bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of Hemolysis in Rapidly Frozen Erythrocytes by Using a Laser Pulse
Authors: FOWLER ALEX J. , TONER MEHMET
Published: February 2006

ABSTRACT: This paper reports on the successful recovery of rapidly frozen unprotected erythrocytes by vitrification of the intracellular solution with a laser pulse prior to thawing. Erythrocytes that were frozen at 10,000 °C/min exhibited 100% hemolysis when thawed unless they were first irradiated by a 7 ns. laser pulse that selectively targeted the intracellular ice so that it was melted and resolidified into a glass phase. Up to 80% of the cells treated in this way remained intact after thawing. Wright's staining confirmed a healthy cell morphology and the retention of hemoglobin in the laser treated cells. While it is well known that small amounts of intracellular ice can be tolerated by cells, the findings of this study are the first to indicate that intracellular ice may be innocuous even when formed in substantial quantities provided that crystal growth and coalescence can somehow be avoided during warming.
Measurement of Cell Volume Loss in the Liquid Region Preceding an Advancing Phase Change Interfacea
Authors: HARMISON HEATHER R. , DILLER KENNETH R. , WALSH JOHN R. , NEILS CHRISTOPHER M. , BRAND JERRY J.
Published: February 2006

ABSTRACT: It is well understood that the solidification of a solution results in a redistribution of solute in the liquid zone. For the freezing of suspensions of cells it is anticipated that accumulation of solute in the region leading a growing ice phase will cause an osmotic response in cells before the ice phase reaches the cells. To measure this phenomenon in a specific algal species, the volume changes in Chlorococcum texanum during freezing were studied using directional solidification cryomicroscopy. The relative cell volume was tracked continuously as a function of temperature and position as cells encountered the moving phase front. The loss of cell volume was measured in the liquid region containing concentrated solute ahead of the growing solid phase.
Sensitivity of Kidney Perfusion Protocol Design to Physical and Physiological Parametersa
Authors: LACHENBRUCH CHARLES A. , DILLER KENNETH R. , PEGG DAVID E.
Published: February 2006

ABSTRACT: The introduction and removal of cryoprotective agents (CPA) to a kidney via vascualar perfusion may induce changes in cell volume that are destructive to the tubular epithelial or capillary endothelial cells as well as causing significant increases in vascular resistance that compromise the perfusion process. A network thermodynamic model of the coupled osmotic, hydrodynamic and elastic properties of the kidney was applied to evaluate the sensitivity of these critical outputs to a set of physiological and perfusion variables. Simulation results suggest that in the design of perfusion protocols for CPAs such as glycerol, it may be advantageous to: (a) select a CPA with as high a cell membrane permeability as possible; (b) increase the concentration of mannitol in the perfusate to about 200 mos/kg, beyond which there is no discernible benefit; (c) when glycerol is the CPA, limit the rate of reduction in the perfusate during removal to 30 mM/min or less; (d) limit the perfusion pressure to 20 – 30 mm Hg, within the practical constraints of the perfusion system; (e) increase the concentration of impermeant in the perfusate to as high as 400 mos/kg, although it is recognized that this departure from plasma‐like composition might impose other problems that are not considered in this model. Further, it was observed that the vascular membrane permeability plays a relatively minor role in controlling cellular osmotic injury and vascular perfusion resistance and is therefore not a critical parameter in the perfusion design process.
Convective heat transfer around an infant heada
Authors: VUILLERME VALERY , FOHR JEAN‐PAUL , SAULNIER JEAN‐PASCAL , ORIOT DENIS , SAULNIER JEAN‐BERNARD , BLAY DOMINIQUE
Published: February 2006

ABSTRACT: Knowledge of heat and mass transfer between an infant and his environment is necessary to control hospital care conditions, or understand a pathology as Sudden Infant Death Syndrome (SIDS). This paper precises the particular importance of the heat transfer over the head and quantifies the influence of various parameters on natural convection heat transfer.
Genetic Variation in Human 5‐HT Receptors: Potential Pathogenetic and Pharmacological Rolea
Authors: GÖTHERT M. , PROPPING P. , BÖNISCH H. , BRÜSS M. , NÖTHEN M. M.
Published: February 2006

ABSTRACT: Mutation screening identified variants of h5‐HT1A (Gly‐22‐Ser, Ile‐28‐Val, Arg‐219‐Leu), h5‐HT1B (Phe‐124‐Cys), h5‐HT2A (Thr‐25‐Asn, His‐452‐Tyr), h5‐HT2C (Cys‐23‐Ser) and h5‐HT7 (Thr‐92‐Lys, Pro‐279‐Leu) receptors. Screening of h5‐HT1D, h5‐ht1e, h5‐ht1f and h5‐ht5 receptor genes failed to detect any significant mutations. No differences in radioligand binding properties were observed between the h5‐HT1A Ile‐28‐Val variant receptor (VR) and the wildtype receptor (WTR). Binding profiles of the h5‐HT1A Gly‐22‐Val variant and the WTR were also very similar, but the 8‐OH‐DPAT‐induced down‐regulation and desensitization of the VR was attenuated. The h5‐HT1B Phe‐124‐Cys variant leads to considerable changes in [3H]5‐carboxamidotryptamine binding: Bmax was decreased and the affinity of various h5‐HT1B ligands was modified (usually increased; e.g., in the case of sumatriptan). The h5‐HT2A His‐452‐Tyr variant causes an alteration of the amplitude and timing of intracellular calcium mobilization in platelets from 452‐His/452‐Tyr heterozygous compared to 452‐His/452‐His homozygous individuals. Most, but not all, of the VRs listed above were examined for association with, e.g., bipolar depression and schizoprenia, yet no relation was observed. The most consistent finding was an association between a silent mutation (102T/C) in the h5‐HT2A receptor gene and schizophrenia; this association may be explained by linkage disequilibrium with a functional variant in the regulatory region of the gene. Studies of the therapeutic response to clozapine produced no homogeneous results with respect to the pharmacogenetic significance of the various mutations in the h5‐HT2A and h5‐HT2C receptor genes.
Gs Protein‐Coupled Serotonin Receptors: Receptor Isoforms and Functional Differences
Authors: HAMBLIN M. W. , GUTHRIE C. R. , KOHEN R. , HEIDMANN D.E. A.
Published: February 2006

ABSTRACT: Three distinct mammalian Gs coupled serotonin receptor genes have been identified, 5‐HT4, 5‐ht6, and 5‐HT7, which produce at least seven different functional receptors through alternative splicing. One of the chief questions facing workers in this area mirrors that confronting the serotonin receptor field as a whole: why so many subtypes? The answer to this question is made more elusive at present by two further considerations. First, there may well be additional Gs coupled receptor subtypes yet to be described. Secondly, although the various isoforms of 5‐HT4 and 5‐HT7 have been shown to be functional in in vitro assays, it remains to be shown that all isoforms have biological significance.

This paper will summarize some of the differences at the molecular and cellular level that are becoming apparent among the 5‐HT4, 5‐ht6 and 5‐HT7 receptor subtypes and their various isoforms. As an example, it will focus on the 5‐HT7 system, and describe recent developments in ascribing particular functions to differences due to alternative splicing.
Identification and Characterization of RNA Editing Events within the 5‐HT2C Receptora
Authors: NISWENDER C. M. , SANDERS‐BUSH E. , EMESON R. B.
Published: February 2006

ABSTRACT: RNA editing is a post‐transcriptional modification that generates an RNA transcript with a nucleotide sequence different from its gene. We have recently discovered RNA editing events, involving the conversion of adenosine bases to inosine residues, within the RNA encoding the serotonin 2C (5‐HT2C) receptor. 1 Editing events at four major positions, termed A, B, C and D, as well as one minor site termed C′, are predicted to alter amino acids within the second intracellular loop of the G‐protein coupled 5‐HT2C receptor. Editing is mediated by at least two members of a family of adenosine deaminases and is contingent upon the presence of an extensive RNA duplex structure formed by exonic and intronic sequences of 5‐HT2C receptor precursor messenger RNA (pre‐mRNA). This critical secondary structure has been observed within brain pre‐mRNA derived from four species; the isolation of edited 5‐HT2C receptor transcripts from these samples further confirms the evolutionary conservation of this RNA processing event. Among members of the 5‐HT2 receptor family, editing within second intracellular loop RNA is unique to the 5‐HT2C receptor. Editing within the 5‐HT2C receptor generates receptor isoforms that differ in their ability to interact with the phospholipase C signaling cascade in a transfected cell line, suggesting that this RNA processing event may contribute to the modulation of serotonergic neurotransmission in the central nervous system.
5‐HT4 Receptors: Cloning and Expression of New Splice Variants
Authors: CLAEYSEN S. , FAYE P. , SEBBEN M. , TAVIAUX S. , BOCKAERT J. , DUMUIS A.
Published: February 2006

ABSTRACT: On the basis of differences in the potencies and intrinsic activity of 5‐HT4 receptor agonists in different biological models it has been suggested that there is heterogeneity among 5‐HT4 receptors. Here, we report the molecular cloning of several 5‐HT4 receptor splice variants in mouse, rat, and human brain. Our data suggest that the differences in efficacy of 5‐HT4 ligands on 5‐HT4 receptor‐mediated responses in several tissues is due to differences in coupling efficiency rather than to the presence of different 5‐HT4 receptor isoforms.
Inducible Knockout Strategies to Probe the Functions of 5‐HT Receptorsa
Authors: STARK K. L. , OOSTING R. S. , HEN R.
Published: February 2006

ABSTRACT: Gene targeting has proven to be extremely powerful in various fields of biological research. Through this technique, knockout mice lacking a particular gene and thus a particular protein, can be generated. One limitation to this technique is the fact that mice develop without the protein of interest and therefore, developmental compensations may have taken place, contributing to an observed phenotype. Inducible strategies, those which allow the timing of expression of a gene to be regulated, are currently being developed and should prove useful when applied to gene targeting technology. In order to begin to apply such new technologies to the field of gene targeting, we first created and tested several reporter constructions using the tetracycline inducible system. Here we describe the creation of several β‐galactosidase reporter constructions and the results of in vitro testing in Cos‐7 cells. We then discuss future knockout strategies based upon our observations.
Epilepsy and Obesity in Serotonin 5‐HT2C Receptor Mutant Mice
Authors: HEISLER L. K. , CHU H.‐M , TECOTT L. H.
Published: February 2006

ABSTRACT: Serotonin 5‐HT2C receptor null mutant mice were generated to assess the contribution of this receptor to the actions of serotonin. Mutant mice displayed both an epilepsy and obesity phenotype. The epilepsy syndrome was characterized by spontaneous seizures, lowered seizure threshold, enhanced seizure propagation and sound‐induced seizure susceptibility. These findings implicate 5‐HT2C receptors in the regulation of neuronal network excitability. It was also observed that body weight and adipose tissue deposition were elevated in adult mutant mice relative to their wild type littermates. Paired‐feeding studies suggest that the obesity syndrome is a result of increased food intake. In addition, mutants displayed reduced sensitivity to the appetite suppressant actions of nonspecific serotonergic agonists. These studies establish a role for 5‐HT2C receptors in the serotonergic regulation of body weight and food intake.
The Putative 5‐ht6 Receptor: Localization and Function
Authors: SLEIGHT A. J. , BOESS F. G. , BÖS M. , BOURSON A.
Published: February 2006

ABSTRACT: Until recently, the majority of actions of the neurotransmitter 5‐hydroxytryptamine (5‐HT) were generally believed to be mediated by members of the 5‐HT1, 5‐HT2, 5‐HT3 and 5‐HT4 receptor families. The application of molecular cloning techniques has revealed the existence of gene products encoding several novel, putative 5‐HT receptors for which little or no prior pharmacological or functional data presently exist. The present challenge to pharmacologists is to determine the physiological relevance of these gene products, establish whether or not they function as endogenous receptors, find selective agents and determine potential therapeutic uses of these compounds. Here we review work detailing the cloning and characterization of the recombinant 5‐ht6 receptor, its distribution and evidence for functional responses mediated by naturally occurring 5‐ht6 receptors.
A Three‐State Receptor Model: Predictions of Multiple Agonist Pharmacology for the Same Receptor Typea
Authors: CARAMELLINI C. , LEFF P.
Published: February 2006

ABSTRACT: Recent studies have demonstrated that activation of the same G‐protein coupled receptor can generate different agonist pharmacology depending on the signaling pathway(s) to which it couples. Two types of behavior have been exemplified; differences in affinity order, and differences in efficacy order with the same affinity order. The two‐state model of receptor activation cannot explain these data, since a single active receptor state cannot couple differently to the two response pathways for different ligands. We have therefore extended the two‐state model to a three‐state model in which receptors exist in three states: an inactive state, R, and two different active states, R* and R**.

The model has two modes, the ‘intact mode’, in which all the equilibria are linked; and the ‘isolated mode’ in which the two response pathways are isolated from each other, giving effectively two separate two‐state systems. In the ‘intact mode’ the same agonist affinity order is predicted for both response pathways, but a different efficacy order. In the ‘isolated mode’, since the equilibria are no longer linked, the model predicts that a different affinity order may be obtained for the two pathways.

Owing to the linkage of all the equilibria in the intact three‐state model the level of constitutive activity through one pathway can affect the direction of agonism through the other pathway, resulting in the conversion of an inverse agonist into a positive agonist. This change in the direction of agonism is also predicted to occur when the two response pathways are isolated.

The three‐state model therefore predicts that agonists, acting at the same receptor, may show different affinity orders and different efficacy orders depending upon which response is measured and the assay system used, and also predicts that inverse agonism may be system dependent.
Interactions between Effectors Linked to Serotonin Receptors
Authors: BERG K. A. , MAAYANI S. , CLARKE W. P.
Published: February 2006

ABSTRACT: In general, there are two types of interactions between effector signaling pathways. “Homologous” interactions are those that occur within a receptor system to alter its own responsiveness, for example the loss of responsiveness (desensitization) that can occur upon agonist occupancy of a receptor. “Heterologous” interactions are those that occur between different receptor systems where the responsiveness of one receptor system is regulated (positively or negatively) by activation of another receptor system (i.e., “cross‐talk”). Many, if not all receptors, couple to multiple cellular effector pathways and alterations in the responsiveness of a receptor system can be effector pathway‐dependent which underscores the importance of studying each effector coupled to a receptor. Regulation of receptor system responsiveness, and consequently the efficacy of drugs, is a highly dynamic process. Perhaps by exploiting these interactions, new targets for pharmacotherapy may be uncovered which will provide for increased efficacy and specificity of drug action.
Creation of a Constitutively Activated State of the 5‐HT2A Receptor by Site‐directed Mutagenesis: Revelation of Inverse Agonist Activity of Antagonists
Authors: EGAN C. , HERRICK‐DAVIS K. , TEITLER M.
Published: February 2006

ABSTRACT: Constitutively active GPCR have revealed novel properties of drugs that exhibit classical competitive antagonism at the native forms of GPCR. These drugs reverse basal levels of constitutive activity, indicating that they have inverse agonist activity. We were interested in determining if competitive antagonists of the native 5‐HT2A receptor, in particular, antipsychotic drugs, exhibit inverse agonist activity at the constitutively active 5‐HT2A receptor. All of the drugs tested reduced basal IP production of constitutively active 5‐HT2A receptors, indicating that they all exhibited inverse agonist activity. Risperidone and ketanserin produced the greatest inhibition of basal IP production resulting in a reduction of basal activity in the C322K mutant receptor of 82% and 80%, respectively. Antipsychotic drugs display inverse agonist activity, indicating that stabilization of the inactive conformation of the 5‐HT2A receptor may be a key component of their mechanism of action.
Pharmacological Characterization of the Constitutively Activated State of the Serotonin 5‐HT2C Receptor
Authors: HERRICK‐DAVIS K. , GRINDE E. , GAUTHIER C. , TEITLER M.
Published: February 2006

ABSTRACT: Previous studies from our laboratory have shown that the 5‐HT2C serotonin receptor can be rendered constitutively active by changing amino acid 312 (third intracellular loop) from serine to lysine (S312K). In the present study, detailed radioligand binding analyses were performed to characterize the constitutively activated state of S312K mutant receptors. All agonists tested displayed high affinity for both [3H]5‐HT and [3H]mesulergine binding to S312K receptors, but displayed low affinity for [3H]mesulergine binding to native 5‐HT2C receptors. [3H]5‐HT labeled the same total number of S312K binding sites as [3H]mesulergine. 5‐HT2C antagonists inhibited S312K basal inositol phosphate production. These results suggest that S312K receptors mimic the active conformation of native 5‐HT2C receptors and provide a good model system for evaluating drugs for inverse agonist activity. Also, S312K receptors may represent a new system for screening 5‐HT2C agonist activity by comparing [3H]mesulergine binding to native and S312K mutant receptors.
Activation of the Mitogen‐activated Protein Kinase Pathwayviathe 5‐HT2AReceptora
Authors: WATTS . W.
Published: February 2006

ABSTRACT:The mitogen‐activated protein kinase (MAPK) pathway, classically associated with cell growth and dependent on tyrosine kinases such as MAPK kinase (MEK), can modulate smooth muscle contractility, and our laboratory has tested the hypothesis that 5‐HT can activate the MAPK pathway in arterial smooth muscle through activation of a 5‐HT2Areceptor. Tyrosine kinase inhibitors including genistein and the specific MEK inhibitor PD098059, but not the inactive tyrosine kinase congener daidzein reduced and shifted 5‐HT‐induced contraction rightward in isolated, endothelium‐denuded rat arteries. Activation of a tyrosine kinase/MEK via the 5‐HT2Areceptor was partially independent of two major signaling pathways typically associated with the 5‐HT2Areceptor‐activation of L‐type voltage gated calcium channels and phospholipase C. Western analyses using antibodies directed against tyrosyl‐phosphorylated‐, activated Erk MAPK, and MEK proteins from cultured aortic smooth muscle cells demonstrated that 5‐HT activated MEK and the Erk MAPKs in a time‐, concentration‐, receptor‐ and tyrosine kinase‐dependent manner. Taken together, these findings provide evidence for a novel pathway of vascular signal transduction‐activation of the MAPK pathway‐for the 5‐HT2Areceptor.
Serotonin Transporter Function in vivo: Assessment by Chronoamperometrya
Authors: FRAZER A. , DAWS L. C.
Published: February 2006

ABSTRACT: Local application of selective serotonin reuptake inhibitors, fluvoxamine and citalopram, prolonged the clearance of exogenously administered serotonin (5‐HT) in both the dentate gyrus and CA3 region of the dorsal hippocampus, as measured using in vivo chronoamperometry. These effects were abolished in rats pretreated with 5,7‐dihydroxytryptamine. The NE uptake inhibitors, desipramine and protriptyline, did not alter the 5‐HT signal in the CA3 region, but prolonged the clearance of 5‐HT in the dentate gyrus; this effect was absent in rats pretreated with 6‐hydroxydopamine. From these data, it is inferred that both the SERT and NET contribute to the active clearance of exogenously applied 5‐HT in the dentate gyrus. In another experiment, cyanopindolol, an antagonist of the serotonin terminal autoreceptor, also prolonged the clearance of 5‐HT from the CA3 region. These and other data have generated a working hypothesis that activation of the terminal serotonin autoreceptor enhances the kinetics of 5‐HT uptake through an effect on the serotonin transporter.
A Toolbox of Recombinant Lipases for Industrial Applications
Authors: SCHMIDT‐DANNERT CLAUDIA , PLEISS JÜRGEN , SCHMID ROLF D.
Published: February 2006

Abstract: We created a toolbox of recombinant, microbial lipases, which allows us in combination with a lipase database to choose among the overexpressed lipases the most appropriate for a specific application and to improve it further via mutagenesis. By systematic comparison of geometry and properties of the scissile fatty acid binding site of five representative lipases of each family of structurally homologous lipases, three subgroups can be defined. Hence, efficient expression systems for the functional production of large amounts of microbial lipases, representing different lipase subgroups, were developed. In particular, recombinant lipases from Bacillus thermocatenulatus and Pseudomonas cepacia were functionally overexpressed in E. coli. The lipase genes from Geotrichum candidum CMICC 335426 and Rhizopus oryzae were overexpressed in Pichia pastoris. Due to an unusual codon usage that prevents heterologous expression, the LIP1 gene (1647 nt) of Candida rugosa was completely synthesized and overexpressed in Pichia pastoris.
New Recognition Elements in Biosensing
Authors: SCHELLER FRIEDER W. , KLEINJUNG FRANK , BIER FRANK F. , MAKOWER ALEXANDER , NEUMANN BARBARA , WOLLENBERGER ULLA , KUROCHKIN ILIYA , EREMENKO ARKADI , BARMIN ANATOLI , KLUβMANN SVEN , FÜRSTE JENS‐PETER , ERDMANN VOLKER A. , MANSUY D.
Published: February 2006

Abstract: The borderline between molecular recognition element (MRE) tools that are biological in nature and synthetic (organic) receptor molecules is no longer well definable; these two classes of recognition elements are merging. This is especially true for enzyme models, polymers imprinted by biomolecules, ionophores (which mimic the function of channels), and (synthetic) oligonucleotides. In this paper, we introduce three new examples of modified recognition elements for biosensing: (i) bienzyme conjugates for quantification of inhibitors of choline esterase; (ii) recognition of l‐adenosine by high‐affinity oligonucleotides (aptamers); (iii) catalyzed conversion of alkanes by hydroperoxides in the presence of porphyrin complexes.
Neural Networks as “Software Sensors” in Enzyme Engineeringa
Authors: LINKO SUSAN , ZHU YI‐HONG , LINKO PEKKA
Published: February 2006

Abstract: Industrial applications of enzyme technology are rapidly increasing. On‐line control of enzyme production processes, however, is difficult owing to the uncertainties typical of biological systems and to the lack of suitable on‐line sensors for key process variables and quality attributes. We demonstrate that well‐trained feedforward backpropagation neural networks with one hidden layer can be employed to overcome such problems with no need for a priori knowledge of the relationships of the process variables involved. Neural network programs were written in Microsoft Visual C++ for Windows and implemented in a personal computer. The goodness of fit of the trained neural network to the reference data was determined by the coefficient of determination, R2. Case studies of β‐galactosidase, glucoamylase, lipase, and xylanase production processes will be used as examples.
Enzymatic Modification of Natural Compounds with Pharmacological Propertiesa
Authors: RIVA SERGIO , MONTI DANIELI , LUISETTI MONICA , DANIELI BRUNO
Published: February 2006

Abstract: Glycosides of various classes of natural products are widely distributed in nature, where they are often present esterified with aliphatic and aromatic acids at specific OH's of their sugar moieties.

Many of these compounds are pharmacologically important molecules or possess other interesting properties. For instance, ginsenosides (e.g., 3) are therapeutic dammarane‐type oligoglycosides isolated from the water‐soluble portion of the dried roots and leaves of Panax ginseng C.A. Meyer (Araliaceae), a plant widely used in traditional Chinese medicine.

In recent years, we have exploited the regioselectivity of lipases and proteases in organic solvents for the synthesis of specific esters of ginsenosides as well as the selectivity of the β‐1,4‐galactosyltransferase from bovine colostrum to obtain new glycosyl derivatives of these compounds.

The application of these two enzymatic methodologies has also been exemplified with other natural compounds with pharmacological properties: digitonin (5), colchicoside (6), and flavonoid glycosides.
Novel Double Bond‐Transferring Hydroxylation Reaction Involved in Microbial Metabolism of Eugenol
Authors: FURUKAWA HIROTAKA , NAGASAWA TORU
Published: February 2006

Abstract: We isolated a eugenol‐degrading bacterium, Pseudomonas fluorescens E118. This strain produced a novel enzyme, eugenol dehydrogenase, which catalyzes the conversion of eugenol into coniferyl alcohol. The enzyme was purified from the eugenol‐induced cells of P. fluorescens E118. The purified enzyme appeared to be homogeneous, judging from the analysis of polyacrylamide gel electrophoresis. The enzyme was a 68‐kDa protein composed of two different subunits (α subunit, 10 kDa; and β subunit, 58 kDa). The enzyme exhibited a cytochrome c‐like absorption spectrum. The a subunit corresponded to cytochrome c. The enzyme catalyzed the dehydrogenation of 4‐alkylphenol into the corresponding alkyl 1‐(4‐hydroxyphenyl)‐alcohol derivatives. The reaction products were isolated and identified physicochemically. The enzyme catalyzed the enantioselective hydroxylation of p‐alkylphenols. p‐Ethylphenol and p‐propylphenol were converted to S‐(−)‐p‐(1‐hydroxyphenyl)ethanol and S‐(−)‐p(1‐hydroxyphenyl)propanol, respectively.
New Strategy of Thrombolysis: Conjunctive Effect of Plasminogen Activators with Different Pharmacokinetic Profilea
Authors: MAKSIMENKO ALEXANDER V.
Published: February 2006

Abstract: Combined actions of native and prolonged thrombolytics allow the use of lower doses and simplified schemes of administration, thus yielding significant results in experimental therapy regarding the efficacy and safety of thrombolysis. Development of prolonged forms of plasminogen activators and testing their effect in combination with the thrombolysis trigger are well founded and of current interest. Thrombolytic compositions on the basis of short‐ and long‐term‐acting plasminogen activators appear to be promising and potentially eligible for bolus administration.
Production of “Neometalloenzymes” by De Novo Biosynthesis: New ELISA Method for Their Characterization
Authors: NEDONCHELLE E. , LEDUC C. , GARCIA X. , GUZMAN R. , VIJAYALAKSHMI M. A.
Published: February 2006

Several approaches known for producing “neometalloenzymes” are classified into two categories: protein engineering using antibodies as starting materials and “de novo” biosynthesis of metal‐binding antibodies with potential catalytic metal‐binding structure. This latter approach is chosen in this study. Polyclonal anti‐zinc‐iminodiacetate [IDA‐Zn(II)] antibodies are produced in rabbits and mice. Because of the absolute need for the unequivocal screening of the hapten [IDA‐Zn(II)] specific antibodies, a new ELISA method was developed using a biheaded polyethylene glycol with biotin on one end and the hapten on the other end.

The parameters for optimizing the immunization and the ELISA technique are discussed and the method is validated with rabbit and mice sera.
Screening of Inhibitory Monoclonal Antibodies: A Critical Step for Producing Anti‐idiotypic Catalytic Antibodies
Authors: AVALLE BÉRANGÈRE , FRIBOULET ALAIN , THOMAS DANIEL
Published: February 2006

Abstract: In accord with the original approach that we proposed, catalytic antibodies may be produced by using the anti‐idiotypic pathway according to antigen/antibody complementarity rules. The generation and screening of the idiotypic Ab1, the central point on which are anchored the interactions with both the antigen (enzyme) and the anti‐idiotypic abzyme, represent a crucial step for the success of this approach. We herein propose to describe a strategy for which we have developed a number of assays, aiming at selecting the proper Ab1, with desired features, likely to elicit an anti‐idiotypic catalytic antibody. β‐lactamase from Bacillus cereus was chosen as the example illustrating our arguments.
Properties of Artificial Proteins with Random Sequencesa
Authors: YOMO TETSUYA , PRIJAMBADA IRFAN D. , YAMAMOTO KEIZO , SHIMA YASUFUMI , NEGORO SEIJI , URABE ITARU
Published: February 2006

Abstract: A library of artificial proteins of 141 amino acid residues, of which 95 are random and which include 20 kinds of amino acids, was prepared. As the properties of the artificial random proteins are free from the evolutionary constraint, they can be used as a standard to discriminate the specialized properties of natural proteins. Out of the 25 identified random proteins, 5 are soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. Therefore, as natural soluble or insoluble proteins can arise from the line of soluble or insoluble ancestry, respectively, solubility does not seem a specialized property of natural proteins. The soluble random proteins RP3‐42 and RP3–45 were purified and their properties were investigated.
A Selenium‐Containing Abzyme, the Activity of Which Surpassed the Level of Native Glutathione Peroxidasea
Authors: LUO GUIMIN , DING LAN , LIU ZHI , YANG TONGSHU , NI JIAZHAN
Published: February 2006

Abstract: Using two different glutathione derivatives as hapten, we have prepared two abzymes, which display glutathione peroxidase (GPX) activity. Their GPX activities are 0.2 and 1.6 times that of natural GPX from rabbit liver, respectively. Selenium content analysis indicates that the activity difference between the two abzymes is possibly attributed to the conformation difference of the abzymes.
Directed Evolution of New Enzymes and Pathways for Environmental Biocatalysisa
Authors: WACKETT LAWRENCE P.
Published: February 2006

Abstract: Biocatalysis is important in both natural and engineered environments. The major global reactions in the biospheric cycling of carbon, nitrogen, and other elements are catalyzed by microorganisms. The global carbon cycle includes millions of organic compounds that are made by plants, microorganisms, and organic chemists. Most of those compounds are transformed by microbial enzymes. Degradative metabolism is known as catabolism and yields principally carbon dioxide, methane, or biomass. Microbial catabolic enzymes are a great resource for biotechnology. They are the building blocks for engineering novel metabolic pathways and evolving improved enzymes in the laboratory. Two multicomponent bacterial oxygenases, cytochrome P450cam and toluene dioxygenase, catalyze the dechlorination of polyhalogenated C2 compounds. Seven genes encoding those functional enzyme complexes were coexpressed in a Pseudomonas and shown to metabolize pentachloroethane to nonhalogenated organic acids that were metabolized further to carbon dioxide. In another example, the enzyme catalyzing the dechlorination of the herbicide atrazine was subjected to iterative DNA shuffling to produce mutations. By using a plate screening assay, mutated atrazine chlorohydrolase that catalyzed a more rapid dechlorination of atrazine was obtained. The mutant genes were sequences and found to encode up to 11 amino acid changes. Atrazine chlorohydrolase is currently being used in a model municipal water treatment system to test the feasibility of using enzymes for atrazine decontamination. These data suggest that the natural diversity of bacterial catabolic enzymes provides the starting point for improved biocatalytic systems that meet the needs of commercial applications.
Multiple Intensified Performance of an Enzyme‐Catalyzed Reaction in Organic Medium
Authors: YAMANE TSUNEO , IWASAKI YUGO , ROXANA ROSU , SHIMIDZU NOBUYOSHI , DOISAKI NOBUSHIGE
Published: February 2006

Abstract: A lipase‐catalyzed glycerolysis reaction (a transesterification between polyunsaturated fatty acid ethyl ester [PUFA] and glycerol) was investigated. Its performance was multiply intensified by (1) using a lipase having high specific activity, high activity in organic solvent, and high tolerance in organic solvent; (2) immobilization on fine CaCO3 powder (cheap and safe material, easy physical adsorption method of immobilization, reusable); (3) reaction in vacuo resulting in 100% conversion and effective avoidance of oxidative deterioration of PUFA; (4) high volumetric productivity because of no use of solvent; and (5) no need of further separation and purification of the oil product. It is emphasized that performance of biocatalytic reactions in organic media should be enhanced manifold for industrial implementation.
Thalassemia in the Next Millennium: Keynote Addressa
Authors: WEATHERALL D. J.
Published: February 2006

Abstract:Over the next decade it will be essential to make the thalassemia problem more visible to governments and international health agencies that are involved in health care in the emerging countries. This will require detailed population surveys to determine the gene frequencies of the important forms of thalassemia, together with a better understanding of their natural history and of the factors that modify their clinical phenotypes. In particular, more needs to be learnt about the natural history and ways of managing the intermediate forms of β thalassemia. While research should continue towards definitive forms of treatment it is important, in the meantime, to pursue the development of cheap and safe oral chelating agents and to carry out clinical trials of drugs that may interact one with another to elevate the level of fetal hemoglobin, particularly in patients with different types of β thalassemia intermedia. The partial control of the disease by carrier detection and prenatal diagnosis will only be feasible in emerging countries if it is possible to obtain the financial support of the major international agencies and the cooperation of their governments and communities; the remarkable success of this approach in some of the Mediterranean islands is a good example of what can be achieved.
Developmental Control of ɛ‐ and γ‐ Globin Genesa
Authors: LI Q. , PETERSON K. R. , STAMATOYANNOPOULOS G.
Published: February 2006

Abstract: In the last few years there have been considerable advances in the understanding of the molecular control of globin genes during development. Several insights have been obtained with studies using transgenic mice. The 5′ to 3′ order of the genes in the β locus, the proximity of the genes to the locus control region and the availability of transcriptional factors have been implicated in the developmental activation of globin genes. Globin genes are turned off by two general mechanisms, autonomous gene silencing involving sequences located in the proximal and distal promoters and competition between genes for interaction with the locus control region. The current understanding of the control of embryonic (ɛ) and fetal (γ) globin genes is reviewed.
β‐YAC Transgenic Mice for Studying LCR Functiona
Authors: PETERSON KENNETH R. , NAVAS PATRICK A. , STAMATOYANNOPOULOS GEORGE
Published: February 2006

Abstract: We have developed methods to produce transgenic mice using yeast artificial chromosomes (YACs) and have applied these methods to the analysis of globin gene regulation using 248 kb β‐globin locus YACs (β‐YACs). The advantages of YAC transgenics are: 1) developmental regulation can be studied in the context of the whole locus, 2) mutations may be readily introduced into the YAC, and 3) the effect of these mutations on gene expression can be analyzed. Mice containing the wild‐type β‐YAC show proper regulation of globin gene expression during development. Transgenics carrying a β‐YAC bearing a −117 Aγ mutation showed the anticipated phenotype of Greek HPFH, demonstrating that mutant β‐YACs can be used to generate mice that recreate human globin developmental mutants. Transgenic mice with YACs have also been used to examine the function of the LCR. Transgenic mice were generated with a β‐YAC containing a deletion of LCR DNAse I‐hypersensitive site 3 (5′HS3). Our results suggest that: 1) the LCR contains functionally redundant elements, 2) the formation of a LCR complex does not require all of the HSs, 3) the individual HSs may modulate the interaction of the LCR with specific globin genes during development, and 4) that most of the HS activity is confined to the core region.
Molecular Basis of Hereditary Persistence of Fetal Hemoglobin
Authors: FORGET BERNARD G.
Published: February 2006

Abstract: Increased levels of fetal hemoglobin (HbF) can ameliorate the clinical course of inherited disorders of β‐globin gene expression, such as β thalassemia and sickle cell anemia. In a group of disorders called hereditary persistence of fetal hemoglobin (HPFH), expression of the γ‐globin gene of HbF persists at high levels in adult erythroid cells. Molecular studies of the HPFH syndromes have identified several important regulatory elements for the normal pattern of γ‐globin gene expression. Deletion as well as nondeletion types of HPFH have been identified. The nondeletion types of HPFH are characterized by the presence of point mutations, in the promoter region of one or another γ‐globin gene, that are thought to alter interactions between various transcription factors and the promoter. The deletion types of HPFH are thought to deregulate the normal developmental pattern of γ‐globin gene expression due to the juxtaposition of normally distant cis‐acting factors into the vicinity of the γ genes. These findings have provided us with a more sophisticated understanding of the molecular basis for the persistent γ‐gene expression in these syndromes and point to certain strategies for potential future attempts at gene therapy for β‐globin gene disorders.
Transcriptional Factors for Specific Globin Genesa
Authors: BIEKER J. J. , OUYANG L. , CHEN X.
Published: February 2006

Abstract: Correct temporal control of the β‐like globin cluster is generated in part by the binding of tissue‐restricted transcriptional regulators to their cognate sites. Erythroid Krüppel‐like Factor (EKLF) is one of these red cell‐specific activators that is particularly important for switching on adult β‐globin gene expression. However, its simple presence is not sufficient to activate the β‐globin promoter, as primitive erythroid cells and a number of erythroid cell lines express EKLF yet do not express adult β‐globin. One explanation that may account for these observations is that post‐translational modification of EKLF differs within these cell populations. To address this issue, we are investigating whether phosphorylation plays a role in modulating EKLF activity. In vitro and in vivo approaches have been used to demonstrate that EKLF is a phosphoprotein whose ability to bind DNA and transcriptionally activate an adjacent promoter is critically dependent on its phosphorylation status. Of particular interest is a casein kinase II site within the EKLF minimal transactivation domain.
Hemoglobin Switching Protocols in Thalassemia: Experience with Sodium Phenylbutyrate and Hydroxyureaa
Authors: DOVER GEORGE J.
Published: February 2006

Abstract: Homozygous β thalassemia affects thousands of people around the world. Current management of this condition includes regular transfusion of red cells, which leads to transfusional iron overload requiring chelation therapy: increasing hemoglobin levels while decreasing or eliminating iron overload is therefore a major therapeutic goal in the treatment of thalassemia. Bone marrow transplantation may achieve this goal, but it is not an option for most patients. This study reports on efforts to increase γ‐globin transcription and HbF production using sodium phenylbutyrate (SPB) and hydroxyurea (HU). It was found that 36% (4/11) of all patients or 50% (4/8) of non‐transfused patients responded to SPB (increase in Hb levels of 1g/dL). A positive correlation between baseline serum erythropoietin level and likelihood of response to SPB was observed. Since HU may also increase HbF production, evaluation of combination therapy with these drugs is underway and preliminary results are reported.
Elimination of Transfusions Through Induction of Fetal Hemoglobin Synthesis in Cooley's Anemiaa
Authors: OLIVIERI NANCY F. , REES DAVID C. , GINDER GORDON D. , THEIN SWEE LAY , WAYE JOHN S. , CHANG LEBE , BRITTENHAM GARY M. , WEATHERALL DAVID J.
Published: February 2006

Pharmacological stimulation of fetal hemoglobin production is of considerable interest as an alternative approach to therapy for Cooley's anemia. While intravenous compounds have been effective in inducing short‐term increases in fetal hemoglobin in a few patients, long‐term elimination of transfusion requirement has not been reported. In patients with Cooley's anemia, treatment with oral sodium phenylbutyrate alone, sodium phenylbytyrate combined with hydroxyurea, and hydroxyurea alone, has augmented fetal hemoglobin production and increased total hemoglobin concentration as much as 5 g/dl over baseline eliminating transfusion requirement in two patients. Parallel declines in circulating nucleated red cell count, and concentrations of serum transform receptor and erythropoietin. are consistent with more effective erythropoiesis. Over extended periods of treatment, no induction of other fetal proteins and no adverse effects were observed. Particular disease mutations and other genetic factors may be of prime importanec in determining the response to agents that induce production of fetal hemoglobin.
Hydroxyurea Therapy in Thalassemiaa
Authors: LOUKOPOULOS DIMITRIS , VOSKARIDOU ERSI , STAMOULAKATOU ALEXANDRA , PAPASSOTIRIOU YANNIS , KALOTYCHOU VASILIKI , LOUTRADI APHRODITE , COZMA GABRIEL , TSIARTA HELENI , PAVLIDES NIKOS
Published: February 2006

Abstract: The clinical effectiveness of Hydroxyurea in thalassemia is still controversial. The present paper puts together the authors' experience in two groups of patients with thalassemia intermedia and sickle cell/β‐thalassemia treated with varying dosages of hydroxyurea over several months. A third group received hydroxyurea along with recombinant human erythropoietin. Our observations are summarized in that treatment with hydroxyrea results in a significant increase of fetal hemoglobin with no change of the total hemoglobin levels. The drug causes also a considerable increase of the erythrocyte volume and hemoglobin content while the MCHC values remain unchanged. As a rule, and without objective criteria so far, patients state feeling better and having more energy. The authors postulate that this feeling may reflect the significant decrease of ineffective erythropoiesis resulting by the replacement of the poorly hemoglobinized, prematurely dying erythroid progenitor and red cell population by another population of cells with higher hemoglobin content and longer survival, the regeneration of which requires less energy and consumption. As expected, patients with sickle cell/β‐thalassemia have also fewer crises and painful episodes. The above findings are in keeping with the few available reports in the literature.
Retroviral Vectors Aimed at the Gene Therapy of Human β‐Globin Gene Disordersa
Authors: PAWLIUK ROBERT , BACHELOT THOMAS , RAFTOPOULOS HARRY , KALBERER CHRISTIAN , HUMPHRIES R. KEITH , BANK ARTHUR , LEBOULCH PHILIPPE
Published: February 2006

Abstract: We are focusing on the development of complex retroviral vectors containing human β‐globin gene and β‐LCR for the gene therapy of sickle cell disease and β‐thalassemias. First generation vectors containing mutated splice‐sites to insure stability of proviral transfer enabled long‐term reconstitution in 10/12 transplanted mice for a least 8 months with high expression levels in 2 out of 3 mice analyzed (5% and 20% murine β). Transfer and expression were also achieved in secondary recipients (range: 3–11% murine β). Position independent expression was not observed. In an effort to increase the efficiency of gene transfer and obtain complete reconstitution of recipient mice with exclusively transduced cells while enriching for proviral integration into active chromatin regions, we have incorporated a cassette expressing CD24 or the green fluorescent protein (GFP). Stable transfer to murine bone marrow cells allowed efficient FACS‐sorting of pure populations of transduced cells. A family of vectors based on these principles and containing segments of γ‐ or δ‐globin genes were also designed for systematic analysis of their anti‐sickling properties.
High‐Level Transfer and Long‐Term Expression of the Human β‐Globin Gene in a Mouse Transplant Modela
Authors: RAFTOPOULOS HARRY , WARD MAUREEN , BANK ARTHUR
Published: February 2006

Abstract: Insertion of a normally functioning human β‐globin gene into the hematopoietic stem cells (HSC) of patients with β‐thalassemia may be an effective approach to the therapy of this disorder. Safe, efficient gene transfer and long‐term, high‐level expression of the transferred human β‐globin gene in animal models are prerequisites for HSC somatic gene therapy. We have recently shown for the first time that, using a modified β‐globin retroviral vector in a mouse transplant model, long‐term, high‐level expression of a transferred human β‐globin gene is possible. The human β‐globin gene continues to be detected up to eight months post‐transplantation of β‐globin‐transduced hematopoietic cells into lethally irradiated mice. The transferred human β‐globin gene is detected in three of five mice surviving long‐term (>4 months) transplanted with bone marrow cells transduced with high‐titer virus. The unrearranged 5.1 kb human β‐globin gene‐containing provirus is seen by Southern blotting in two of these mice. More importantly, long‐term expression of the transferred gene is seen in two mice at levels of 5% and 20% that of endogenous murine β‐globin. We document stem cell transduction by showing continued high‐level expression of the human β‐globin gene in secondarily transplanted recipient mice. These results provide evidence of HSC transduction with a human β‐globin gene in animals and demonstrate that retroviral‐mediated unrearranged human β‐globin gene transfer leads to a high level of human β‐globin gene expression in the long term for the first time. A gene therapy strategy may be a feasible therapeutic approach to the β‐thalassemias if consistent human β‐globin gene transfer and expression into HSC can be achieved.