocinolone acetonide; hydrocortisone, at concentrations of 10/sup -5/ to 10/sup -8/ M inhibits by 90-30% and fluocinolone acetonide at concentrations of 10/sup -8/ to 10/sup -11/ M shows a greater than 90% inhibition. At these concentrations the hormones have no effect on cell growth or viability. In this study it has been shown that there is a group of proteins, including the ..cap alpha..- and ..beta..-globins, whose regulation is associated with the induction of Friend erythroleukemia cell differentiation, and that the expression of these, in addition to ..cap alpha..- and ..beta..-globin, is affected by glucocorticoids. It is concluded that, although the translation of ..cap alpha..- and ..beta..-globin mRNA is a major site of inhibition by glucocorticoids, there is a detectable amount of ..cap alpha..- and ..beta..-globin mRNA translation which results in unequal amounts of globin synthesis and an overall more potent inhibition of hemoglobin formation.

  18. A New Intergenic α-Globin Deletion (α-α(Δ125)) Found in a Kabyle Population.

    PubMed

    Rabbind Singh, Amrathlal; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-03-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact. PMID:26911300

  19. Role of the GATA-1/FOG-1/NuRD Pathway in the Expression of Human β-Like Globin Genes▿

    PubMed Central

    Miccio, Annarita; Blobel, Gerd A.

    2010-01-01

    The human β-globin genes are expressed in a developmentally controlled fashion. Studies on the molecular mechanisms underlying the stage-specific regulation of globin genes have been fueled by the clinical benefit of elevated fetal γ-globin expression in patients with sickle cell anemia and thalassemia. Recent reports suggested a role of the hematopoietic transcription factor GATA-1, its cofactor FOG-1, and the associated chromatin remodeling complex NuRD in the developmental silencing of HBG1 and HBG2 gene expression. To examine whether FOG-1 via NuRD controls HBG1 and HBG2 silencing in vivo, we created mice in which the FOG-1/NuRD complex is disrupted (A. Miccio et al., EMBO J. 29:442-456, 2010) and crossed these with animals carrying the entire human β-globin gene locus as a transgene. We found that the FOG-1/NuRD interaction is dispensable for the silencing of human HBG1 and HBG2 expression. In addition, mutant animals displayed normal silencing of the endogenous embryonic globin genes. In contrast, a significant reduction of adult-type human and murine globin gene expression was found in adult bone marrows of mutant animals. These results suggest that, unexpectedly, NuRD is required for FOG-1-dependent activation of adult-type globin gene expression but is dispensable for human γ-globin silencing in vivo. PMID:20439494

  20. Erythroid activator NF-E2, TAL1 and KLF1 play roles in forming the LCR HSs in the human adult β-globin locus.

    PubMed

    Kim, Yea Woon; Yun, Won Ju; Kim, AeRi

    2016-06-01

    The β-like globin genes are developmental stage specifically transcribed in erythroid cells. The transcription of the β-like globin genes requires erythroid specific activators such as GATA-1, NF-E2, TAL1 and KLF1. However, the roles of these activators have not fully elucidated in transcription of the human adult β-globin gene. Here we employed hybrid MEL cells (MEL/ch11) where a human chromosome containing the β-globin locus is present and the adult β-globin gene is highly transcribed by induction. The roles of erythroid specific activators were analyzed by inhibiting the expression of NF-E2, TAL1 or KLF1 in MEL/ch11 cells. The loss of each activator decreased the transcription of human β-globin gene, locus wide histone hyperacetylation and the binding of other erythroid specific activators including GATA-1, even though not affecting the expression of other activators. Notably, sensitivity to DNase I was reduced in the locus control region (LCR) hypersensitive sites (HSs) with the depletion of activators. These results indicate that NF-E2, TAL1 and KLF1, all activators play a primary role in HSs formation in the LCR. It might contribute to the transcription of human adult β-globin gene by allowing the access of activators and cofactors. The roles of activators in the adult β-globin locus appear to be different from the roles in the early fetal locus. PMID:27026582

  1. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    PubMed Central

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin muα-globin2/huβ-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  2. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

    PubMed

    Aimola, Idowu A; Inuwa, Hajiya M; Nok, Andrew J; Mamman, Aisha I; Bieker, James J

    2016-04-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer. PMID:26879870

  3. S1 nuclease analysis of alpha-globin gene expression in preleukemic patients with acquired hemoglobin H disease after transfer to mouse erythroleukemia cells.

    PubMed

    Helder, J; Deisseroth, A

    1987-04-01

    The loss of alpha-globin gene transcriptional activity rarely occurs as an acquired abnormality during the evolution of myeloproliferative disease or preleukemia. To test whether the mutation responsible for the loss of alpha-globin gene expression (hemoglobin H disease) in these patients is linked with the alpha-globin genes on chromosome 16, we transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human alpha-globin genes. After transfer to mouse erythroleukemia cells, the expression of human alpha-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human alpha-globin genes transferred to mouse erythroleukemia cells from normal donors. These data showed that factor(s) in the mouse erythroleukemia cell can genetically complement the alpha-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the alpha-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients. PMID:3031681

  4. Prucalopride exerts neuroprotection in human enteric neurons.

    PubMed

    Bianco, Francesca; Bonora, Elena; Natarajan, Dipa; Vargiolu, Manuela; Thapar, Nikhil; Torresan, Francesco; Giancola, Fiorella; Boschetti, Elisa; Volta, Umberto; Bazzoli, Franco; Mazzoni, Maurizio; Seri, Marco; Clavenzani, Paolo; Stanghellini, Vincenzo; Sternini, Catia; De Giorgio, Roberto

    2016-05-15

    Serotonin (5-hydroxytryptamine, 5-HT) and its transporters and receptors are involved in a wide array of digestive functions. In particular, 5-HT4 receptors are known to mediate intestinal peristalsis and recent data in experimental animals have shown their role in neuronal maintenance and neurogenesis. This study has been designed to test whether prucalopride, a well-known full 5-HT4 agonist, exerts protective effects on neurons, including enteric neurons, exposed to oxidative stress challenge. Sulforhodamine B assay was used to determine the survival of SH-SY5Y cells, human enteric neurospheres, and ex vivo submucosal neurons following H2O2 exposure in the presence or absence of prucalopride (1 nM). Specificity of 5-HT4-mediated neuroprotection was established by experiments performed in the presence of GR113808, a 5-HT4 antagonist. Prucalopride exhibited a significant neuroprotective effect. SH-SY5Y cells pretreated with prucalopride were protected from the injury elicited by H2O2 as shown by increased survival (73.5 ± 0.1% of neuronal survival vs. 33.3 ± 0.1%, respectively; P < 0.0001) and a significant reduction of proapoptotic caspase-3 and caspase-9 activation in all neurons tested. The protective effect of prucalopride was reversed by the specific 5-HT4 antagonist GR113808. Prucalopride promotes a significant neuroprotection against oxidative-mediated proapoptotic mechanisms. Our data pave the way for novel therapeutic implications of full 5-HT4 agonists in gut dysmotility characterized by neuronal degeneration, which go beyond the well-known enterokinetic effect. PMID:26893157

  5. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts

    PubMed Central

    Vinjamur, Divya S.; Alhashem, Yousef N.; Mohamad, Safa F.; Amin, Parth; Williams, David C.; Lloyd, Joyce A.

    2016-01-01

    In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies. PMID:26840243

  6. Krüppel-Like Transcription Factor KLF1 Is Required for Optimal γ- and β-Globin Expression in Human Fetal Erythroblasts.

    PubMed

    Vinjamur, Divya S; Alhashem, Yousef N; Mohamad, Safa F; Amin, Parth; Williams, David C; Lloyd, Joyce A

    2016-01-01

    In human adult erythroid cells, lower than normal levels of Krüppel-like transcription factor 1 (KLF1) are generally associated with decreased adult β- and increased fetal γ-globin gene expression. KLF1 also regulates BCL11A, a known repressor of adult γ-globin expression. In seeming contrast to the findings in adult cells, lower amounts of KLF1 correlate with both reduced embryonic and reduced fetal β-like globin mRNA in mouse embryonic erythroid cells. The role of KLF1 in primary human fetal erythroid cells, which express both γ- and β-globin mRNA, is less well understood. Therefore, we studied the role of KLF1 in ex vivo differentiated CD34+ umbilical cord blood cells (UCB erythroblasts), representing the fetal milieu. In UCB erythroblasts, KLF1 binds to the β-globin locus control region (LCR), and the β-globin promoter. There is very little KLF1 binding detectable at the γ-globin promoter. Correspondingly, when cultured fetal UCB erythroblasts are subjected to lentiviral KLF1 knockdown, the active histone mark H3K4me3 and RNA pol II recruitment are diminished at the β- but not the γ-globin gene. The amount of KLF1 expression strongly positively correlates with β-globin mRNA and weakly positively correlates with BCL11A mRNA. With modest KLF1 knockdown, mimicking haploinsufficiency, γ-globin mRNA is increased in UCB erythroblasts, as is common in adult cells. However, a threshold level of KLF1 is evidently required, or there is no absolute increase in γ-globin mRNA in UCB erythroblasts. Therefore, the role of KLF1 in γ-globin regulation in fetal erythroblasts is complex, with both positive and negative facets. Furthermore, in UCB erythroblasts, diminished BCL11A is not sufficient to induce γ-globin in the absence of KLF1. These findings have implications for the manipulation of BCL11A and/or KLF1 to induce γ-globin for therapy of the β-hemoglobinopathies. PMID:26840243

  7. Cerium and yttrium oxide nanoparticles are neuroprotective

    SciTech Connect

    Schubert, David . E-mail: schubert@salk.edu; Dargusch, Richard; Raitano, Joan; Chan, S.-W.

    2006-03-31

    The responses of cells exposed to nanoparticles have been studied with regard to toxicity, but very little attention has been paid to the possibility that some types of particles can protect cells from various forms of lethal stress. It is shown here that nanoparticles composed of cerium oxide or yttrium oxide protect nerve cells from oxidative stress and that the neuroprotection is independent of particle size. The ceria and yttria nanoparticles act as direct antioxidants to limit the amount of reactive oxygen species required to kill the cells. It follows that this group of nanoparticles could be used to modulate oxidative stress in biological systems.

  8. Synthesis and Evaluation of Neuroprotective Selenoflavanones

    PubMed Central

    Choi, Yong-Sung; Kim, Dong-Myung; Kim, Yoon-Jung; Yang, Sai; Lee, Kyung-Tae; Ryu, Jong Hoon; Jeong, Jin-Hyun

    2015-01-01

    The physicochemical properties and antioxidant activity of a molecule could be improved by the substitution of an oxygen atom in a molecule with selenium. We synthesized selenoflavanones and flavanones to evaluate their neuroprotective effects. The selenoflavanones showed improved physicochemical properties, suggestive of the ability to pass through the blood-brain barrier (BBB). They showed in vitro antioxidant effects against hydrogen peroxide, and did not result in severe cytotoxicity. Moreover, infarction volumes in a transient ischemia mouse model were significantly reduced by the selenoflavanone treatments. PMID:26690420

  9. Enhanced Delivery of Erythropoietin Across the Blood-Brain Barrier for Neuroprotection against Ischemic Neuronal Injury

    PubMed Central

    Zhang, Feng; Xing, Juan; Liou, Anthony Kian-Fong; Wang, Suping; Gan, Yu; Luo, Yumin; Ji, Xuming; Stetler, R. Anne; Chen, Jun; Cao, Guodong

    2010-01-01

    Due to limited penetration of the BBB, many therapeutic agents in clinical use require higher doses in order to reach effective concentrations in brain. In some instances, these high doses elicit severe side effects. In the case of erythropoietin (EPO), an established neuroprotectant against ischemic brain injury, its low BBB permeability requires such a high therapeutic dose that it can induce dangerous complications such as polycythmia and secondary stroke. The purpose of this study is to generate a modified EPO that has increased facility crossing the BBB without losing its neuroprotective element. We have engineered a fusion protein (EPO-TAT) by tagging a protein transduction domain derived from HIV TAT to the EPO protein. This sequence enhanced the capacity of EPO to cross the BBB in animals at least twofold when IP administered and up to five-fold when IV administered. In vitro experiments showed that this EPO fusion protein retained all its protective properties against neuronal death elicited by oxygen-glucose deprivation and NMDA insults. The needed therapeutic dose of the EPO-TAT was decreased by ~10-fold compared to that of regular EPO to achieve equivalent neuroprotection in terms of reducing volume of infarction induced by middle cerebral artery occlusion in mice. Our results support the approach of using a protein transduction domain coupled to therapeutic agents. In this way, not only can the therapeutic doses be lowered, but agents without BBB permeability may now be available for clinical applications. PMID:20577577

  10. Small-Molecule Anticonvulsant Agents with Potent in vitro Neuroprotection and Favorable Drug-like Properties

    PubMed Central

    Smith, Garry R.; Brenneman, Douglas E.; Zhang, Yan; Du, Yanming; Reitz, Allen B.

    2014-01-01

    Severe seizure activity is associated with reoccurring cycles of excitotoxicity and oxidative stress that result in progressive neuronal damage and death. Intervention with these pathological processes is a compelling disease-modifying strategy for the treatment of seizure disorders. We have optimized a series of small molecules for neuroprotective and anticonvulsant activity as well as altered their physical properties to address potential metabolic liabilities, to improve CNS penetration and to prolong the duration of action in vivo. Utilizing phenotypic screening of hippocampal cultures with nutrient medium depleted of antioxidants as a disease model, cell death and decreased neuronal viability produced by acute treatment with glutamate or hydrogen peroxide were prevented. Modifications to our previously reported proof of concept compounds have resulted in a lead which has full neuroprotective action at < 1 nM and antiseizure activity across six animal models, including the kindled rat, and displays excellent pharmacokinetics including high exposure to the brain. These modifications have also eliminated the requirement for a chiral molecule, removing the possibility of racemization and making large scale synthesis more easily accessible. These studies strengthen our earlier findings which indicate that potent, multifunctional neuroprotective anticonvulsants are feasible within a single molecular entity which also possesses favorable CNS-active drug properties in vitro and in vivo. PMID:24277343

  11. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the βA(T87Q)-Globin Gene

    PubMed Central

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-01-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (βAT87Q-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. βAT87Q-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  12. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene.

    PubMed

    Negre, Olivier; Eggimann, Anne-Virginie; Beuzard, Yves; Ribeil, Jean-Antoine; Bourget, Philippe; Borwornpinyo, Suparerk; Hongeng, Suradej; Hacein-Bey, Salima; Cavazzana, Marina; Leboulch, Philippe; Payen, Emmanuel

    2016-02-01

    β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease. PMID:26886832

  13. [Neuroprotective effects of peptides bioregulators in people of various age].

    PubMed

    Umnov, R S; Lin'kova, N S; Khavinson, V Kh

    2013-01-01

    The review presents comparative characteristics of 2 peptide neuroprotective groups: polypeptide complexes (cortexin, cerebrolizin) and short peptides (semax, kortagen, pinealon). The data of clinical applying of peptides in elderly and old age people and cellular and molecular mechanisms of their neuroprotective activity is described. PMID:24738258

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

    PubMed Central

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

    1995-01-01

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

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

    SciTech Connect

    Beraud-Colomb, E. |; Maroc, N.; Roubin, R.

    1995-12-01

    Analyzing the nuclear DNA from ancient human bones is an essential step to the understanding of genetic diversity in current populations, provided that such systematic studies are experimentally feasible. This article reports the successful extraction and amplification of nuclear DNA from the P-globin region from 5 of 10 bone specimens up to 12,000 years old. These have been typed for P-globin frameworks by sequencing through two variable positions and for a polymorphic (AT){sub x}(T){sub y} microsatellite 500 bp upstream of the P-globin gene. These specimens of human remains are somewhat older than those analyzed in previous nuclear gene sequencing reports and considerably older than those used to study high-copy-number human mtDNA. These results show that the systematic study of nuclear DNA polymorphisms of ancient populations is feasible. 34 refs., 3 figs., 2 tabs.

  16. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    PubMed

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-01

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. PMID:24814285

  17. Polymorphism and divergence in the beta-globin replication origin initiation region.

    PubMed

    Fullerton, S M; Bond, J; Schneider, J A; Hamilton, B; Harding, R M; Boyce, A J; Clegg, J B

    2000-01-01

    DNA sequence polymorphism and divergence was examined in the vicinity of the human beta-globin gene cluster origin of replication initiation region (IR), a 1.3-kb genomic region located immediately 5' of the adult-expressed beta-globin gene. DNA sequence variation in the replication origin IR and 5 kb of flanking DNA was surveyed in samples drawn from two populations, one African (from the Gambia, West Africa) and the other European (from Oxford, England). In these samples, levels of nucleotide and length polymorphism in the IR were found to be more than two times as high as adjacent non-IR-associated regions (estimates of per-nucleotide heterozygosity were 0.30% and 0.12%, respectively). Most polymorphic positions identified in the origin IR fall within or just adjacent to a 52-bp alternating purine-pyrimidine ((RY)n) sequence repeat. Within- and between-populations divergence is highest in this portion of the IR, and interspecific divergence in the same region, determined by comparison with an orthologous sequence from the chimpanzee, is also pronounced. Higher levels of diversity in this subregion are not, however, primarily attributable to slippage-mediated repeat unit changes, as nucleotide substitution contributes disproportionately to allelic heterogeneity. An estimate of helical stability in the sequenced region suggests that the hypervariable (RY)n constitutes the major DNA unwinding element (DUE) of the replication origin IR, the location at which the DNA duplex first unwinds and new strand synthesis begins. These findings suggest that the beta-globin IR experiences a higher underlying rate of neutral mutation than do adjacent genomic regions and that enzyme fidelity associated with the initiation of DNA replication at this origin may be compromised. The significance of these findings for our understanding of eukaryotic replication origin biology is discussed. PMID:10666717

  18. Repeated Evolution of Chimeric Fusion Genes in the β-Globin Gene Family of Laurasiatherian Mammals

    PubMed Central

    Gaudry, Michael J.; Storz, Jay F.; Butts, Gary Tyler; Campbell, Kevin L.; Hoffmann, Federico G.

    2014-01-01

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB “Lepore” deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived “anti-Lepore” duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20–100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion. PMID:24814285

  19. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    SciTech Connect

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-06-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes.

  20. A globin domain in a neuronal transmembrane receptor of Caenorhabditis elegans and Ascaris suum: molecular modeling and functional properties.

    PubMed

    Tilleman, Lesley; Germani, Francesca; De Henau, Sasha; Helbo, Signe; Desmet, Filip; Berghmans, Herald; Van Doorslaer, Sabine; Hoogewijs, David; Schoofs, Liliane; Braeckman, Bart P; Moens, Luc; Fago, Angela; Dewilde, Sylvia

    2015-04-17

    We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O2 with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor. PMID:25666609

  1. Transfer of 5′-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro

    PubMed Central

    Plotch, Stephen J.; Bouloy, Michele; Krug, Robert M.

    1979-01-01

    We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, 4886-4890]. Here, we present direct evidence that the 5′-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (β-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the β-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing 32P label only in the cap (m7G32pppm6Am-) were prepared by recapping β-eliminated globin mRNAs with the vaccinia virus enzymes, [α-32P]GTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a 32P-labeled 5′-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5′ end than ApG-primed cRNA segments, which initiate exactly at the 3′ end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed. Images PMID:287003

  2. Compounds of the anthracycline family of antibiotics elevate human gamma-globin expression both in erythroid cultures and in a transgenic mouse model.

    PubMed

    Spyrou, Pandelis; Phylactides, Marios; Lederer, Carsten W; Kithreotis, Lucas; Kirri, Andriani; Christou, Soteroulla; Kkolou, Elena; Kanavakis, Emanuel; Anagnou, Nicholas P; Stamatoyannopoulos, George; Kleanthous, Marina

    2010-01-01

    We examined the effect of the anthracyclines aclarubicin, bleomycin, daunorubicin, doxorubicin and idarubicin on human gamma- and beta-globin promoter activity in an in vitro luciferase assay, ex vivo in erythroid cultures and in vivo in transgenic mice carrying the human gamma-globin gene. Effects in erythroid liquid cultures derived from healthy donors were assayed by evaluating HbF production with high performance liquid chromatography and by measuring mRNA levels of the globin genes and the proportion of erythroblasts containing HbF. Compounds testing positive in the in vitro and ex vivo assays were applied to erythroid cultures derived from thalassaemic patients. Doxorubicin, idarubicin and daunorubicin increased HbF production in cultures of both, healthy and thalassaemic donors. Daunorubicin induced HbF in thalassaemic cells ex vivo with the highest statistical significance and, importantly and in contrast to the clinical HbF inducer hydroxyurea, showed specific induction of gamma-globin without associated induction of alpha-globin. Daunorubicin was screened in transgenic mice carrying the human (A)gamma-globin gene, and it resulted in increased (A)gamma-globin mRNA levels. Our results indicate that anthracyclines are a promising group of compounds with the potential to provide lead substances for the synthesis of new agents with clinical applications as gamma-globin gene inducers. In parallel, future studies of the epigenetic effects of the five anthracyclines on the beta-globin locus will generate possible mechanistic leads on the regulation of the globin genes. PMID:19914848

  3. Can we switch microglia's phenotype to foster neuroprotection? Focus on multiple sclerosis

    PubMed Central

    Giunti, Debora; Parodi, Benedetta; Cordano, Christian; Uccelli, Antonio; Kerlero de Rosbo, Nicole

    2014-01-01

    Microglia cells, the resident innate immune cells in the brain, are highly active, extending and retracting highly motile processes through which they continuously survey their microenvironment for ‘danger signals’ and interact dynamically with surrounding cells. Upon sensing changes in their central nervous system microenvironment, microglia become activated, undergoing morphological and functional changes. Microglia activation is not an ‘all-or-none’ process, but rather a continuum depending on encountered stimuli, which is expressed through a spectrum of molecular and functional phenotypes ranging from so-called ‘classically activated’, with a highly pro-inflammatory profile, to ‘alternatively activated’ associated with a beneficial, less inflammatory, neuroprotective profile. Microglia activation has been demonstrated in most neurological diseases of diverse aetiology and has been implicated as a contributor to neurodegeneration. The possibility to promote microglia’s neuroprotective phenotype has therefore become a therapeutic goal. We have focused our discussion on the role of microglia in multiple sclerosis, a prototype of inflammatory, demyelinating, neurodegenerative disease, and on the effect of currently approved or on-trial anti-inflammatory therapeutic strategies that might mediate neuroprotection at least in part through their effect on microglia by modifying their behaviour via a switch of their functional phenotype from a detrimental to a protective one. In addition to pharmaceutical approaches, such as treatment with glatiramer acetate, interferon-β, fingolimod or dimethyl fumarate, we address the alternative therapeutic approach of treatment with mesenchymal stem cells and their potential role in neuroprotection through their ‘calming’ effect on microglia. PMID:24116890

  4. Synthesis and characterization of brain penetrant prodrug of neuroprotective D-264: Potential therapeutic application in the treatment of Parkinson's disease.

    PubMed

    Dholkawala, Fahd; Voshavar, Chandrashekhar; Dutta, Aloke K

    2016-06-01

    Parkinson's disease (PD) is one of the major debilitating neurodegenerative disorders affecting millions of people worldwide. Progressive loss of dopamine neurons resulting in development of motor dysfunction and other related non-motor symptoms is the hallmark of PD. Previously, we have reported on the neuroprotective property of a potent D3 preferring agonist D-264. In our goal to increase the bioavailability of D-264 in the brain, we have synthesized a modified cysteine based prodrug of D-264 and evaluated its potential in crossing the blood-brain barrier. Herein, we report the synthesis of a novel modified cysteine conjugated prodrug of potent neuroprotective D3 preferring agonist D-264 and systematic evaluation of the hydrolysis pattern of the prodrug to yield D-264 at different time intervals in rat plasma and brain homogenates using HPLC analysis. Furthermore, we have also performed in vivo experiments with the prodrug to evaluate its enhanced brain penetration ability. PMID:26994936

  5. Biophysical Characterisation of Globins and Multi-Heme Cytochromes Using Electron Paramagnetic Resonance and Optical Spectroscopy

    NASA Astrophysics Data System (ADS)

    Desmet, Filip

    Heme proteins of different families were investigated in this work, using a combination of pulsed and continuous-wave electron paramagnetic resonance (EPR) spectroscopy, optical absorption spectroscopy, resonance Raman spectroscopy and laser flash photolysis. The first class of proteins that were investigated, were the globins. The globin-domain of the globin-coupled sensor of the bacterium Geobacter sulfurreducens was studied in detail using different pulsed EPR techniques (HYSCORE and Mims ENDOR). The results of this pulsed EPR study are compared with the results of the optical investigation and the crystal structure of the protein. The second globin, which was studied, is the Protoglobin of Methanosarcina acetivorans, various mutants of this protein were studied using laser flash photolysis and Raman spectroscopy to unravel the link between this protein's unusual structure and its ligand-binding kinetics. In addition to this, the CN -bound form of this protein was investigated using EPR and the influence of the strong deformation of the heme on the unusual low gz values is discussed. Finally, the neuroglobins of three species of fishes, Danio rerio, Dissostichus mawsoni and Chaenocephalus aceratus are studied. The influence of the presence or absence of two cysteine residues in the C-D and D-region of the protein on the EPR spectrum, and the possible formation of a disulfide bond is studied. The second group of proteins that were studied in this thesis belong to the family of the cytochromes. First the Mouse tumor suppressor cytochrome b561 was studied, the results of a Raman and EPR investigation are compared to the Human orthologue of the protein. Secondly, the tonoplast cytochrome b561 of Arabidopsis was investigated in its natural form and in two double-mutant forms, in which the heme at the extravesicular side was removed. The results of this investigation are then compared with two models in literature that predict the localisation of the hemes in this

  6. Same. beta. -globin gene mutation is present on nine different. beta. -thalassemia chromosomes in a Sardinian population

    SciTech Connect

    Pirastu, M.; Galanello, R.; Doherty, M.A.; Tuveri, T.; Cao, A.; Kan, Y.W.

    1987-05-01

    The predominant ..beta..-thalassemia in Sardinia is the ..beta../sup 0/ type in which no ..beta..-globin chains are synthesized in the homozygous state. The authors determined the ..beta..-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same ..beta../sup 39(CAG..-->..TAG)/ nonsense mutation was found on nine different chromosome haplotypes. Although this mutation may have arisen more than once, the multiple haplotypes could also be generated by crossing over and gene conversion events. These findings underscore the frequency of mutational events in the ..beta..-globin gene region.

  7. Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

    PubMed

    Mushtaq, Gohar; Greig, Nigel H; Anwar, Firoz; Al-Abbasi, Fahad A; Zamzami, Mazin A; Al-Talhi, Hasan A; Kamal, Mohammad A

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  8. Thrombolysis and neuroprotection in cerebral ischemia.

    PubMed

    Gutiérrez, M; Díez Tejedor, E; Alonso de Leciñana, M; Fuentes, B; Carceller, F; Roda, J M

    2006-01-01

    Stroke is a major cause of death and disability worldwide. The resulting burden on society grows with the increase in the incidence of stroke. The term brain attack was introduced to describe the acute presentation of stroke and emphasize the need for urgent action to remedy the situation. Though a large number of therapeutic agents, like thrombolytics, NMDA receptor antagonists, calcium channel blockers and antioxidants, have been used or are being evaluated, there is still a large gap between the benefits of these agents and the properties of an ideal drug for stroke. So far, only thrombolysis with rtPA within a 3-hour time window has been shown to improve the outcome of patients with ischemic stroke. Understanding the mechanisms of injury and neuroprotection in these diseases is important to target news sites for treating ischemia. Better evaluation of the drugs and increased similarity between the results of animal experimentation and in the clinical setting requires critical assessment of the selection of animal models and the parameters to be evaluated. Our laboratory has employed a rat embolic stroke model to investigate the combination of rtPA with citicoline as compared to monotherapy alone and investigated whether neuroprotection should be provided before or after thrombolysis in order to achieve a greater reduction of ischemic brain damage. PMID:16651822

  9. Nutraceutical Antioxidants as Novel Neuroprotective Agents

    PubMed Central

    Kelsey, Natalie A.; Wilkins, Heather M.; Linseman, Daniel A.

    2015-01-01

    A variety of antioxidant compounds derived from natural products (nutraceuticals) have demonstrated neuroprotective activity in either in vitro or in vivo models of neuronal cell death or neurodegeneration, respectively. These natural antioxidants fall into several distinct groups based on their chemical structures: (1) flavonoid polyphenols like epigallocatechin 3-gallate (EGCG) from green tea and quercetin from apples; (2) non-flavonoid polyphenols such as curcumin from tumeric and resveratrol from grapes; (3) phenolic acids or phenolic diterpenes such as rosmarinic acid or carnosic acid, respectively, both from rosemary; and (4) organosulfur compounds including the isothiocyanate, L-sulforaphane, from broccoli and the thiosulfonate allicin, from garlic. All of these compounds are generally considered to be antioxidants. They may be classified this way either because they directly scavenge free radicals or they indirectly increase endogenous cellular antioxidant defenses, for example, via activation of the nuclear factor erythroid-derived 2-related factor 2 (Nrf2) transcription factor pathway. Alternative mechanisms of action have also been suggested for the neuroprotective effects of these compounds such as modulation of signal transduction cascades or effects on gene expression. Here, we review the literature pertaining to these various classes of nutraceutical antioxidants and discuss their potential therapeutic value in neurodegenerative diseases. PMID:21060289

  10. Nutraceutical antioxidants as novel neuroprotective agents.

    PubMed

    Kelsey, Natalie A; Wilkins, Heather M; Linseman, Daniel A

    2010-11-01

    A variety of antioxidant compounds derived from natural products (nutraceuticals) have demonstrated neuroprotective activity in either in vitro or in vivo models of neuronal cell death or neurodegeneration, respectively. These natural antioxidants fall into several distinct groups based on their chemical structures: (1) flavonoid polyphenols like epigallocatechin 3-gallate (EGCG) from green tea and quercetin from apples; (2) non-flavonoid polyphenols such as curcumin from tumeric and resveratrol from grapes; (3) phenolic acids or phenolic diterpenes such as rosmarinic acid or carnosic acid, respectively, both from rosemary; and (4) organosulfur compounds including the isothiocyanate, L-sulforaphane, from broccoli and the thiosulfonate allicin, from garlic. All of these compounds are generally considered to be antioxidants. They may be classified this way either because they directly scavenge free radicals or they indirectly increase endogenous cellular antioxidant defenses, for example, via activation of the nuclear factor erythroid-derived 2-related factor 2 (Nrf2) transcription factor pathway. Alternative mechanisms of action have also been suggested for the neuroprotective effects of these compounds such as modulation of signal transduction cascades or effects on gene expression. Here, we review the literature pertaining to these various classes of nutraceutical antioxidants and discuss their potential therapeutic value in neurodegenerative diseases. PMID:21060289

  11. Neuro-protective Mechanisms of Lycium barbarum.

    PubMed

    Xing, Xiwen; Liu, Fenyong; Xiao, Jia; So, Kwok Fai

    2016-09-01

    Neuronal diseases, including retinal disorders, stroke, Alzheimer's disease, Parkinson's disease and spinal cord injury, affect a large number of people worldwide and cause heavy social and economic burdens. Although many efforts have been made by scientists and clinicians to develop novel drug and healthcare strategies, few of them received satisfactory outcomes to date. Lycium barbarum is a traditional homology of medicine and food in Chinese medicine, with the capability to nourish the eyes, liver and kidneys. Recent studies have also explored its powerful neuro-protective effects on a number of neuronal diseases. In the current review, we collected key recent findings regarding the neuro-protective effects and mechanisms of L. barbarum derivatives, primarily its polysaccharide (LBP) , in some common diseases of the nervous system. A comprehensive comparison with currently available drugs has also been discussed. In general, LBP is a promising neuronal protector with potent ameliorative effects on key pathological events, such as oxidative stress, inflammation, apoptosis and cell death with minimal side effects. PMID:27033360

  12. Neuroprotective Mechanisms Mediated by CDK5 Inhibition

    PubMed Central

    Mushtaq, Gohar; Greig, Nigel H.; Anwar, Firoz; Al-Abbasi, Fahad A.; Zamzami, Mazin A.; Al-Talhi, Hasan A.; Kamal, Mohammad A.

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not well-understood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine-induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  13. Neuroprotective strategies against calpain-mediated neurodegeneration.

    PubMed

    Yildiz-Unal, Aysegul; Korulu, Sirin; Karabay, Arzu

    2015-01-01

    Calpains are calcium-dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over-activation. According to the results of numerous studies to date, there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration, leading to neuronal death. Thus, it is very crucial to unravel all the aspects of calpain-mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects. Protecting neurons against calpain-activated apoptosis basically requires developing effective, reliable, and most importantly, therapeutically applicable approaches to succeed. From this aspect, the most significant studies focusing on preventing calpain-mediated neurodegeneration include blocking the N-methyl-d-aspartate (NMDA)-type glutamate receptor activities, which are closely related to calpain activation; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors, or inhibiting its downstream processes; and utilizing the neuroprotectant steroid hormone estrogen and its receptors. In this review, the most remarkable neuroprotective strategies for calpain-mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another, in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases. PMID:25709452

  14. Ocular neuroprotection by siRNA targeting caspase-2

    PubMed Central

    Ahmed, Z; Kalinski, H; Berry, M; Almasieh, M; Ashush, H; Slager, N; Brafman, A; Spivak, I; Prasad, N; Mett, I; Shalom, E; Alpert, E; Di Polo, A; Feinstein, E; Logan, A

    2011-01-01

    Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss. PMID:21677688

  15. Distribution of beta-globin haplotypes among the tribes of southern Gujarat, India.

    PubMed

    Aggarwal, Aastha; Khurana, Priyanka; Mitra, Siuli; Raicha, Bhavesh; Saraswathy, K N; Italia, Yazdi M; Kshatriya, Gautam K

    2013-06-01

    The present study was carried out in Indo-European speaking tribal population groups of southern Gujarat (India) to elucidate the allelic and haplotypic content of β-globin system in individuals with HbAA genotypes. 6 neutral restriction sites of the β-globin system were analysed and various statistical parameters were estimated to draw meaningful interpretations. All the 6 sites were found to be polymorphic and most were in Hardy-Weinberg Equilibrium in the studied group. Haplotypes were constructed using two different combinations of the 6 restriction sites analysed. Analysis of the 5 sites revealed a set of three predominant haplotypes, '+----', '-++-+' and '-+-++'; and haplotypes '+--', '++-' and '+++' were found to be the most frequent when the 3 sites were used to construct the haplotypes. Haplotypic heterozygosity levels (>83%) observed in the present study group were comparable to those observed in African and Afro-American populations and greater than other world populations. All the ancestral haplotypes, +-----, -++-+, -+-++ and ----+ were found in the study group. The distribution pattern of various haplotypes was consistent with the global pattern. The paucity of comparable data from other Indian populations restricted one from making interpretations about the study group's relationships with other Indian populations but the results were indicative of older population histories or experience of gene flow by the study group and their affinities with populations of southern India. PMID:23500448

  16. Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

    PubMed Central

    Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

    2003-01-01

    Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

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

    PubMed

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

    2016-01-01

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

  18. Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia.

    PubMed

    Origa, Raffaella; Paglietti, Maria E; Sollaino, Maria C; Desogus, Maria F; Barella, Susanna; Loi, Daniela; Galanello, Renzo

    2014-01-01

    α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype-phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present. PMID:23896219

  19. Determination of ligand pathways in globins: apolar tunnels versus polar gates.

    PubMed

    Salter, Mallory D; Blouin, George C; Soman, Jayashree; Singleton, Eileen W; Dewilde, Sylvia; Moens, Luc; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Olson, John S

    2012-09-28

    Although molecular dynamics simulations suggest multiple interior pathways for O(2) entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands. PMID:22859299

  20. Effect of AGM and fetal liver-derived stromal cell lines on globin expression in adult baboon (P. anubis) bone marrow-derived erythroid progenitors.

    PubMed

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  1. Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

    PubMed Central

    Lavelle, Donald; Vaitkus, Kestutis; Ruiz, Maria Armila; Ibanez, Vinzon; Kouznetsova, Tatiana; Saunthararajah, Yogen; Mahmud, Nadim; DeSimone, Joseph

    2012-01-01

    This study was performed to investigate the hypothesis that the erythroid micro-environment plays a role in regulation of globin gene expression during adult erythroid differentiation. Adult baboon bone marrow and human cord blood CD34+ progenitors were grown in methylcellulose, liquid media, and in co-culture with stromal cell lines derived from different developmental stages in identical media supporting erythroid differentiation to examine the effect of the micro-environment on globin gene expression. Adult progenitors express high levels of γ-globin in liquid and methylcellulose media but low, physiological levels in stromal cell co-cultures. In contrast, γ-globin expression remained high in cord blood progenitors in stromal cell line co-cultures. Differences in γ-globin gene expression between adult progenitors in stromal cell line co-cultures and liquid media required cell-cell contact and were associated with differences in rate of differentiation and γ-globin promoter DNA methylation. We conclude that γ-globin expression in adult-derived erythroid cells can be influenced by the micro-environment, suggesting new potential targets for HbF induction. PMID:22693559

  2. Duplication of the gamma-globin gene mediated by L1 long interspersed repetitive elements in an early ancestor of simian primates.

    PubMed Central

    Fitch, D H; Bailey, W J; Tagle, D A; Goodman, M; Sieu, L; Slightom, J L

    1991-01-01

    Regions surrounding the single gamma-globin gene of galago and the duplicated gamma 1- and gamma 2-globin genes of gibbon, rhesus monkey, and spider monkey were sequenced and aligned with those from humans. Contrary to previous studies, spider monkey was found to have not one but two gamma-globin genes, only one of which (gamma 2) is functional. The reconstructed evolutionary history of the gamma-globin genes and their flanking sequences traces their origin to a tandem duplication of a DNA segment approximately 5.5 kilobases long that occurred before catarrhine primates (humans, apes, and Old World monkeys) diverged from platyrrhines (New World monkeys), much earlier than previously thought. This reconstructed molecular history also reveals that the duplication resulted from an unequal homologous crossover between two related L1 long interspersed repetitive elements, one upstream and one downstream of the single ancestral gamma-globin gene. Perhaps facilitated by the redundancy resulting from the duplication, the gamma-globin genes escaped the selective constraints of embryonically functioning genes and evolved into fetally functioning genes. This view is supported by the finding that a burst of nonsynonymous substitutions occurred in the gamma-globin genes while they became restructured for fetal expression in the common ancestor of platyrrhines and catarrhines. PMID:1908094

  3. Revisiting the Term Neuroprotection in Chronic and Degenerative Diseases.

    PubMed

    Orsini, Marco; Nascimento, Osvaldo J M; Matta, Andre P C; Reis, Carlos Henrique Melo; de Souza, Olivia Gameiro; Bastos, Victor Hugo; Moreira, Rayele; Ribeiro, Pedro; Fiorelli, Stenio; Novellino, Pietro; Pessoa, Bruno; Cunha, Mariana; Pupe, Camila; Morales, Pedro S; Filho, Pedro F Moreira; Trajano, Eduardo Lima; Oliveira, Acary Bulle

    2016-04-01

    Thanks to the development of several new researches, the lifetime presented a significant increase, even so, we still have many obstacles to overcome - among them, manage and get responses regarding neurodegenerative diseases. Where we are in the understanding of neuroprotection? Do we really have protective therapies for diseases considered degeneratives such as amyotrophic lateral sclerosis and its variants, Parkinson's disease, Alzheimer's disease and many others? Neuroprotection is defined by many researches as interactions and interventions that can slow down or even inhibit the progression of neuronal degeneration process. We make some considerations on this neuroprotective effect. PMID:27127599

  4. Revisiting the Term Neuroprotection in Chronic and Degenerative Diseases

    PubMed Central

    Orsini, Marco; Nascimento, Osvaldo J.M.; Matta, Andre P.C.; Reis, Carlos Henrique Melo; de Souza, Olivia Gameiro; Bastos, Victor Hugo; Moreira, Rayele; Ribeiro, Pedro; Fiorelli, Stenio; Novellino, Pietro; Pessoa, Bruno; Cunha, Mariana; Pupe, Camila; Morales, Pedro S.; Filho, Pedro F. Moreira; Trajano, Eduardo Lima; Oliveira, Acary Bulle

    2016-01-01

    Thanks to the development of several new researches, the lifetime presented a significant increase, even so, we still have many obstacles to overcome – among them, manage and get responses regarding neurodegenerative diseases. Where we are in the understanding of neuroprotection? Do we really have protective therapies for diseases considered degeneratives such as amyotrophic lateral sclerosis and its variants, Parkinson’s disease, Alzheimer’s disease and many others? Neuroprotection is defined by many researches as interactions and interventions that can slow down or even inhibit the progression of neuronal degeneration process. We make some considerations on this neuroprotective effect. PMID:27127599

  5. Neuroprotective dibenzylbutyrolactone lignans of Torreya nucifera.

    PubMed

    Jang, Y P; Kim, S R; Kim, Y C

    2001-07-01

    The methanolic extract of the bark of Torreya nucifera Sieb. et Zucc. (Taxaceae) significantly protected primary cultures of rat cortical cells exposed to the excitotoxic amino acid, L-glutamate. (-)-Arctigenin (1), (-)-traxillagenin (2), arctiin (4), traxillaside (5), and a newly-reported compound 3 (-)-4'-demethyltraxillagenin [(2R,3R)-2-(4''-hydroxy-3''-methoxybenzyl)-3-(4'-hydroxy-3',5'-dimethoxybenzyl)-butyrolactone] were isolated by bioactivity-guided fractionation and further separation using chromatographic techniques. These lignans and their glycosides had significant neuroprotective activities against glutamate-induced toxicity in primary cultures of rat cortical cells at concentrations ranging from 0.01 microM to 10.0 microM. PMID:11488466

  6. [Amides of creatine: perspectives of neuroprotection].

    PubMed

    Vlasov, T D; Chefu, S G; Baĭsa, A E; Leko, M V; Burov, S V; Veselkina, O S

    2011-07-01

    We evaluated the efficacy of derivatives of creatine and amino acids (CrAA) for decreasing cerebral injury in rats with transient middle cerebral artery occlusion (MCAO). Neuroprotective effects of amides of creatine and glycine (CrGlyOEt), phenylalanine (CrPheNH2), thyrosine (CrTyrNH2), and GABA (CrGABAOEt) were investigated. Brain injury was evaluated on day 2 after transient MCAO using a TTC staining of brain slices. Compared with the MCAO control group, all the CrAms showed decreased cerebral injury (p < 0.05). However CrPheNH2, CrTyrNH2, and CrGABAOEt were toxic after intravenous administration and investigated only after intraperitoneal injection. CrGlyOEt did not show any toxicity at dose of 1 mmol/kg. These data evidenced that creatinyl amides can represent promising candidates for the development of new drugs useful in brain ischemia treatment. PMID:21961295

  7. Modeling Emergence in Neuroprotective Regulatory Networks

    SciTech Connect

    Sanfilippo, Antonio P.; Haack, Jereme N.; McDermott, Jason E.; Stevens, S.L.; Stenzel-Poore, Mary

    2013-01-05

    The use of predictive modeling in the analysis of gene expression data can greatly accelerate the pace of scientific discovery in biomedical research by enabling in silico experimentation to test disease triggers and potential drug therapies. Techniques that focus on modeling emergence, such as agent-based modeling and multi-agent simulations, are of particular interest as they support the discovery of pathways that may have never been observed in the past. Thus far, these techniques have been primarily applied at the multi-cellular level, or have focused on signaling and metabolic networks. We present an approach where emergence modeling is extended to regulatory networks and demonstrate its application to the discovery of neuroprotective pathways. An initial evaluation of the approach indicates that emergence modeling provides novel insights for the analysis of regulatory networks that can advance the discovery of acute treatments for stroke and other diseases.

  8. The relationship between the presence of extra α-globin genes and blood cell traits in Altamurana sheep

    PubMed Central

    2003-01-01

    Additional α-globin genes in sheep might produce extra α-globin chains and, consequently, the subject carrying triplicated (ααα) or quadruplicated (αααα) haplotypes may exhibit different hematological phenotypes when compared to the normal duplicated (αα) homozygotes (NN). Both ααα and αααα heterozygous (ND) and ααα and αααα homozygous (DD) individuals were obtained by selection and inbreeding. Chromatographic RP-HPLC analyses of the globin chains of 65 subjects (15 DD, 20 ND and 30 NN) were performed. A highly significant linear regression (r2 = 0.967) of the α/β ratio on the number of α-globin genes was found, and the α/β ratio ranged on average from 1.0 in NN individuals to 1.2 in the ND and 1.6 in the DD subjects. Values for blood fell within the range of normality but were rather peculiar as a whole. When the erythrocytes of individuals carrying normal arrangements were compared with those of subjects with extra α-genes, the latter had fewer erythrocytes that were bigger in size and had a higher Hb content and a greater osmotic fragility. This hematological picture is consistent with the existence of an unbalanced α/β ratio. PMID:12927085

  9. HbVar: A relational database of human hemoglobin variants and thalassemia mutations at the globin gene server.

    PubMed

    Hardison, Ross C; Chui, David H K; Giardine, Belinda; Riemer, Cathy; Patrinos, George P; Anagnou, Nicholas; Miller, Webb; Wajcman, Henri

    2002-03-01

    We have constructed a relational database of hemoglobin variants and thalassemia mutations, called HbVar, which can be accessed on the web at http://globin.cse.psu.edu. Extensive information is recorded for each variant and mutation, including a description of the variant and associated pathology, hematology, electrophoretic mobility, methods of isolation, stability information, ethnic occurrence, structure studies, functional studies, and references. The initial information was derived from books by Dr. Titus Huisman and colleagues [Huisman et al., 1996, 1997, 1998]. The current database is updated regularly with the addition of new data and corrections to previous data. Queries can be formulated based on fields in the database. Tables of common categories of variants, such as all those involving the alpha1-globin gene (HBA1) or all those that result in high oxygen affinity, are maintained by automated queries on the database. Users can formulate more precise queries, such as identifying "all beta-globin variants associated with instability and found in Scottish populations." This new database should be useful for clinical diagnosis as well as in fundamental studies of hemoglobin biochemistry, globin gene regulation, and human sequence variation at these loci. PMID:11857738

  10. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    NASA Astrophysics Data System (ADS)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.