bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.
Authors: Mancini M , Hadchouel M , Davis H L , Whalen R G , Tiollais P , Michel M L
Published: July 2002

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.
In vitroandin vivodifferentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded >100-fold by coculture with a clonal yolk sac endothelial cell line
Authors: Lu Li-Sheng , Wang Shur-Jen , Auerbach Robert
Published: July 2002

The yolk sac, first site of hematopoiesis during mammalian development, contains not only hematopoietic stem cells but also the earliest precursors of endothelial cells. We have previously shown that a nonadherent yolk sac cell population (WGA+, density <1.077, AA4.1+) can give rise to B cells, T cells, and myeloid cells bothin vitroandin vivo. We now report on the ability of a yolk sac-derived cloned endothelial cell line (C166) to provide a suitable microenvironment for expansion of these early precursor cells. Single day 10 embryonic mouse yolk sac hematopoietic stem cells were expanded >100 fold within 8 days by coculture with irradiated C166 cells. Colony-forming ability was retained for at least three passagesin vitro, with retention of the ability to differentiate into T-cell, B-cell, and myeloid lineages. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype than the initial yolk sac stem cells. When reintroduced into adult allogeneic immunocompromised (scid) hosts, they were able to give rise to all of the leukocyte lineages, including T cells, B cells, and myeloid cells. We conclude that yolk sac endothelial cells can support the stable proliferation of multipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent development and differentiation, forin vivohematopoietic restitution, and for potential use as a vehicle for gene transfer.
Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex
Authors: Trowbridge Janet M. , Rogatsky Inez , Garabedian Michael J.
Published: July 2002

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor
in vivo
in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27
KIP1
, or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121
in vitro
by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A
in vivo
. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.
Developmental disorder associated with increased cellular nucleotidase activity
Authors: Page Theodore , Yu Alice , Fontanesi John , Nyhan William L.
Published: July 2002

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine.
De novo
synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.
Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: A fluorescence resonance energy transfer study
Authors: Damjanovich Sándor , Bene László , Matkó János , Alileche Abdelkrim , Goldman Carolyn K. , Sharrow Susan , Waldmann Thomas A.
Published: July 2002

Assembly and mutual proximities of α, β, and γcsubunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate- and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2Rα, IL-2Rβ, and γcsubunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2–10 nm) by FRET on a cell-by-cell basis. The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15. FRET data from donor- and acceptor-labeled IL-2Rβ-α, γ-α, and γ-β pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells. These mutual proximities do not appear to represent mAb-induced microaggregation, because FRET measurements with Fab fragments of the mAbs gave similar results. The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15. Based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a “triangular model” in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact. IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific α receptors with the β and/or γcsubunits of the IL-2R complex. These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution. This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.
Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase
Authors: Thurmond Robin L. , Creuzenet Carole , Reeves Philip J. , Khorana H. Gobind
Published: July 2002

Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1–3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in
V
max
for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in
V
max
relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the
K
m
by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.
Alprazolam dependence prevented by substituting with the β-carboline abecarnil
Authors: Pinna Graziano , Galici Ruggero , Schneider Herbert H. , Stephens David N. , Turski Lechoslaw
Published: July 2002

Abrupt termination of the treatment of humans with benzodiazepines (BDZs) leads to a rapid onset of discontinuation syndrome characterized by anxiety, muscle spasms, and occasionally convulsions. For this reason, it is recommended in clinical practice to reduce the dose of the BDZs gradually at the end of treatment. Nevertheless, many clinicians report signs of dependence even during gradual reduction of doses (tapering) of the BDZs in a large proportion of patients. Thus, there is considerable interest in discovering means of weaning patients away from BDZs without the risk of discontinuation syndrome. In the present study, mice withdrawn from chronic treatment with alprazolam showed anxiety, muscle rigidity, and seizures between days 1 and 28 after termination of the treatment. Replacement of alprazolam with the β-carboline abecarnil for 7 days prevented the occurrence of the signs of dependence. In contrast, substitution of the β-carboline antagonist ethyl-5-isopropoxy-4-methyl-β-carboline-3-carboxylate (ZK93426) for alprazolam worsened the discontinuation syndrome. Replacement therapy with abecarnil after long-term treatment with the BDZs offers a novel method for rapid tapering.
Increased rates of CD4+and CD8+T lymphocyte turnover in simian immunodeficiency virus-infected macaques
Authors: Rosenzweig Michael , DeMaria MaryAnn , Harper David M. , Friedrich Stuart , Jain Rakesh K. , Johnson R. Paul
Published: July 2002

Defining the rate at which T cells turn over has important implications for our understanding of T lymphocyte homeostasis and AIDS pathogenesis, yet little information on T cell turnover is available. We used the nucleoside analogue bromodeoxyuridine (BrdUrd) in combination with five-color flow cytometric analysis to evaluate T lymphocyte turnover rates in normal and simian immunodeficiency virus (SIV)-infected rhesus macaques. T cells in normal animals turned over at relatively rapid rates, with memory cells turning over more quickly than naive cells. In SIV-infected animals, the labeling and elimination rates of both CD4+and CD8+BrdUrd-labeled cells were increased by 2- to 3-fold as compared with normal controls. In normal and SIV-infected animals, the rates of CD4+T cell BrdUrd-labeling and decay were closely correlated with those of CD8+T cells. The elimination rate of BrdUrd-labeled cells was accelerated in both naive and memory T lymphocytes in SIV-infected animals. Our results provide direct evidence for increased rates of both CD4+and CD8+T cell turnover in AIDS virus infection and have important implications for our understanding of T cell homeostasis and the mechanisms responsible for CD4+T cell depletion in AIDS.
Mutagenesis associated with nitric oxide production in macrophages
Authors: Zhuang John C. , Lin Charley , Lin Doris , Wogan Gerald N.
Published: July 2002

To better understand the mechanisms through which persistent infections/inflammation increase cancer risks, we assessed the potential genotoxic properties of NO produced by macrophages. We recently showed that mouse macrophage RAW264.7 cells were capable of resuming exponential growth after stimulation for NO production by interferon-γ (IFN-γ) and/or lipopolysaccharide. Here, we report that increases in mutant fraction (MF) in the endogenous, X-linked,
hprt
gene of the cells are associated with NO exposure. Cells stimulated with 100 units/ml IFN-γ continuously for 14 and 23 days produced a total of 9.8 and 14 μmol of NO per 10
7
cells, respectively. MFs in the
hprt
gene of NO-producing cells were 16.6 and 31.3 × 10
−5
, respectively, compared with 2.2 and 2.5 × 10
−5
in untreated cells. Addition of an NO synthase inhibitor,
N
-monomethyl-
l
-arginine, to the culture medium decreased NO production and MF by 90% and 85%, respectively. Reverse transcription-PCR and DNA sequencing revealed that NO-associated
hprt
mutations did not differ significantly from those arising spontaneously, with the exception that certain small deletions/insertions and multiple exon deletions were observed only in the former. MF also increased significantly in cells stimulated for only 4 days with lipopolysaccharide plus IFN-γ for higher rates of NO production. The types and proportion of
hprt
mutations induced under these conditions were strikingly similar to those associated with long-term NO exposure. These results indicate that NO exposure results in gene mutations in RAW264.7 cells through mechanisms yet to be identified and may also contribute to spontaneous mutagenesis.
Neurotrophins induce release of neurotrophins by the regulated secretory pathway
Authors: Krüttgen Alex , Möller J. Carsten , Heymach John V. , Shooter Eric M.
Published: July 2002

Recent studies have established that neurotrophin synthesis and secretion are regulated by activity and that these factors are involved in activity-dependent processes in the nervous system. Neurotrophins also are known to induce increases in intracellular calcium, a trigger for regulated secretion. This finding raises the possibility that neurotrophins themselves may stimulate regulated secretion of neurotrophins. To address this question, we studied the release of neurotrophins from transfected PC12 cells, a widely used model for neuronal secretion and neurotrophin signal transduction. We found that neurotrophins induced the regulated secretion of brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and neurotrophin-4/5. The effect of brain-derived neurotrophic factor on release of NT-3 could be abolished by REX, a p75 blocking antibody, but not by K252a, an inhibitor of neurotrophin tyrosine kinase receptor (Trk) signaling. The nerve growth factor effect on release of NT-3 could be blocked only by simultaneous application of REX and K252a, suggesting that they are mediated by TrkA as well as p75. Our data show that neurotrophins are able to induce the regulated secretion of neurotrophins and suggest a signal-transducing role for both TrkA and p75 in this process. The neurotrophin-induced release of neurotrophins may be relevant for activity-dependent processes such as synaptic plasticity and memory formation.
Synthesis, biophysical properties, and nuclease resistance properties of mixed backbone oligodeoxynucleotides containing cationic internucleoside guanidinium linkages: Deoxynucleic guanidine/DNA chimeras
Authors: Barawkar Dinesh A. , Bruice Thomas C.
Published: July 2002

The synthesis of mixed backbone oligodeoxynucleotides (18-mers) consisting of positively charged guanidinium linkages along with negatively charged phosphodiester linkages is carried out. The use of a base labile-protecting group for guanidinium linkage offers a synthetic strategy similar to standard oligonucleotide synthesis. The nuclease resistance of the oligodeoxyribonucleotides capped with guanidinium linkages at 5′ and 3′ ends are reported. The hybridization properties and sequence specificity of binding of these deoxynucleic guanidine/DNA chimeras with complementary DNA or RNA are described.
p19
Arf
induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation
Authors: Radfar Arash , Unnikrishnan Indira , Lee Han-Woong , DePinho Ronald A. , Rosenberg Naomi
Published: July 2002

The
Ink4a/Arf
locus encodes p16
Ink4a
and p19
Arf
and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19
Arf
and the p53 tumor-suppressor protein. Because
Tp53
mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the
Ink4a/Arf
locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16
Ink4a
and p19
Arf
are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from
Ink4a/Arf
null mice revealed that these cells bypassed crisis. Because expression of p19
Arf
but not p16
Ink4a
induced apoptosis in Ab-MLV-transformed pre-B cells, p19
Arf
appears to be responsible for these events. Consistent with the link between p19
Arf
and p53,
Ink4a/Arf
expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19
Arf
is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19
Arf
–p53 regulatory loop plays an important role in lymphoma induction.
Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb β
s
-globin yeast artificial chromosome: A mouse model of sickle cell anemia
Authors: Chang Judy C. , Lu Ronghua , Lin Chin , Xu Shan-Mei , Kan Yuet Wai , Porcu Susanna , Carlson Elaine , Kitamura Michael , Yang Suya , Flebbe-Rehwaldt Linda , Gaensler Karin M. L.
Published: July 2002

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and β
s
transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb β
s
yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.
In vivopriming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen
Authors: Drakesmith Hal , O’Neil Deborah , Schneider Susanne C. , Binks Mike , Medd Patrick , Sercarz Eli , Beverley Peter , Chain Benjamin
Published: July 2002

T cells recognizing poorly displayed self determinants escape tolerance mechanisms and persist in the adult repertoire. The process by which these T cells are primed is not clear, but once activated, they can cause autoimmunity. Here, we show that dendritic cells treated with interleukin 6 (IL-6) process and present determinants from a model native antigen in a qualitatively altered hierarchy, activating T cellsin vitroandin vivoagainst determinants that were previously cryptic because of poor display. IL-6 does not induce conventional maturation of dendritic cells but alters the pH of peripheral, early endosomal compartments and renders the cells more susceptible to killing by chloroquine. Acidification of endosomes by ouabain mimics the effect of IL-6 and allows processing of the same cryptic determinant. These results suggest that cytokines such as IL-6 could initiate and help to propagate an autoimmune disease process by differentiating dendritic cells into a state distinct from that induced by normal maturation.
The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines
Authors: Tsatsanis Christos , Patriotis Christos , Bear Susan E. , Tsichlis Philip N.
Published: July 2002

Tpl-2expression is induced within 30–60 min after ConA stimulation of rat splenocytes, suggesting that it may contribute to the induction of IL-2 during T cell activation. Herein we show that wild-type and carboxyl-terminally truncated (activated) Tpl-2 activate the nuclear factor of activated T cells (NFAT) and induce interleukin 2 (IL-2) expression in EL4 cells. In Jurkat cells the truncated Tpl-2 activates NFAT and induces IL-2, whereas wild-type Tpl-2 activates NFAT only when cotransfected with NFAT expression constructs, suggesting that Tpl-2 may induce NFAT activation signals. Experiments in NIH 3T3 cells revealed that the NFATp isoform, but not the NFATc or NFATx isoform, undergoes nuclear translocation when coexpressed with wild-type Tpl-2 and confirmed this hypothesis. Activation of NFAT by anti-CD3 stimulation but not by phorbol 12-myristate 13-acetate and ionomycin in Jurkat cells was inhibited by the kinase-dead Tpl-2K167M, suggesting that Tpl-2 contributes to the transduction of NFAT activation signals originating in the T cell receptor. The Tpl-2-mediated induction of IL-2 was not observed in T cell lymphoma lines other than EL4 and Jurkat, as well as in normal T cells. NFAT activation by Tpl-2, however, was observed in several cell lines including some of nonhematopoietic origin. The activation of NFAT by Tpl-2 in different cell types defines a molecular mechanism that may contribute to its oncogenic potential.
Human
XIST
yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells
Authors: Heard Edith , Mongelard Fabien , Arnaud Danielle , Chureau Corinne , Vourc’h Claire , Avner Philip
Published: July 2002

Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The
Xist
gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse
Xist
gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human
XIST
gene was introduced into mouse embryonic stem (ES) cells. The human
XIST
transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human
XIST
expression was found in only a proportion of differentiated cells, suggesting that the events downstream of
XIST
RNA coating that culminate in stable inactivation may require species-specific factors. Human
XIST
RNA appears to coat mouse autosomes in ES cells before
in vitro
differentiation, in contrast to the behavior of the mouse
Xist
gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in
Xist
regulation but also provides evidence that factors implicated in
Xist
RNA chromosome coating may already be present in undifferentiated ES cells.
Osteoblast-specific gene expression after transplantation of marrow cells: Implications for skeletal gene therapy
Authors: Hou Zhen , Nguyen Que , Frenkel Baruch , Nilsson Susan K. , Milne Moira , van Wijnen André J. , Stein Janet L. , Quesenberry Peter , Lian Jane B. , Stein Gary S.
Published: July 2002

Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded
ex vivo
. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton.
Validation of the single-stranded channel conformation of gramicidin A by solid-state NMR
Authors: Kovacs F. , Quine J. , Cross T. A.
Published: July 2002

The monovalent cation selective channel formed by a dimer of the polypeptide gramicidin A has a single-stranded, right-handed helical motif with 6.5 residues per turn forming a 4-Å diameter pore. The structure has been refined to high resolution against 120 orientational constraints obtained from samples in a liquid-crystalline phase lipid bilayer. These structural constraints from solid-state NMR reflect the orientation of spin interaction tensors with respect to a unique molecular axis. Because these tensors are fixed in the molecular frame and because the samples are uniformly aligned with respect to the magnetic field of the NMR spectrometer, each constraint restricts the orientation of internuclear vectors with respect to the laboratory frame of reference. The structural motif of this channel has been validated, and the high-resolution structure has led to precise models for cation binding, cation selectivity, and cation conductance efficiency. The structure is consistent with the electrophysiological data and numerous biophysical studies. Contrary to a recent claim [Burkhart, B. M., Li, N., Langs, D. A., Pangborn, W. A. & Duax, W. L. (1998)
Proc. Natl. Acad. Sci. USA
95, 12950–12955], the solid-state NMR constraints for gramicidin A in a lipid bilayer are not consistent with an x-ray crystallographic structure for gramicidin having a double-stranded, right-handed helix with 7.2 residues per turn.
Guest exchange in an encapsulation complex: A supramolecular substitution reaction
Authors: Santamaría Javier , Martín Tomás , Hilmersson Göran , Craig Stephen L. , Rebek Julius
Published: July 2002

Encapsulation complexes are reversibly formed assemblies in which small molecule guests are completely surrounded by large molecule hosts. The assemblies are held together by weak intermolecular forces and are dynamic: they form and dissipate on time scales ranging from milliseconds to days—long enough for many interactions, even reactions, to take place within them. Little information is available on the exchange process, how guests get in and out of these complexes. Here we report that these events can be slow enough for conventional kinetic studies, and reactive intermediates can be detected. Guest exchange has much in common with familiar chemical substitution reactions, but differs in some respects: no covalent bonds are made or broken, the substrate is an assembly rather than a single molecule, and at least four molecules are involved in multiple rate-determining steps.
In vivoproliferation of naïve and memory influenza-specific CD8+T cells
Authors: Flynn Kirsten J. , Riberdy Janice M. , Christensen Jan P. , Altman John D. , Doherty Peter C.
Published: July 2002

The virus-specific CD8+T cell response has been analyzed through the development, effector, and recovery phases of primary and secondary influenza pneumonia. Apparently, most, if not all, memory T cells expressing clonotypic receptors that bind a tetrameric complex of influenza nucleoprotein (NP)366–374peptide+H-2Db(NPP) are induced to divide during the course of this localized respiratory infection. The replicative phase of the recall response ends about the time that virus can no longer be recovered from the lung, whereas some primary CD8+NPP+T cells may proliferate for a few more days. The greatly expanded population of CD8+NPP+memory T cells in the lymphoid tissue of secondarily challenged mice declines progressively in mean prevalence over the ensuing 100 days, despite the fact that at least some of these lymphocytes continue to cycle. The recall of cell-mediated immunity thus is characterized by massive proliferation of the antigen-specific CD8+set, whereas the extent of lymphocyte turnover in the absence of cognate peptide is variable, at a low level, and can be influenced by intercurrent infection.
Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler–Najjar syndrome type I with a chimeric oligonucleotide
Authors: Kren Betsy T. , Parashar Bhupesh , Bandyopadhyay Paramita , Chowdhury Namita Roy , Chowdhury Jayanta Roy , Steer Clifford J.
Published: July 2002

Crigler–Najjar syndrome type I is characterized by unconjugated hyperbilirubinemia resulting from an autosomal recessive inherited deficiency of hepatic UDP-glucuronosyltransferase (UGT) 1A1 activity. The enzyme is essential for glucuronidation and biliary excretion of bilirubin, and its absence can be fatal. The Gunn rat is an excellent animal model of this disease, exhibiting a single guanosine (G) base deletion within the
UGT1A1
gene. The defect results in a frameshift and a premature stop codon, absence of enzyme activity, and hyperbilirubinemia. Here, we show permanent correction of the
UGT1A1
genetic defect in Gunn rat liver with site-specific replacement of the absent G residue at nucleotide 1206 by using an RNA/DNA oligonucleotide designed to promote endogenous repair of genomic DNA. The chimeric oligonucleotide was either complexed with polyethylenimine or encapsulated in anionic liposomes, administered i.v., and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, and DNA sequencing, and confirmed by genomic Southern blot analysis. DNA repair was specific, efficient, stable throughout the 6-month observation period, and associated with reduction of serum bilirubin levels. Our results indicate that correction of the
UGT1A1
genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, with consequent improvement in the metabolic abnormality.
CDC
45
and
DPB
11
are required for processive DNA replication and resistance to DNA topoisomerase I-mediated DNA damage
Authors: Reid Robert J. D. , Fiorani Paola , Sugawara Mizuno , Bjornsti Mary-Ann
Published: July 2002

The antitumor agent camptothecin targets DNA topoisomerase I by reversibly stabilizing a covalent enzyme-DNA intermediate. The subsequent collision of DNA replication forks with these drug-enzyme-DNA complexes produces the cytotoxic DNA lesions that signal cell cycle arrest and ultimately lead to cell death. Despite intense investigation, the character of the lesions produced and the repair processes that resolve the damage remain poorly defined. A yeast genetic screen was implemented to isolate conditional mutants with enhanced sensitivity to DNA topoisomerase I-mediated DNA damage. Cells exhibiting temperature-sensitive growth in the presence of the DNA topoisomerase I mutant, Top1T722Ap, were selected. Substitution of Ala for Thr722 increases the stability of the covalent Top1T722Ap-DNA intermediate, mimicking the cytotoxic action of camptothecin. Two mutants isolated,
cdc45
-
10
and
dpb11
-
10
, exhibited specific defects in DNA replication and a synthetic lethal phenotype in the absence of DNA damaging agents. The accumulation of Okazaki fragments under nonpermissive conditions suggests a common function in promoting processive DNA replication through polymerase switching. These results provide a mechanistic basis for understanding the cellular processes involved in the resolution of DNA damage induced by camptothecin and DNA topoisomerase I.
Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene
Lhx8
Authors: Zhao Yangu , Guo Yi-Jun , Tomac Andreas C. , Taylor Nora R. , Grinberg Alexander , Lee Eric J. , Huang SingPing , Westphal Heiner
Published: July 2002

Formation of the mammalian secondary palate is a highly regulated and complex process whose impairment often results in cleft palate, a common birth defect in both humans and animals. Loss-of-function analysis has linked a growing number of genes to this process. Here we report that
Lhx8
, a recently identified LIM homeobox gene, is expressed in the mesenchyme of the mouse palatal structures throughout their development. To test the function of
Lhx8 in vivo
, we generated a mutant mouse with a targeted deletion of the
Lhx8
gene. Our analysis of the mutant animals revealed a crucial role for
Lhx8
in palatogenesis. In
Lhx8
homozygous mutant embryos, the bilateral primordial palatal shelves formed and elevated normally, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. Because development of other craniofacial structures appeared normal, the impaired palatal formation in
Lhx8
-mutant mice was most likely caused by an intrinsic primary defect in the mesenchyme of the palatal shelves. The cleft palate phenotype observed in
Lhx8
-mutant mice suggests that
Lhx8
is a candidate gene for the isolated nonsyndromic form of cleft palate in humans.
Prolactin is not a juvenile hormone in
Xenopus
laevis
metamorphosis
Authors: Huang Haochu , Brown Donald D.
Published: July 2002

Prolactin (PRL) is widely considered to be the juvenile hormone of anuran tadpoles and to counteract the effects of thyroid hormone (TH), the hormone that controls amphibian metamorphosis. This putative function was concluded mainly from experiments in which mammalian PRL was injected into tadpoles or added to cultured tadpole tissues. In this study, we show that overexpression of ovine or
Xenopus laevis
PRL in transgenic
X. laevis
does not prolong tadpole life, establishing that PRL does not play a role in the life cycle of amphibians that is equivalent to that of juvenile hormone in insect metamorphosis. However, overexpression of PRL produces tailed frogs by reversing specifically some but not all of the programs of tail resorption and stimulating growth of fibroblasts in the tail. Whereas TH induces muscle resorption in tails of these transgenics, the tail fibroblasts continue to proliferate resulting in a fibrotic tail that is resistant to TH.
Adaptive variation in lactate dehydrogenase-B gene expression: Role of a stress-responsive regulatory element
Authors: Schulte Patricia M. , Glémet Hélène C. , Fiebig Aline A. , Powers Dennis A.
Published: July 2002

Although changes in gene regulation may play an important role in adaptive evolution, there have been few attempts to investigate the molecular mechanisms responsible for adaptively significant variation in gene expression. Here we describe the mechanism underlying an adaptive difference in the expression of the lactate dehydrogenase-B gene (
Ldh-B
) between northern and southern populations of the fish
Fundulus heteroclitus. Ldh-B
regulatory sequences from northern and southern individuals, coupled to a luciferase reporter gene, were introduced into the livers of live fish. Deletion studies indicated that sequence changes between 400 and 500 bp upstream of the transcription start site resulted in a 2-fold difference in reporter gene transcription. These sequence changes can account for the previously observed 2-fold difference in
Ldh-B
transcription between populations. Variation in transcription factors did not play an important role. Sequences within the functionally important region resemble a mammary tumor virus glucocorticoid responsive element (MTV-GRE) in southern alleles, whereas northern alleles differ from the consensus by 1 bp. To test the hypothesis that this element is involved in the variation between populations of
F. heteroclitus
, we exposed transiently transgenic fish containing
Ldh-B
regulatory sequence/reporter gene constructs to handling stress or injected cortisol. Both treatments increased reporter gene transcription driven by southern alleles but not northern alleles, as expected if an MTV-GRE sequence were involved. This finding suggests that sequence variation in a GRE is the cause of the adaptive differences in
Ldh-B
gene expression between populations and demonstrates that small changes in gene regulatory sequences can have important evolutionary consequences.
Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior
Authors: Auger Anthony P. , Tetel Marc J. , McCarthy Margaret M.
Published: July 2002

Steroid hormone action during brain development exerts profound effects on reproductive physiology and behavior that last into adulthood. A variety of
in vitro
studies indicate that steroid receptors require nuclear receptor coactivators for efficient transcriptional activity. To determine the functional significance of the nuclear receptor coactivator SRC-1 in developing brain, we investigated the consequence of reducing SRC-1 protein during sexual differentiation of the brain. We report that reducing SRC-1 protein interferes with the defeminizing actions of estrogen in neonatal rat brain. Our data indicate that SRC-1 protein expression is critically involved in the hormone-dependent development of normal male reproductive behavior and brain morphology.
Gene inactivation by multiphoton-targeted photochemistry
Authors: Berns Michael W. , Wang Zifu , Dunn Andrew , Wallace Vincent , Venugopalan Vasan
Published: July 2002

Multiphoton-targeted photochemistry was used to selectively inactivate the expression of genes in vertebrate cells. A membrane permeable DNA-associating vital dye, ethidium bromide monoacetate (visible wavelength single photon absorption peak at 530 nm) was used to photosensitize chromosomes in dividing cells. A 100-ps infrared laser beam operating at 1.06 microns was focused onto a selected region of a mitotic chromosome corresponding to the sites of the nucleolar (ribosomal) genes. Individual cells followed through mitosis demonstrated a reduction in the number of nucleoli formed in daughter cells that corresponded to the number of nucleolar genes sites irradiated. These results demonstrate the ability to selectively manipulate genes by using the focal point specificity characteristic of multiphoton microscopy. This technique should have wide biotechnology applications both
in vitro
and
in vivo
.
Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: Isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens
Authors: Wang Z.-G. , Williams L. J. , Zhang X.-F. , Zatorski A. , Kudryashov V. , Ragupathi G. , Spassova M. , Bornmann W. , Slovin S. F. , Scher H. I. , Livingston P. O. , Lloyd K. O. , Danishefsky S. J.
Published: July 2002

We have previously reported on a carbohydrate-based vaccine program for immunotherapy in cancer patients. One such vaccine, based on the globo H antigen conjugated to the protein keyhole limpet hemocyanin (KLH), has been in clinical evaluation. Although this and other carbohydrate vaccines have been shown to induce antibody responses, there are currently no quantitative data on the antibody levels achieved in immunized patients by these or other anti-cancer vaccines. We report herein an efficient route to complex synthetic oligosaccharides attached to an affinity matrix for identifying and isolating antibodies elicited against such a carbohydrate-based vaccine in humans. Pre- and postvaccination profiles from serum samples of patients immunized with globo H-KLH were compared. All anti-globo H antibody activity was efficiently separated from other serum constituents. The isolated antibodies were readily quantified, and their specificities were analyzed. Since no comparable data were available on antibodies resulting from the vaccination of other cancer patients, we compared the observed levels with those quoted in studies with bacterial polysaccharide vaccines that had been quantified. Remarkably, cancer patients immunized with globo H-KLH produce anti-globo H antibody levels often exceeding those formed by immunization with bacterial polysaccharides. In addition, substantial quantities of both IgG and IgM antibodies were elicited, clearly indicating a class switch to IgG. Taken together, these analyses serve to clarify several aspects of the immune response to the vaccine and give several new insights to the carbohydrate-based vaccination strategy. Furthermore, antibodies so isolated could well have applications in clinical therapy.
Cross‐cognitive Aspects of Discourse Comprehension: Semantic Post‐representation
Authors: Le Ny Jean‐François
Published: July 2002

Four related sciences, cognitive psychology, artificial intelligence, linguistics and neurobiology, are compared in a cross‐cognitive way with respect to their approaches in the study of discourse comprehension, in particular its final product, semantic post‐representations. The nature and structure of these, as they are built in a human mind after processing a short piece of discourse (one or a few sentences), seem to be best described in the framework of activation models, a family in which the basic processes of comprehension are considered to be activation of semantic units from long‐term memory, predication and construction of higher‐level propositional constituents. The notion of “activation level”, applied to such representational units in working memory, is particularly fruitful in this framework. Besides, a satisfying neural interpretation of this psychological type of model can be proposed. The paper shortly presents a series of experiments, involving a semantic probing technique and three main categories of factors, with results that support the semantic post‐representation view, in addition to others. A critical comparison of this analysis with neurofunctional imagery data confirms the necessity of cross‐cognitive exchanges.
Phosphatidylinositol 3-Kinase Is a Negative Regulator of Cellular Differentiation
Authors: Ptasznik Andrzej , Beattie Gillian M. , Mally Martin I. , Cirulli Vincenzo , Lopez Ana , Hayek Alberto
Published: July 2002

Phosphatidylinositol 3-kinase (PI3K) has been shown to be an important mediator of intracellular signal transduction in mammalian cells. We show here, for the first time, that the blockade of PI3K activity in human fetal undifferentiated cells induced morphological and functional endocrine differentiation. This was associated with an increase in mRNA levels of insulin, glucagon, and somatostatin, as well as an increase in the insulin protein content and secretion in response to secretagogues. Blockade of PI3K also increased the proportion of pluripotent precursor cells coexpressing multiple hormones and the total number of terminally differentiated cells originating from these precursor cells. We examined whether any of the recently described modulators of endocrine differentiation could participate in regulating PI3K activity in fetal islet cells. The activity of PI3K was inversely correlated with the hepatocyte growth factor/scatter factor–induced downregulation or nicotinamideinduced upregulation of islet-specific gene expression, giving support to the role of PI3K, as a negative regulator of endocrine differentiation. In conclusion, our results provide a mechanism for the regulation of hormone-specific gene expression during human fetal neogenesis. They also suggest a novel function for PI3K, as a negative regulator of cellular differentiation.
Disruption of the NF-H Gene Increases Axonal Microtubule Content and Velocity of Neurofilament Transport: Relief of Axonopathy Resulting from the Toxin β,β′-Iminodipropionitrile
Authors: Zhu Qinzhang , Lindenbaum Michael , Levavasseur Françoise , Jacomy Hélène , Julien Jean-Pierre
Published: July 2002

To investigate the role of the neurofilament heavy (NF-H) subunit in neuronal function, we generated mice bearing a targeted disruption of the gene coding for the NF-H subunit. Surprisingly, the lack of NF-H subunits had little effect on axonal calibers and electron microscopy revealed no significant changes in the number and packing density of neurofilaments made up of only the neurofilament light (NF-L) and neurofilament medium (NF-M) subunits. However, our analysis of NF-H knockout mice revealed an ∼2.4-fold increase of microtubule density in their large ventral root axons. This finding was further corroborated by a corresponding increase in the ratio of assembled tubulin to NF-L protein in insoluble cytoskeletal preparations from the sciatic nerve. Axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice. When treated with β,β′-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates. These results indicate that the NF-H subunit is a key mediator of IDPN-induced axonopathy.