bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐HT4 Receptors: Gene, Transduction and Effects on Olfactory Memory
Authors: BOCKAERT J. , CLAEYSEN S. , SEBBEN M. , DUMUIS A.
Published: February 2006

ABSTRACT: In this paper we discuss 1) the primary structures, pharmacology, and brain distribution of cloned 5‐HT4 receptors; 2) the chromosomal localization of the h5‐HT4 receptor; 3) whether benzamides are full or partial agonists because of a species or a coupling difference; 4) the intrinsic activity of 5‐HT4 receptors and inverse agonism of GR125487 in COS‐7 cells but not in colliculi neurons; 5) the modulation of 5‐HT4 receptor binding and activity; and 6) the long‐term blockade of K+ channels by 5‐HT4 agonists and its effect on olfactory memory. We conclude that 1) the cloning of 5‐HT4 receptors in different species using RT‐PCR from different tissues reveals the presence of several splice variants for 5‐HT4 receptors differing in the C‐terminal part, downstream from the amino acid L358; 2) the pharmacological properties of 5‐HT4 receptors are dependent on the cellular context in which they are expressed; and 3) 5‐HT4 agonists can be added to the list of compounds having pro‐cognitive properties.
Serotonin Receptor Variants in Disease: New Therapeutic Opportunities?
Authors: PEROUTKA S. J.
Published: February 2006

ABSTRACT: This review describes a novel genetic approach to the assessment of receptor function that is based on association studies of polymorphisms within human genes. The realization that variations within human genes may significantly affect gene function has led to increased use of this approach in recent years. Analysis of polymorphisms within the human 5‐HT2A receptor is used as a specific example of the application of association genetics to elucidate gene function. The interaction of many neuroleptics and antidepressants with 5‐HT2A receptors points up the potential importance of this receptor for understanding and treating neuropsychiatric disorders such as schizophrenia and depression.
Mouse 5‐HT2B Receptor‐mediated Serotonin Trophic Functionsa
Authors: CHOI D.‐S. , KELLERMANN O. , RICHARD S. , COLAS J.‐F. , BOLAÑOS‐JIMENEZ F. , TOURNOIS C. , LAUNAY J.‐M. , MAROTEAUX L.
Published: February 2006

ABSTRACT: 5‐HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto‐oncogene product p21ras. During mouse embryogenesis, a peak of 5‐HT2B receptor expression is detected at the neurulation stage; we localized the 5‐HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5‐HT2B receptors shortly after gastrulation, is supported by culture of embryos exposed to 5‐HT2B‐high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube.

Functional 5‐HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma‐derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5‐HT2B receptors become functional, and on day 4, the appearance of 5‐HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin suggesting autoreceptor functions for 5‐HT2B receptors.
Putative 5‐ht5 Receptors: Localization in the Mouse CNS and Lack of Effect in the Inhibition of Dural Protein Extravasation
Authors: WAEBER C. , GRAILHE R. , YU X‐J. , HEN R. , MOSKOWITZ M. A.
Published: February 2006

ABSTRACT: Putative 5‐ht5 receptor binding sites were visualized by in vitro autoradiography using [125I]LSD (in the presence of clozapine and spiperone) or [3H]5‐carboxamidotryptamine (in the presence 8‐OH‐DPAT, GR127935 and spiperone). Under these conditions, no [3H]5‐carboxamidotryptamine labeling was detected in the brain of mice lacking the gene encoding the putative 5‐ht5a receptor (knockout mice), whereas intermediate densities of binding sites were seen in the olfactory bulb and neocortex of wild‐type mice. [125I]LSD labeled the same areas as [3H]5‐carboxamidotryptamine in wild‐type mice. High densities of [125I]LSD binding sites were observed in the medial habenula of wild type and knockout mice. 5‐CT competed for [125I]LSD binding sites with an affinity of 2 nM in the olfactory bulb and neocortex of wild‐type mice and an affinity of 30 nM in the habenula of knockout mice, suggesting that habenular labeling might be accounted for by putative 5‐ht5b receptors. In the presence of 5′‐guanylylimidodiphosphate, 5‐CT displaced [125I]LSD from putative 5‐ht5a and 5‐ht5b sites with a 6‐times and 3‐times lower affinity, respectively, suggesting that both receptor subtypes are coupled to G proteins in brain. We also studied the inhibitory effect of 5‐CT on dural neurogenic inflammation in knockout mice. In wild type mice, 3 ng/kg 5‐CT inhibited dural protein extravasation by 60%. A similar effect was observed in knockout mice, even in the presence of the 5‐HT1B receptor antagonist GR127935. These results suggest that the inhibitory effects of 5‐CT are not mediated by a site with the characteristics of the putative 5‐ht5 receptor.
Pleiotropic Behavior of 5‐HT2A and 5‐HT2C Receptor Agonists
Authors: BERG K. A. , CLARKE W. P. , MAAYANI S. , GOLDFARB J.
Published: February 2006

ABSTRACT: There is now considerable evidence that a single receptor subtype can couple to multiple effector pathways within a cell. Recently, Kenakin proposed a new concept, termed “agonist‐directed trafficking of receptor stimulus”, that suggests that agonists may be able to selectively activate a subset of multiple signaling pathways coupled to a single receptor subtype. 5‐HT2A and 5‐HT2C receptors couple to phospholipase C‐(PLC) mediated inositol phosphate (IP) accumulation and PLA2‐mediated arachidonic acid (AA) release. Relative efficacies of agonists (referenced to 5‐HT) differed depending upon whether IP accumulation or AA release was measured. For the 5‐HT2C receptor system, some agonists (e.g. TFMPP) preferentially activated the PLC‐IP pathway, whereas others (e.g. LSD) favored PLA2‐AA. As expected, EC50's of agonists did not differ between pathways. For the 5‐HT2A receptor system, all agonists tested had greater relative efficacy for PLA2‐AA than for PLC‐IP. In contrast, relative efficacies were not different for 5‐HT2A agonists when sequential effects in a pathway were measured (IP accumulation vs. calcium mobilization). These data strongly support the agonist‐directed trafficking hypothesis.
Serotonergic Antagonist Effects on Trafficking of Serotonin 5‐HT2A Receptors in Vitro and in Vivoa
Authors: WILLINS D. L. , ALSAYEGH L. , BERRY S. A. , BACKSTROM J. R. , SANDERS‐BUSH E. , FRIEDMAN L. , KHAN N. , ROTH B. L.
Published: February 2006

ABSTRACT: The mechanism by which antagonists down‐regulate 5‐HT2A receptors is unknown. We here report that a variety of 5‐HT2A antagonists induce a change in the subcellular distribution of 5‐HT2A receptors both in vitro and in vivo. In a stably transfected NIH 3T3 cell‐line, brief exposure to 1 μM clozapine caused a 2.5‐fold increase in intracellular 5‐HT2A‐like immunoreactivity, as measured by confocal microscopy. Confirmatory studies utilizing a biotin‐trap technique, demonstrated that the increase in intracellular immunoreactivity results from internalization of receptor from the cell surface. Exposure of transfected cells to other 5‐HT2A receptor antagonists produced similar increases in intracellular 5‐HT2A‐like immunoreactivity. In vivo administration of clozapine (20 mg/kg, sc, ×7 days) caused a greater than twofold increase in intracellular immunoreactivity in cell bodies of cortical pyramidal neurons. Additionally, chronic clozapine administration was associated with a decrease in labeling of apical dendrites on pyramidal cells. These results show that clozapine causes a change in subcellular distribution of 5‐HT2A receptors in vitro and in vivo.
Regulation of Levels of 5‐HT2A Receptor mRNA
Authors: WOHLPART K. L. , MOLINOFF P. B.
Published: February 2006

ABSTRACT: We have been able to identify two separate pathways present in P11 cells that can increase the levels and stability of 5‐HT2A receptor mRNA. One pathway is activated following exposure of cells to serotonin and is dependent upon activation of a PKC isoform that is susceptible to downregulation by a 24‐h pretreatment with PMA; another pathway is activated following treatment of cells with a calcium ionophore. This pathway is not dependent on activation of PKC isoforms that can be downregulated by a 24‐h treatment with phorbol ester. Such heterologous regulation of levels of 5‐HT2A receptor mRNA may be important in understanding in vivo events where multiple factors contribute to the modulation of levels of cell surface receptors.
A Putative α‐helical Gβγ‐coupling Domain in the Second Intracellular Loop of the 5‐HT1A Receptora
Authors: ALBERT P. R. , MORRIS S. J. , GHAHREMANI M. H. , STORRING J. M. , LEMBO P. M. C.
Published: February 2006

ABSTRACT: We have identified a conserved threonine residue in the second intracellular (i2) loop of the 5‐HT1A receptor that when mutated to alanine prevents coupling to Gβγ‐mediated signaling, while preserving Gαi‐induced actions. 48 In this review, we investigate the characteristics and potential role of the i2 domain in the coupling of the 5‐HT1A receptor and other receptors to G proteins. The i2 domain, as well as portions of the i3 domain, is predicted to form an amphipathic α‐helix with a positively charged face and a hydrophobic face. Mutagenesis experiments support a model in which the hydrophobic faces of these α‐helical domains form an intracellular binding “pocket” for interaction with G proteins. Embedded in the hydrophobic face, Thr149 is crucial for signaling through Gβγ subunits, perhaps via interaction with its hydroxyl side‐chain. Mutation of other residues of the i2 domain of Gi‐coupled receptors is required to substantiate the importance of the α‐helical i2 domain in receptor‐Gβγ signaling. If confirmed in other receptors, these results support a general model in which activated receptor and Gβγ subunits remain associated to interact with effectors in a receptor‐specific manner.
Serotonin 5‐HT2A Receptor Activation Inhibits Cytokine‐stimulated Inducible Nitric Oxide Synthase in C6 Glioma Cellsa
Authors: MILLER K. J. , GONZALEZ H. A.
Published: February 2006

ABSTRACT: C6‐glioma cells endogenously express both 5‐HT2A receptors and inducible nitric oxide synthase (iNOS). iNOS can be induced by transcriptional activation to produce nitric oxide (NO) in response to a challenge with the pro‐inflammatory cytokines TNF‐α and IFN‐γ. Experiments were conducted to determine whether 5‐HT2A receptor activation could modify the production of NO in response to the inducing agents. 1 μM DOI produced a dose‐dependent inhibition of the cytokine‐inducted nitrite levels of 40% which was inhibited by spiperone and ritanserin. In addition, the DOI‐mediated decrease was prevented by the PKC inhibitor chelerythrine (100 nM). The effectiveness of DOI was lost when added more than two hours after the addition of inducing agent, suggesting that DOI was regulating iNOS at the level of transcription rather than post‐translationally. We suggest that there is a link between the serotonergic system and NO‐mediated immune responses in the brain.
Molecular, Cellular and Physiological Characteristics of 5‐HT‐Moduline, a Novel Endogenous Modulator of 5‐HT1B Receptor Subtype
Authors: MASSOT O. , ROUSSELLE J.‐C. , GRIMALDI B. , CLÖZ‐TAYARANI I. , FILLION M. P. , PLANTEFOL M. , BONNIN A. , PRUDHOMME N. , FILLION G.
Published: February 2006

ABSTRACT: The serotonergic transmission is considered as a neuromodulatory system in the Central Nervous System. 5‐HT1B receptors play an important role in this modulatory activity. We have purified from mammalian brain an endogenous peptide, LSAL, we called 5‐HT‐moduline, interacting specifically with 5‐HT1B receptors. This interaction is characterized by a high affinity (Ki=10−10M) and a non‐competitive mechanism. Direct [3H]5‐HT‐moduline binding revealed a single population of sites having an apparent affinity constant close to 10−10M. Autoradiographic studies showed a brain distribution of [3H]5‐HT‐moduline binding sites closely related to the 5‐HT1B receptors. In functional studies, the peptide is able to reverse the activity of a 5‐HT1B agonist in the nanomolar range. Furthermore, this antagonist effect is also observed in vivo on mice behavior. Immunocytochemistry revealed an heterogeneous distribution of 5‐HT‐moduline in mouse brain. The labeled structures correspond to cellular profiles with axon‐like prolongations. Moreover, in vitro, LSAL is released in a Ca++,K+‐dependent manner. Therefore, 5‐HT‐moduline behaves as a neurotransmitter. The fact that 5‐HT‐moduline induces the desensitization of 5‐HT1B receptors reflects the existence of a novel and efficient mechanism able to rapidly modulate the serotonergic activity.
Oleamide‐induced Modulation of 5‐Hydroxytryptamine Receptor‐mediated Signalinga
Authors: THOMAS E. A. , CARSON M. J. , SUTCLIFFE J. G.
Published: February 2006

ABSTRACT: We investigated the effects of oleamide, a fatty acid amide isolated from the cerebrospinal fluid of sleep‐deprived cats, on serotonin receptor‐mediated signaling in cultured mammalian cells. Oleamide demonstrated opposing effects on 5‐HT2A and 5‐HT7 receptors, in rat pituitary cells and transfected HeLa cells, respectively. Oleamide caused a potentiation of 5‐HT‐elicited inositol phosphate formation mediated by the 5‐HT2A receptor, but inhibited the effects of 5‐HT on cAMP production mediated by the 5‐HT7 receptor. In addition, oleamide alone caused a significant increase in cAMP accumulation that was dependent on the presence of the 5‐HT7 receptor, but was not blocked by clozapine. These results demonstrate that oleamide can have diverse effects on 5‐HT‐mediated signal transduction at different subtypes of mammalian 5‐HT receptors. Additionally, our data suggest that oleamide may act at an allosteric site on the 5‐HT7 receptor and can elicit functional responses via activation of this site.
Regulation of Membrane Excitability in the Central Nervous System by Serotonin Receptor Subtypesa
Authors: ANDRADE R.
Published: February 2006

ABSTRACT:Serotonin exerts multiple electrophysiological effects on neurons of the central nervous system. It is now known that this diversity reflects at least in part the existence of multiple serotonin receptor subtypes. An example of this occurs in the CA1 region of the hippocampus where as many as ten different serotonin receptor subtypes appear to be expressed. Recent electrophysiological studies have been able to assign specific functional roles to at least 5 of these receptors. These receptors are differentially expressed in the two different cell types present in this region, pyramidal cells and GABAergic interneurons, and mediate different effects on membrane excitability. This distribution is consistent with the different functional roles played by these cells in hippocampus. Thus the differential expression of serotonin receptor subtypes in the CA1 region allows serotonin to modify the function of hippocampal neuronal networks in a manner that is both selective and precise.
Role of Somatodendritic 5‐HT Autoreceptors in Modulating 5‐HT Neurotransmissiona
Authors: BLIER P. , PIÑEYRO G. , EL MANSARI M. , BERGERON R. , DE MONTIGNY C.
Published: February 2006

ABSTRACT: A very important element controlling serotonin (5‐HT) release throughout the brain is the 5‐HT1A autoreceptor present on the soma and dendrites of 5‐HT neurons since it exerts a negative feedback influence on their firing activity. This 5‐HT1A autoreceptor receives an increased activation by endogenous 5‐HT at the beginning of a treatment with a selective 5‐HT reuptake inhibitor (SSRI) and, consequently, a decreased 5‐HT neuronal firing activity is obtained. As the SSRI treatment is prolonged, the 5‐HT1A autoreceptor desensitizes and firing activity is restored in the presence of the SSRI. That this adaptive change underlies, at least in part, the delayed therapeutic effect of SSRI in major depression is supported by the acceleration of the antidepressant response by the concomitant administration of the 5‐HT1A autoreceptor antagonist pindolol with SSRIs.
Active Site Flexibility in Enzyme Catalysisa
Authors: TSOU CHEN‐LU
Published: February 2006

Abstract: The inactivation of a number of enzymes during denaturation by physical and chemical factors precedes detectable global conformational changes of the molecules as monitored by conventional methods. It was suggested that the enzyme active site is more flexible and more sensitive to denaturation than the molecule as a whole. The well‐known “induced fit” hypothesis by Koshland implies multiconformational states of enzymes in equilibrium with one another easily perturbed by ligands. Each intermediary step during the entire catalytic process may require the molecule to be in a particular conformation state; rapid interconversion between the different conformation states may well be involved in the catalytic process. As a relative fragile and consequently flexible active site has now been envisaged, it appears that a rapid cycling of the different active site conformation states is essential for the full expression of enzyme activity.
Protein Disulfide Isomerase Assists Protein Folding as Both an Isomerase and a Chaperonea
Authors: WANG CHIH‐CHEN
Published: February 2006

Abstract: Protein disulfide isomerase (PDI) is the physiological catalyst of native disulfide bond formation of nascent peptides in the cells. As a foldase, PDI has both isomerase and chaperone activities. The chaperone activity is intrinsic and independent of its isomerase activity. Both chaperone and isomerase activities are required for PDI to assist folding of denatured and reduced disulfide‐containing proteins. PDI may have great applications in protein production by bioengineering for its function as a foldase.
Biosensors for Environmental Monitoring
Authors: KARUBE ISAO , YANO KAZUYOSHI , SASAKI SATOSHI , NOMURA YOKO , IKEBUKURO KAZUNORI
Published: February 2006

Abstract: Environmental monitoring is of great importance for its protection. Conventional monitoring methods are often slow and complex and require expensive equipment, making them unsuitable for in situ, real‐time monitoring of pollutants. Biosensors based on a combination of a biological sensing element and an electronic signal‐transducing element are alternative methods to conventional ones. Biosensors have a number of advantages, such as high selectivity, high stability, and short response time. Various kinds of biosensors have been developed and employed for detection of pollutants such as phosphate, cyanide, and herbicides. Some of these have already been exploited as real‐time monitoring in situ. In this article, some of the applications of biosensors for environmental control are described.
Biosensors for Fermentation Process Controla
Authors: LIU JIANGUO , LI GAOXIANG , XIE SHUNZHEN , HAN RUSHUI , CHEN KEMIN
Published: February 2006

Abstract: Two biosensors for fermentation process control have been introduced, which were developed in our lab recently. One is an enzyme electrode‐based on‐line monitoring system for glutamate fermentation process control and the other is an H+‐ISFET‐based ENFET for penicillin G fermentation process control.
Chiral Alcohol Synthesis with Microbial Carbonyl Reductases in a Water‐Organic Solvent Two‐Phase System
Authors: SHIMIZU SAKAYU , KATAOKA MICHIHIKO , KITA KEIKO
Published: February 2006

Abstract: Production of chiral 4‐chloro‐3‐hydroxybutanoate ethyl esters (CHBE) was performed through microbial asymmetric reduction of 4‐chloroacetoacetate ethyl ester (CAAE). The enzymes reducing CAAE to (R)‐ and (S)‐CHBE were found to be produced by Sporobolomyces salmonicolor and Candida magnoliae, respectively. The enzyme of S. salmonicolor is a novel NADPH‐dependent aldehyde reductase (AR) belonging to the aldo‐keto reductase superfamily. When AR‐overproducing Escherichia coli transformant cells or C. magnoliae cells were incubated in an organic solvent‐water two‐phase system, 300 or 90 mg/mL of CAAE was almost stoichiometrically converted to (R)‐ or (S)‐CHBE (>92% ee), respectively.
Fighting Nerve Agent Chemical Weapons with Enzyme Technology
Authors: LEJEUNE KEITH E. , DRAVIS BRYAN C. , YANG FANGXIAO , HETRO AMY D. , DOCTOR BHUPENDRA P. , RUSSELL ALAN J.
Published: February 2006

Abstract: The extreme toxicity of organophosphorous‐based compounds has been known since the late 1930s. Starting in the mid‐1940s, many nations throughout the world have been producing large quantities of organophosphorous (OP) nerve agents. Huge stockpiles of nerve agents have since developed. There are reportedly more than 200,000 tons of nerve agents in existence worldwide. There is an obvious need for protective clothing capable of guarding an individual from exposure to OP chemical weapons. Also, chemical processes that can effectively demilitarize and detoxify stored nerve agents are in great demand. The new and widely publicized Chemical Weapons Treaty requires such processes to soon be in place throughout the world. Biotechnology may provide the tools necessary to make such processes not only possible, but quite efficient in reducing the nerve agent dilemma.

The following paper discusses some of the history in developing enzyme technology against nerve agents. Our laboratory has interest in enhancing the productivity and potential utility of these systems in both demilitarization and decontamination applications. Freeze‐dried nerve agent‐hydrolyzing enzyme preparations have been shown to be effective in decontaminating gaseous nerve agents. The direct incorporation of nerve agent‐hydrolyzing enzymes within cross‐linked polyurethane foam matrices during polymer synthesis has been shown to dramatically enhance the productivity of two different enzyme systems. The future goal of such work lies in building a bridge between the clinical application of nerve agent‐hydrolyzing enzymes and practical processing techniques that may take advantage of the initial results already achieved in the laboratory.
The Dynamics of Globin Gene Expression and Gene Therapy Vectors
Authors: GROSVELD FRANK , DE BOER ERNIE , DILLON NIALL , GRIBNAU JOOST , MILOT ERIC , TRIMBORN TOLLEIV , WIJGERDE MARK , FRASERA PETER
Published: February 2006

Abstract:The most important level of regulation of the β‐globin genes is by activation of all of the genes by the Locus Control Region (LCR) and repression of the early genes by an as yet unknown factor acting on sequences flanking the genes. Superimposed on this is a mechanism in which the early genes (ɛ and γ) suppress the late genes (δ and β) by competition for the interaction with the LCR. Although this extra level of gene regulation is quantitatively of less importance than the direct repression mechanism, it has important implications and has provided an excellent assay system to probe the regulation of transcription at the single cell level.

These studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. The levels of expression of each of the genes appear to depend on: 1) the frequency of interaction which is itself dependednt on the distance of the gene to the LCR, 2) the affinity of the LCR for the gene and 3) the stability of the LCR/gene complex. The latter two are dependent on the balance of transcription factors. We conclude that transcription only appears to take place while the LCR and gene interact and that the level of transcription is determined by the frequency and duration of such interaction rather than by changes in the rate of transcription of promoters.
Reduced β‐Globin Gene Expression in Adult Mice Containing Deletions of Locus Control Region 5′ HS‐2 or 5′ HS‐3a
Authors: LEY TIMOTHY J. , HUG BRUCE , FIERING STEVEN , EPNER ELLIOT , BENDER M. A. , GROUDINE MARK
Published: February 2006

Abstract: To gain insights into the functions of individual DNA'se hypersensitive sites within the β globin locus control region (LCR), we deleted the endogenous 5′ HS‐2 and HS‐3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5′ HS‐2 or 5′ HS‐3 reduced the expression of the embryonic ɛy and βh1 globin genes minimally in yolk sac‐derived erythrocytes, but that both knockouts reduced the output of the adult β (β‐Major +β‐Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK‐Neo cassette was retained within either the HS‐2 or HS‐3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK‐Neo was shown to be expressed in an erythroid‐specific fashion when it was retained in the HS‐3 position. These results show that neither 5′ HS‐2 nor HS‐3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult β‐globin gene expression in adult red blood cells. Each site therefore contains some non‐redundant information that contributes to adult globin gene function.
Expression and Developmental Control of the Human α‐Globin Gene Cluster
Authors: LIEBHABER STEPHEN A. , RUSSELL J. ERIC
Published: February 2006

Abstract: The human α‐globin gene cluster contains three functional genes ζ, α2 and α1. The ζ‐globin gene is expressed exclusively in the primitive erythroblasts of the embryonic yolk sac and is selectively silenced during the transition from primitive to definitive erythropoesis. The two α‐globin genes are expressed through development; they are expressed at equivalent levels in embryonic cells at a 2.6: 1 ratio of α2 : α1 in fetal and adult cells. The dominant contribution of the α2‐globin locus to overall expression of adult α‐globin is reflected in the more severe phenotype resulting from mutations that affect this locus. Developmental silencing of the ζ‐globin gene reflects both transcriptional and posttranscriptional mechanisms. Transcriptional silencing is mediated by an interaction between the ζ‐globin gene promoter and a silencer located in the 3′ flanking region. This transcriptional silencing is only partial, and residual levels of ζ‐globin mRNA are subject to subsequent degredation. This instability of ζ‐globin mRNA relative to that of α‐globin mRNA reflects differences in their respective 3′UTR segments; the ζ‐globin mRNA 3′UTR has a lower affinity for a sequence‐specific mRNP stability complex which assembles at this site. The α‐globin mRNA assembles this complex at a higher efficiency and mutations which interfere with 3′UTR function result in corresponding loss of α‐globin gene expression. These data outline a developmental pathway for the α‐globin gene cluster which reflects transcriptional and posttranscriptional controls.
Silencing and Activation of Embryonic Globin Gene Expression
Authors: GINDER GORDON D. , SINGAL RAKESH , LITTLE JANE A. , DEMPSEY NANCY , FERRIS RICHARD , WANG SHOU ZHEN
Published: February 2006

Abstract: An understanding of the mechanisms that control developmental stage‐specific transcription of globin genes offers the promise of successful therapeutic activation of fetal or embryonic β‐type genes in β‐thalassemia syndromes. A large body of evidence supports the notion of conservation of such mechanisms across vertebrate species and validates the use of pre‐clinical studies of silencing and activation of fetal or embryonic globin genes in animals. Using globin gene transfections into primary avian erythroid cells and cultured murine erythroleukemia cells, we have studied mechanisms involved in stage‐specific embryonic β‐type globin gene silencing and activation. These studies show that 1) methylation of the exact CpG nucleotides that are methylated in normal adult erythroid cells in vivo is capable of blocking transcription of a transfected embryonic globin gene promoter via binding of a methyl DNA binding protein in primary erythroid cells. 2) Activation of embryonic β‐type globin gene transcription in adult erythroid cells by short chain fatty acids is mediated through specific DNA sequences both in the promoter and downstream of the promoter.
Cellular and Molecular Effects of a Pulse Butyrate Regimen and New Inducers of Globin Gene Expression and Hematopoiesis
Authors: IKUTA TOHRU , ATWEH GEORGE , BOOSALIS VASSILIKI , WHITE GARY L. , DA FONSECA SILVANA , BOOSALIS MICHAEL , FALLER DOUGLAS V. , PERRINE SUSAN P.
Published: February 2006

Abstract: Cooley's anemia is characterized by a deficiency of β‐globin chains, a relative excess of α‐globin chains, and consequent accelerated programmed death of developing erythroid cells in the bone marrow. Increasing expression of the γ‐globin genes to adequately balance excess α‐globin chains can ameliorate this disorder. Butyrates induce γ‐globin experimentally, but can also cause cell growth arrest with prolonged exposure or high concentrations, which in turn can accelerate apoptosis. To determine if these potentially opposing effects can be balanced to enhance therapeutic efficacy, an intermittent “pulsed” regimen of butyrate was evaluated. Following induction of γ‐globin mRNA and protein synthesis, total hemoglobin increased in β‐thalassemia patients by more than 2 g/dl above baseline, and Hb F increased above 20% in 5/8 sickle cell patients from baseline levels of 2% Hb F. Specific regulatory regions were identified in the γ‐ and β‐globin gene promoters to which new binding of transcription factors, including αCP2 (an activator of γ globin) occur during therapy solely in the butyrate‐responsive patients. Other compounds which induce γ globin, derivatives of acetic, phenoxyacetic, propionic, and cinnamic acids, and dimethylbutyrate, are under investigation. Some of these newer γ‐globin inducers (designated hemokines) provide better potential as therapeutics by also acting to increase hematopoietic cell viability and proliferation. Pharmacologic induction of expression of the endogenous γ‐globin genes is a realistic approach to therapy of the β‐globin disorders for many patients, with some effective agents available now and new therapeutics, with enhanced activities, under development.
Butyrate Trialsa
Authors: CAPPELLINI MARIA DOMENICA , GRAZIADEI GIOVANNA , CICERI LAURA , COMINO ALESSIA , BIANCHI PAOLO , POMATI MAURO , FIORELLI GEMINO
Published: February 2006

Abstract: The aims of this study were to ascertain tolerability, safety and efficacy of oral isobutyramide (150 mg/kg bw/day) in stimulating fetal hemoglobin production in twelve thalassemia intermedia patients. Patients were treated for 28 days and followed for a further 28 days. Efficacy was monitored by non‐α/α globin chain ratio and percentage of HbF. Five patients experienced increases of non‐α/α ratio ranging between 5.3 and 100% at the end of treatment. Five patients show an increase of HbF ranging between 4.4 and 26%. Their HbF% continues to increase during follow‐up period. The analysis of variance for HbF showed a time effect close to significance both in treatment period (p= 0.06) and in follow‐up period (p= 0.08). Moreover, to evaluate a possible erythropoietic modification, serum Erythropoietin (sEpo) and serum Transferrin Receptor (sTfR) were evaluated. Serum Epo and sTfR levels were significantly increased during treatment (p < 0.05 vs baseline).
The Role of Recombinant Human Erythropoietin in the Treatment of Thalassemia
Authors: RACHMILEWITZ ELIEZER A. , AKER MEMET
Published: February 2006

Abstract: The rationale for treatment with recombinant human erythropoietin (rHuEPO) in thalassemia came from studies in baboons, thalassemic mice and in erythroid cultures. The results demonstrated an increase in γ globin synthesis and consequently in fetal Hb (Hb F) resulting in improvement in erythropoietic parameters. In addition, endogenous serum Epo levels in various forms of thalassemia were inconsistent and not related to the severity of the anemia. Therefore, several preliminary studies with rHuEPO were performed, mainly on patients with β thalassemia intermedia. The results indicate: a) a significant, dose‐related (500 u/kg to 1000 u/kg × 3/week) increase in thalassemia erythropoiesis without changes in % of Hb F, MCV and MCH, mainly in splenectomized patients; b) the minimum effective dose is 500 u/kg × 3/week; c) there were no major side effects during the continuous treatment period of 9 months. In order to improve both quantitative and qualitative thalassemia erythropoiesis, several trials were undertaken combining rHuEPO with hydroxyurea (HU), which is known to increase % Hb F, MCV and MCH without a major effect on Hb levels. The designed trial included 3 to 6 months of HU alone (20 mg/kg × 4/week), or with rHuEPO alone (500 u/kg × 3/week or 375 u/kg × 2/week) or a combination of the two drugs. The results show an additive effect of the two drugs, in some of the patients. It is not known whether the addition of oral iron to rHuEPO is warranted for maximal erythropoietic response. The major limiting factor in designing large scale clinical trials is the relatively high cost of the drug. Nevertheless rHuEPO alone or in combination with other Hb F modulating drugs may have a positive effect in thalassemia with resulting improvement in the quality of life.
Targeted Integration of a Recombinant Globin Gene Adeno‐Associated Viral Vector into Human Chromosome 19a
Authors: BERTRAN JOAN , YANG YANPING , HARGROVE PHILLIP , VANIN ELIO F. , NIENHUIS ARTHUR W.
Published: February 2006

Abstract: Transfer of a globin gene into stem cells along with the regulatory elements required to achieve high level expression in maturing erythroid cells would provide effective gene therapy for Cooley's Anemia. We have explored the use of recombinant adeno‐associated viral (rAAV) vectors for this purpose. A vector designated rHS32Aγ*3′RE that contains regulatory elements from the locus control and flanking regions, integrates as a stable head‐to‐tail concatamer in erythroleukemia cells at a high multiplicity of infection and exhibits high level, regulated γ globin gene expression. Inducible expression of the non‐structural Rep proteins of wild‐type AAV in HeLa cells transduced with rAAV vectors does not increase overall integration frequency, but targeted integration of rHS32Aγ*3′RE into human chromosome 19 was documented.
Pathophysiology of Iron Overloada
Authors: HERSHKO CHAIM , LINK GABRIELA , CABANTCHIK IOAV
Published: February 2006

Abstract: In thalassemia, iron overload is the joint outcome of excessive iron absorption and transfusional siderosis. While iron absorption is limited by a physiologic ceiling of about 3 mg/d, plasma iron turnover in thalassemia may be 10 to 15 times normal, caused by the wasteful, ineffective erythropoiesis of an enormously expanded erythroid marrow. This outpouring of catabolic iron exceeds the iron‐binding capacity of transferrin and appears in plasma as non‐transferrin‐plasma iron (NTPI). The toxicity of NTPI is much higher than of transferrin‐iron as judged by its ability to promote hydroxyl radical formation resulting in peroxidative damage to membrane lipids and proteins. In the heart, this results in impaired function of the mitochrondrial respiratory chain and abnormal energy metabolism manifested clinically in fatal hemosiderotic cardiomyopathy. Ascorbate increases the efficacy of iron chelators by expanding the intracellular chelatable iron pool, but, at suboptimal concentrations is a pro‐oxidant, enhancing the catalytic effect of iron in free radical formation. NTPI is removed by i.v. DFO in a biphasic manner and reappears rapidly upon cessation of DFO, lending support to the continuous, rather than intermittent, use of chelators. Unlike DFO and other hexadentate chelators, bidentate chelators such as L1 may produce incomplete intermediate iron complexes at suboptimal drug concentrations.
Long‐Term Trials of Deferiprone in Cooley's Anemiaa
Authors: OLIVIERI NANCY F. , BRITTENHAM GARY M.
Published: February 2006

Abstract: Deferoxamine is the currently available agent for the iron‐chelation therapy required by Cooley's anemia patients. The difficulties associated with parenteral administration have mandated a search for alternative therapies, especially orally active iron chelators, to remove excess iron that results in damage to the liver, endocrine organs, and heart. Four orally active agents have reached clinical trials in the last decade. The agent under consideration in this paper, deferiprone (1,2‐dimethyl‐3‐hydroxypyridin‐4‐one), has shown some promise, but, according to the studies discussed here, may not provide adequate sustained control of body iron in a substantial proportion of Cooley's anemia patients.
Survival and Disease Complications in Thalassemia Major
Authors: BORGNA‐PIGNATTI CATERINA , RUGOLOTTO SIMONE , DE STEFANO PIERO , PIGA ANTONIO , DI GREGORIO FELICIA , GAMBERINI MARIA RITA , SABATO VINCENZO , MELEVENDI CATERINA , CAPPELLINI MARIA DOMENICA , VERLATO GIUSEPPE
Published: February 2006

Abstract: We studied survival and disease complications in 1,146 patients with thalassemia major, born from January 1, 1960 to December 31, 1987. At last follow‐up, in March 1997, probability of survival to age 20 years was 89% and to age 25 years was 82% for patients born in the years 1970‐1974. Patients who died had a serum ferritin level, measured the year before death, significantly higher than those who survived. Diabetes was present in 5.4% of the patients; heart failure in 6.4%; arrhythmias in 5.0%, thrombosis in 1.1%, hypothyroidism in 11.6%, HIV infection in 1.8%. Hypogonadism was diagnosed in 55% of 578 patients who had reached pubertal age: 83.5% of hypogonadic females and 78.6% of males were receiving substitutive hormonal therapy. In conclusion, the survival of patients with thalassemia major is good and improving, but the prevalence of severe complications is still high.
New Approaches to the Management of Hepatitis and Endocrine Disorders in Cooley's Anemia
Authors: WONKE BEATRIX , HOFFBRAND A. V. , BOULOUX P. , JENSEN C. , TELFER P.
Published: February 2006

Abstract: Hepatitis C infection is common in patients receiving life‐long blood transfusion therapy. Interferon‐α induces long‐term viral clearance in 25‐30% of patients suffering from Cooley's anemia. Ribavirin, an orally active guanoside analogue together with interferon‐α produces a sustained response in up to 40% of patients with cirrhosis, who had previously failed single agent treatment. Growth retardation in iron‐overloaded patients is the result of growth hormone deficiency in up to 30% of patients. Height gain can be sucessfully achieved in these patients with growth hormone treatment. Pregnancy in women with Cooley's anemia is now a reality, and over 100 pregnancies have been documented. Conception may be spontaneous or the result of ovulation induction. Cardiomyopathy and diabetes require careful assessment in these patients before a decision is made to treat with gonadotrophins to induce ovulation.
Diagnosis and Management of Iron‐induced Heart Disease in Cooley's Anemia
Authors: JESSUP MARIELL , MANNO CATHERINE S.
Published: February 2006

Abstract: Patients with homozygous β‐thalassemia are chronically transfused and, if not assiduously chelated, are at risk for cardiac dysfunction. Available data suggest that even in optimally chelated patients, cardiac pathology is abnormal secondary to iron deposition, fibrosis, hypertrophy, and the structural effects of chronic anemia. Evidence of myopericarditis may also be found. Cardiac performance is usually only subtly affected, primarily with diastolic abnormalities not routinely detected on echocardiograms or nuclear scan. In poorly chelated patients, severe heart failure occurs and is easily predictable but invariably fatal, despite treatment with diuretics, vasodilators, inotropes, and antiarrhythmics. Based on successful prevention of heart failure with ACE inhibitors in other forms of cardiomyopathy, we suggest multicenter trials to explore methods to stabilize cardiac function in patients at risk for iron‐induced heart disease. Long‐term adverse effects of iron deposition, diastolic dysfunction, and abnormal hormone regulation need to be quantitated in patients reaching their third and fourth decades when the potential for ischemic cardiac disease could compound cardiac dysfunction.
Bone Marrow Transplantation in Thalassemia: The Experience of Pesaro
Authors: LUCARELLI GUIDO , GALIMBERTI MARIA , GIARDINI CLAUDIO , POLCHI PAOLA , ANGELUCCI EMANUELE , BARONCIANI DONATELLA , ERER BUKET , GAZIEV DJAVID
Published: February 2006

Abstract: Early trials of allogenic bone marrow transplantation (BMT) for homozygous β thalassemia and the analyses of results of transplantation in patients under 17 years of age have allowed us to identify 3 classes of risk using the criteria of degree of hepatomegaly, the degree of portal fibrosis, and the quality of the chelation treatment given before the transplant. Patients for whom all 3 criteria were adverse constituted Class 3, patients with none of the adverse criteria constituted Class 1, and patients with 1 or various associations of 2 of the adverse criteria formed Class 2. Most patients older than 16 years have disease characteristics that place them in Class 3, with very few in Class 2. For all the patients with an HLA identical donor we are actually using 2 protocols to which the patient is assigned on the basis of the Class he belongs to at the time of BMT and independently from the age of the patient. For 104 patients in Class 1 and for 262 patients in Class 2 prepared for the transplant with busulfan 14mg/kg, cyclophosphamide 200mg/kg and cyclosporine alone, the probabilities of survival and of event‐free survival are 95% and 90% for Class 1 and 87% and 84% for Class 2. For 33 Class 3 patients prepared for the transplant with busulfan 14 mg/kg, cyclophosphamide reduced to 160 mg/kg, cyclosporine, and “short” methotrexate, the probabilities of survival and event‐free survival are 89% and 64%. For 57 adult patients (17 to 35 years), who underwent the transplant after preparation with the same protocol used for Class 3, the probabilities of survival and of event‐free survival are 70% and 68%, respectively. BMT remains the only form of radical treatment for thalassemia in those patients with an HLA‐identical donor.
Late Effects of Bone Marrow Transplantation for Thalassemiaa
Authors: PIGA ANTONIO , LONGO FILOMENA , VOI VINCENZO , FACELLO SILVIA , MINIERO ROBERTO , DRESOW BERND
Published: February 2006

Abstract: Long term effects of BMT in thalassemia were monitored in 33 patients transplanted between 1987 and 1995 and compared with 155 patients matched for age and treated during the same period with conventional therapy (CT). The incidence of fulminant sepsis and growth impairment was significantly higher in transplanted patients, whereas the occurrence of hypothyroidism, hypogonadism, and cardiopathy was higher in CT patients. For diabetes, liver disease, and severe infections, the differences were not statistically significant. After BMT we performed monthly erythrocytaferesis for iron removal in 23 (70%) patients, obtaining a complete normalization of iron stores in 91% of cases; among untreated patients, 60% had evidence of iron up to 8.3 years after BMT. Protection against poliovirus, tetanus, diphtheria, and hepatitis B has been lost in 74%, 47%, 78%, and 44%, respectively. After BMT a careful follow‐up is needed to monitor and treat late transplant‐related and thalassemia‐related complications.
Relationship between Genotype and Phenotype: Thalassemia Intermediaa
Authors: GALANELLO RENZO , CAO ANTONIO
Published: February 2006

Abstract: Thalassemia intermedia encompasses a number of clinical conditions ranging in severity from β‐thalassemia carrier state to transfusion‐dependent thalassemia major. The molecular bases of thalassemia intermedia, only partially defined, are very heterogeneous, but in general any factor able to reduce the globin‐chain imbalance results in a milder form of thalassemia. These factors are the presence of a silent or mild β‐thalassemia allele, associated with a high residual β‐globin production, and the coinheritance of α‐thalassemia or of genetic determinants that increase the γ‐chain production. Less frequent mechanisms are double heterozygosity for β‐thalassemia and triplicated α genes, and the presence of a hyperunstable hemoglobin variant. However, for a consistent number of β0‐thalassemia homozygotes with a thalassemia intermedia phenotype the modifying factor has not been defined yet. In contrast, there are simple β‐thalassemia carriers who, for unknown reasons, have an unusually severe clinical phenotype.
The Morbidity of Bone Disease in Thalassemia
Authors: VICHINSKY ELLIOT P.
Published: February 2006

Abstract: As thalassemia patients age, bone disease becomes a serious cause of morbidity. The frequency and type of bone disease is affected by the underlying type of thalassemia and its treatment. Problems include rickets, scoliosis, spinal deformities, nerve compression, fractures and severe osteoporosis. In early stages, patients may be asymptomatic but can present with back pain, a limp, dyspnea, neurological emergencies, or sudden fractures. The etiologies are often multifactorial, culminating with increased bone resorption and remodeling. They include hormonal deficiency, bone marrow expansion, nutritional deficiency, or desferal toxicity. Particular risk factors include older patients, low baseline hemoglobin, delayed puberty, hormonal failure, and high iron stores. Nutritional deficiencies may further compound the patient's risk for bone disease. Increasing evidence suggests that these complications and their associated long‐term morbidity can be prevented if an annual screening is done, followed by long‐term intervention. Patients treated with amino biphosphonates inhibit bone resorption and may demonstrate rapid healing. Intra‐nasal calcitonin has also been successful in treating osteopenia. Early use of estrogen and testosterone appears to markedly lower the risk for selective patients. Both transfused and non‐transfused patients should be educated about risk factors and early symptoms. All patients should be screened annually for bone disease. Once adolescence occurs, annual testing in selected cases should include bone density studies with X‐ray absorptiometry.
Future Orientation and Life Expectations of Adolescents and Young Adults with Thalassemia Majora
Authors: BUSH SHERRY , MANDEL FRANCINE S. , GIARDINA PATRICIA J.
Published: February 2006

Abstract: Until recently, Thalassemia Major was considered a fatal disease and patients did not usually live into adulthood. Advances in the medical management of the disease have greatly increased the life expectancy of these patients. The present study aims to evaluate the future orientation and other aspects of psychosocial functioning of thalassemics compared to healthy controls. Thirty patients and 33 healthy subjects of similar age, ethnicity, education, and geographic area were compared on measures of future expectations, perceived social support, life orientation, health locus of control and hopelessness. Results show no significant differences between thalassemics and controls on all measures except for higher levels of internal health locus of control among the patient group. Results and implications of perceptions of thalassemics' future orientation relevant to patient care are discussed.