bluuebunny/crossref_metadata_2025 huggingface dataset:
Mice deficient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus.
Authors: Lledo P M , Tremblay P , DeArmond S J , Prusiner S B , Nicoll R A
Published: July 2002

We recorded in the CA1 region from hippocampal slices of prion protein (PrP) gene knockout mice to investigate whether the loss of the normal form of prion protein (PrPC) affects neuronal excitability as well as synaptic transmission in the central nervous system. No deficit in synaptic inhibition was found using field potential recordings because (i) responses induced by stimulation in stratum radiatum consisted of a single population spike in PrP gene knockout mice similar to that recorded from control mice and (ii) the plot of field excitatory postsynaptic potential slope versus the population spike amplitude showed no difference between the two groups of mice. Intracellular recordings also failed to detect any difference in cell excitability and the reversal potential for inhibitory postsynaptic potentials. Analysis of the kinetics of inhibitory postsynaptic current revealed no modification. Finally, we examined whether synaptic plasticity was altered and found no difference in long-term potentiation between control and PrP gene knockout mice. On the basis of our findings, we propose that the loss of the normal form of prion protein does not alter the physiology of the CA1 region of the hippocampus.
CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.
Authors: Klinman D M , Yi A K , Beaucage S L , Conover J , Krieg A M
Published: July 2002

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is 20 times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (IL) 6, IL-12, and interferon gamma (but not IL-2, IL-3, IL-4, IL-5, or IL-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.
The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.
Authors: Sadowski C L , Henry R W , Kobayashi R , Hernandez N
Published: July 2002

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.
Two-state models of protein folding kinetics
Authors: Zwanzig Robert
Published: July 2002

The folding of some proteins appears to be a two-state kinetic process. A two-state kinetic model is justified if protein molecules rapidly equilibrate between different unfolded conformations prior to complete folding. Here I show that this rapid equilibration is a natural consequence of reasonable assumptions about reaction rate constants and folding thermodynamics.
Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2
Authors: Eichmann Anne , Corbel Catherine , Nataf Valérie , Vaigot Pierre , Bréant Christiane , Le Douarin Nicole M.
Published: July 2002

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages. However, it was not possible to exclude the hypothesis that hemopoietic failure was a secondary effect resulting from the absence of an endothelial cell microenvironment. To distinguish between these two hypotheses, we have produced a mAb directed against the extracellular domain of avian VEGFR2 and isolated VEGFR2+ cells from the mesoderm of chicken embryos at the gastrulation stage. We have found that in clonal cultures, a VEGFR2+ cell gives rise to either a hemopoietic or an endothelial cell colony. The developmental decision appears to be regulated by the binding of two different VEGFR2 ligands. Thus, endothelial differentiation requires VEGF, whereas hemopoietic differentiation occurs in the absence of VEGF and is significantly reduced by soluble VEGFR2, showing that this process could be mediated by a second, yet unidentified, VEGFR2 ligand. These observations thus suggest strongly that in the absence of the VEGFR2 gene product, the precursors of both hemopoietic and vascular endothelial lineages cannot survive. These cells therefore might be the initial targets of the VEGFR2 null mutation.
Chaperonin filaments: The archaeal cytoskeleton?
Authors: Trent Jonathan D. , Kagawa Hiromi K. , Yaoi Takuro , Olle Eric , Zaluzec Nestor J.
Published: July 2002

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon
Sulfolobus shibatae
the two chaperonin proteins represent ≈4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations ≥ 0.5 mg/ml purified chaperonins form filaments in the presence of Mg
2+
and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist
in vivo
and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained
S. shibatae
cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments
in vivo
suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.
Selective amplification via biotin- and restriction-mediated enrichment (SABRE), a novel selective amplification procedure for detection of differentially expressed mRNAs
Authors: Lavery Daniel J. , Lopez-Molina Luis , Fleury-Olela Fabienne , Schibler Ueli
Published: July 2002

We present a novel subtractive enrichment protocol for the identification of differentially expressed mRNA species. This procedure, called SABRE (selective amplification via biotin- and restriction-mediated enrichment), uses selective streptavidin-biotin affinity and restriction enzyme site reconstitution to enrich for cDNA species more abundant in one population than in another. Analysis of liver cDNA from a mouse strain expressing the neomycin resistance gene demonstrated that this procedure is capable of identifying species present in one population but absent from another. Furthermore, experiments to identify genes with circadian expression patterns in mouse liver demonstrated that SABRE is capable of detecting even modest 2- to 10-fold differences in accumulation of moderately rare mRNA species, representing as little as 0.03% of total mRNA. These experiments identified the gene encoding coumarin 7-hydroxylase as displaying circadian expression in mouse liver.
Distinct roles for E2F proteins in cell growth control and apoptosis
Authors: DeGregori James , Leone Gustavo , Miron Alexander , Jakoi Laszlo , Nevins Joseph R.
Published: July 2002

E2F transcription activity is composed of a family of heterodimers encoded by distinct genes. Through the overproduction of each of the five known E2F proteins in mammalian cells, we demonstrate that a large number of genes encoding proteins important for cell cycle regulation and DNA replication can be activated by the E2F proteins and that there are distinct specificities in the activation of these genes by individual E2F family members. Coexpression of each E2F protein with the DP1 heterodimeric partner does not significantly alter this specificity. We also find that only E2F1 overexpression induces cells to undergo apoptosis, despite the fact that at least two other E2F family members, E2F2 and E2F3, are equally capable of inducing S phase. The ability of E2F1 to induce apoptosis appears to result from the specific induction of an apoptosis-promoting activity rather than the lack of induction of a survival activity, because co-expression of E2F2 and E2F3 does not rescue cells from E2F1-mediated apoptosis. We conclude that E2F family members play distinct roles in cell cycle control and that E2F1 may function as a specific signal for the initiation of an apoptosis pathway that must normally be blocked for a productive proliferation event.
Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis
Authors: Perron H. , Garson J. A. , Bedin F. , Beseme F. , Paranhos-Baccala G. , Komurian-Pradel F. , Mallet F. , Tuke P. W. , Voisset C. , Blond J. L. , Lalande B. , Seigneurin J. M. , Mandrand B. , Sclerosis the Collaborative Research Group on Multiple
Published: July 2002

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein–Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial
pol
fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a
pol
region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.
Nitrergic control of peripheral sympathetic responses in the human corpus cavernosum: A comparison with other species
Authors: Cellek Selim , Moncada Salvador
Published: July 2002

Noradrenergic contractions induced by electrical field stimulation (EFS) of the rabbit anococcygeus muscle and the human and rabbit corpus cavernosum did not occur until termination of stimulation, even when EFS was applied for long periods (10 min). After treatment with a nitric oxide synthase inhibitor, a scavenger of NO, or a specific inhibitor of the soluble guanylate cyclase, EFS-induced contraction began as soon as stimulation commenced and its magnitude and duration were increased. In the presence of a cGMP-phosphodiesterase inhibitor, the lag period between the end of EFS and the onset of contraction was longer, and the response was smaller. Even when the concentration of endogenous noradrenaline was increased with cocaine, the contraction still did not occur during EFS and the lag period was unchanged, although the response was enhanced. When tissue tone was elevated, relaxation occurred during EFS followed by a contraction. After blockade of neuronal noradrenaline release with guanethidine, contractions of the tissues to increasing concentrations of exogenous noradrenaline were significantly reduced by EFS, an effect that was reversible by inhibition of NO synthase. In contrast, in the rat and mouse anococcygeus muscles contraction began immediately with EFS, and nitrergic stimulation by EFS did not affect the responses elicited by high concentrations of exogenous noradrenaline. These results suggest that the human and rabbit genitourinary organs have a powerful nitrergic innervation that does not merely modulate, but actually controls, the sympathetic responses. Our observations may increase understanding of the balance between nitrergic and sympathetic systems in humans, disruption of which may contribute to certain pathological conditions.
Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation
Authors: Rossi Fabio , Charlton Carol A. , Blau Helen M.
Published: July 2002

We present an approach for monitoring protein–protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing β-galactosidase (β-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the β-gal activity resulting from the forced interaction of nonfunctional weakly complementing β-gal peptides (Δα and Δω) serves as a measure of the extent of interaction of the non-β-gal portions of the chimeras. To test this application of
lacZ
intracistronic complementation, proteins that form a complex in the presence of rapamycin were used. These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Δα and Δω, respectively. Enzymatic β-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays. This approach may prove to be a valuable adjunct to
in vitro
immunoprecipitation and crosslinking methods and
in vivo
yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.
RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2
Authors: Li Hui , Gomes Paulo J. , Chen J. Don
Published: July 2002

Steroids, thyroid hormones, vitamin D
3
, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.
Functionality of major histocompatibility complex class II molecules in mice doubly deficient for invariant chain and H-2M complexes
Authors: Tourne Sylvie , Miyazaki Toru , Wolf Paula , Ploegh Hidde , Benoist Christophe , Mathis Diane
Published: July 2002

By combining two previously generated null mutations, Ii° and M°, we produced mice lacking the invariant chain and H-2M complexes, both required for normal cell-surface expression of major histocompatibility complex class II molecules loaded with the usual diverse array of peptides. As expected, the maturation and transport of class II molecules, their expression at the cell surface, and their capacity to present antigens were quite similar for cells from Ii°M° double-mutant mice and from animals carrying just the Ii° mutation. More surprising were certain features of the CD4+T cell repertoire selected in Ii°M° mice: many fewer cells were selected than in Ii+M° animals, and these had been purged of self-reactive specificities, unlike their counterparts in Ii+M° animals. These findings suggest (i) that the peptides carried by class II molecules on stromal cells lacking H-2M complexes may almost all derive from invariant chain and (ii) that H-2M complexes edit the peptide array displayed on thymic stromal cells in the absence of invariant chain, showing that it can edit,in vivo, peptides other than CLIP.
RETRACTED: Lipochitooligosaccharide-induced tobacco cells release a peptide as mediator of the glycolipid signal
Authors: John Michael , Schmidt Jürgen , Walden Richard , Czaja Inge , Dülz Marion , Schell Jeff , Röhrig Horst
Published: July 2002

Lipochitooligosaccharides (LCOs) are plant growth regulators that promote at subfemtomolar concentrations cell division in tobacco protoplasts. In response to LCO treatment, tobacco cells release a second growth factor that fully mediates the growth-promoting activities of the initial extracellular LCO stimulus. This diffusible growth factor was isolated from the protoplasts’ culture filtrate and shown to be a peptide. We report that the LCO-induced mitogen released by tobacco cells and a synthetic heptadecapeptide derived from region 2 of the tobacco homolog of the early nodulin gene
ENOD40
are antigenically related and qualitatively indistinguishable in their ability to stimulate cell division.
Functional consequences of NR2 subunit composition in single recombinant
N
-methyl-
d
-aspartate receptors
Authors: Brimecombe Jessica C. , Boeckman Faye A. , Aizenman Elias
Published: July 2002

Single-channel recordings were obtained from Chinese hamster ovary cells transfected with the
N
-methyl-
d
-aspartate (NMDA) receptor subunit NR1 in combination with NR2A, NR2B, NR2C, or NR2A/NR2B. NMDA-activated currents were recorded under control conditions and in the presence of a thiol reductant (DTT), an oxidant (5,5′-dithio-
bis
[2-nitrobenzoic acid], DTNB), or the noncompetitive antagonist CP101,606 (CP). For all subunit combinations, DTT increased the frequency of channel opening when compared with DTNB. In addition, channels obtained from NR1/NR2A-transfected cells also exhibited a pronounced difference in mean open dwell-time between redox conditions. CP dramatically reduced both the open dwell-time and frequency of channel opening of NR1/NR2B-containing receptors, but only modestly inhibited NR1/NR2A and NR1/NR2C channel activity. A small number of patches obtained from cells transfected with NR1/NR2A/NR2B had channels with properties intermediate to NR1/NR2A and NR1/NR2B receptors, including insensitivity to CP block but redox properties similar to NR1/NR2B, consistent with the coassembly of NR2A with NR2B. Hence, NMDA receptors containing multiple types of NR2 subunits can have functionally distinguishable attributes.
A functionally defined model for the M
2
proton channel of influenza A virus suggests a mechanism for its ion selectivity
Authors: Pinto Lawrence H. , Dieckmann Gregg R. , Gandhi Chris S. , Papworth Carol G. , Braman Jeffrey , Shaughnessy Margaret A. , Lear James D. , Lamb Robert A. , DeGrado William F.
Published: July 2002

The M
2
protein from influenza A virus forms proton-selective channels that are essential to viral function and are the target of the drug amantadine. Cys scanning was used to generate a series of mutants with successive substitutions in the transmembrane segment of the protein, and the mutants were expressed in
Xenopus laevis
oocytes. The effect of the mutations on reversal potential, ion currents, and amantadine resistance were measured. Fourier analysis revealed a periodicity consistent with a four-stranded coiled coil or helical bundle. A three-dimensional model of this structure suggests a possible mechanism for the proton selectivity of the M
2
channel of influenza virus.
Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: A novel mechanism of growth hormone receptor regulation
Authors: Leung Kin-Chuen , Waters Michael J. , Markus Irit , Baumbach William R. , Ho Ken K. Y.
Published: July 2002

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 ± 2.9% and 23.0 ± 2.7% of control (mean ± SE;
P
< 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (
P
< 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (
P
≤ 0.0005), whereas GHR translocation was completely abolished by 75–90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.
Myc represses transcription of the growth arrest gene
gas1
Authors: Lee Theodore C. , Li Linheng , Philipson Lennart , Ziff Edward B.
Published: July 2002

Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the
ODC
and
cdc25A
genes. We show that c-Myc transcriptionally represses the expression of a growth arrest gene,
gas1
. A conserved Myc structure, Myc box 2, is required for repression of
gas1
, and for Myc induction of proliferation and transformation, but not for activation of
ODC
. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen was sufficient to repress
gas1
gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including
gas1
, is one step in promotion of cell growth by Myc.
The N terminus of the
Drosophila
Numb protein directs membrane association and actin-dependent asymmetric localization
Authors: Knoblich Juergen A. , Jan Lily Y. , Jan Yuh Nung
Published: July 2002

Drosophila
Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in
Drosophila
embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41–118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to β-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and β-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.
Structure of the recombinant full-length hamster prion protein PrP(29–231): The N terminus is highly flexible
Authors: Donne David G. , Viles John H. , Groth Darlene , Mehlhorn Ingrid , James Thomas L. , Cohen Fred E. , Prusiner Stanley B. , Wright Peter E. , Dyson H. Jane
Published: July 2002

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrP
C
to the infectious scrapie form PrP
Sc
; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29–231 [PrP(29–231)] is investigated by multidimensional heteronuclear NMR. Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29–231) contains three helices and possibly one short β-strand. Most striking is the random-coil nature of chemical shifts for residues 30–124 in the full-length PrP. Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121–231), the secondary structure boundaries of PrP(29–231) are different from those in mouse PrP(121–231) but similar to those found in the structure of Syrian hamster PrP(90–231). Comparison of resonance assignments of PrP(29–231) and PrP(90–231) indicates that there may be transient interactions between the additional residues and the structured core. Backbone dynamics studies done by using the heteronuclear [
1
H]-
15
N nuclear Overhauser effect indicate that almost half of PrP(29–231), residues 29–124, is highly flexible. This plastic region could feature in the conversion of PrP
C
to PrP
Sc
by template-assisted formation of β-structure.
Chlorophyll precursors are signals of chloroplast origin involved in light induction of nuclear heat-shock genes
Authors: Kropat Janette , Oster Ulrike , Rüdiger Wolfhart , Beck Christoph F.
Published: July 2002

Coordination between the activities of organelles and the nucleus requires the exchange of signals. Using
Chlamydomonas
, we provide evidence that plastid-derived chlorophyll precursors may replace light in the induction of two nuclear heat-shock genes (
HSP70A
and
HSP70B
) and thus qualify as plastidic signal. Mutants defective in the synthesis of Mg-protoporphyrin IX were no longer inducible by light. Feeding of Mg-protoporphyrin IX or its dimethyl ester to wild-type or mutant cells in the dark resulted in induction. The analysis of
HSP70A
promoter mutants that do or do not respond to light revealed that these chlorophyll precursors specifically activate the light signaling pathway. Activation of gene expression was not observed when protoporphyrin IX, protochlorophyllide, or chlorophyllide were added. A specific interaction of defined chlorophyll precursors with factor(s) that regulate nuclear gene expression is suggested.
Virtual combinatorial libraries: Dynamic generation of molecular and supramolecular diversity by self-assembly
Authors: Huc Ivan , Lehn Jean-Marie
Published: July 2002

Molecular and supramolecular diversity may be generated, respectively, by reversible, covalent or noncovalent self-assembly of basic components whose various potential combinations in number and nature represent a virtual combinatorial library. This concept is applied to the induction of inhibitors of carbonic anhydrase (CA) by reversible recombination of aldehyde and amine components. It is found that the presence of CA favors the formation of those condensation compounds that may be expected to present the strongest binding to the CA active site. The virtual combinatorial library approach may represent a powerful methodology for the discovery of substrates, inhibitors, receptors, catalysts, and carriers for a variety of processes.
The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins
Authors: Pause Arnim , Lee Stephen , Worrell Robert A. , Chen David Y. T. , Burgess Wilson H. , Linehan W. Marston , Klausner Richard D.
Published: July 2002

The inactivation of the von Hippel-Lindau (VHL) gene predisposes affected individuals to VHL syndrome and is an early genetic event associated with sporadic renal cell carcinoma and CNS hemangioblastomas. The VHL protein (pVHL) has been shown to form a stable complex with elongin B and elongin C, two factors that stabilize and activate the transcription elongation factor elongin A. Here, Hs-CUL-2, a member of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-elongin B-C (VBC) complex
in vitro
and
in vivo
. Nearly 70% of naturally occurring cancer-predisposing mutations of VHL disrupt this interaction. The pVHL-Hs-CUL-2 association is strictly dependent on the integrity of the trimeric VBC complex. Immunofluorescence studies show Hs-CUL-2 to be a cytosolic protein that can be translocated to the nucleus by pVHL. Recently it has been shown that a yeast Hs-CUL-2 homolog, Cdc53, is part of a ubiquitin protein ligase complex that targets cell cycle proteins for degradation by the ubiquitin proteolytic pathway. In
Caenorhabditis elegans
, a null mutation of another Hs-cul-2 homolog, Ce-cul-1, results in hyperplasia in all tissues and is required for cell cycle exit. Hence, Hs-cul-2 may be required for VHL function and, therefore, may be a candidate human tumor-suppressor gene.
A mutation in autosomal dominant myotonia congenita affects pore properties of the muscle chloride channel
Authors: Fahlke Christoph , Beck Carol L. , George Alfred L.
Published: July 2002

Autosomal dominant myotonia congenita is an inherited disorder of skeletal muscle caused by mutations in a voltage-gated Cl
−
channel gene (
CLCN1
, 7q35). Here, we report that a mutation predicting the substitution of Gly 230 by glutamic acid (G230E) between segments D3 and D4 dramatically alters the pore properties of a recombinant human muscle Cl
−
channel (hClC-1) expressed in a mammalian cell line (tsA201). The G230E mutation causes substantial changes in anion and cation selectivity as well as a fundamental change in rectification of the current–voltage relationship. Whereas wild-type channels are characterized by pronounced inward rectification and a Cl > thiocyanate > Br > NO
3
> I > CH
3
SO
3
selectivity, G230E exhibits outward rectification at positive potentials and a thiocyanate > NO
3
> I > Br > Cl > CH
3
SO
3
selectivity. Furthermore, the cation-to-anion permeability ratio of the mutant is much greater than that of the wild-type channel. Voltage-dependent blocks by intracellular and extracellular iodide help to distinguish two distinct ion binding sites within the hClC-1 conduction pathway. Both binding sites are preserved in the mutant but have decreased affinities for iodide. These findings suggest that Gly 230 is critical for normal ion conductance in hClC-1 and that this residue resides within the channel pore.
Nucleolin is one component of the B cell-specific transcription factor and switch region binding protein, LR1
Authors: Hanakahi L. A. , Dempsey Laurie A. , Li Ming-Jie , Maizels Nancy
Published: July 2002

LR1 is a B cell-specific, sequence-specific DNA binding activity that regulates transcription in activated B cells. LR1 also binds Ig heavy chain switch region sequences and may function in class switch recombination. LR1 contains two polypeptides, of 106 kDa and 45 kDa, and here we report that the 106-kDa component of LR1 is nucleolin. This identification, initially made by microsequence analysis, was verified by showing that (i) LR1–DNA binding activity increased in B cells transfected with a nucleolin cDNA expression construct; (ii) LR1–DNA binding activity was recognized by antibodies raised against recombinant human nucleolin; and (iii) in B cells transfected with epitope-tagged nucleolin expression constructs, the LR1–DNA complex was recognized by the anti-tag antibody. Nucleolin is an abundant nucleolar protein which is believed to play a role in rDNA transcription or organization, or rRNA processing. Homology between nucleolin and histone H1 suggests that nucleolin may alter DNA organization in response to cell cycle controls, and the nucleolin component of LR1 may therefore function to organize switch regions before, during, or after switch recombination. The demonstration that nucleolin is a component of a B cell-specific complex that binds switch region sequences suggests that the G-rich switch regions may have evolved from rDNA.
Cytosol-to-membrane redistribution of Bax and Bcl-XLduring apoptosis
Authors: Hsu Yi-Te , Wolter Keith G. , Youle Richard J.
Published: July 2002

Bcl-2, Bcl-XL, and Bax are members of the Bcl-2 family that play key roles in the regulation of apoptosis. These proteins are believed to be membrane bound and their ability to undergo both homodimerization and heterodimerization has been proposed to regulate apoptosis. Herein we report that in murine thymocytes, Bcl-2 is exclusively membrane-bound, whereas Bax is present predominantly in the cytosol and Bcl-XLis present in both soluble and membrane-bound forms. Induction of apoptosis in murine thymocytes by dexamethasone or γ-irradiation shifts the subcellular locations of Bax and Bcl-XLfrom soluble to membrane-bound forms. A similar shift in the localization of Bax from the cytosol to membranes was observed in HL-60 leukemia cells upon induction of apoptosis by staurosporine. Inhibition of apoptosis with cycloheximide inhibits the movement of Bax and Bcl-XLin thymocytes from the cytosol into membranes induced by dexamethasone treatment. These movements may represent an important step in the pathway by which members of this family regulate apoptosis.
Evolution of antifreeze glycoprotein gene from a trypsinogen gene in Antarctic notothenioid fish
Authors: Chen Liangbiao , DeVries Arthur L. , Cheng Chi-Hing C.
Published: July 2002

Freezing avoidance conferred by different types of antifreeze proteins in various polar and subpolar fishes represents a remarkable example of cold adaptation, but how these unique proteins arose is unknown. We have found that the antifreeze glycoproteins (AFGPs) of the predominant Antarctic fish taxon, the notothenioids, evolved from a pancreatic trypsinogen. We have determined the likely evolutionary process by which this occurred through characterization and analyses of notothenioid AFGP and trypsinogen genes. The primordial AFGP gene apparently arose through recruitment of the 5′ and 3′ ends of an ancestral trypsinogen gene, which provided the secretory signal and the 3′ untranslated region, respectively, plusde novoamplification of a 9-nt Thr-Ala-Ala coding element from the trypsinogen progenitor to create a new protein coding region for the repetitive tripeptide backbone of the antifreeze protein. The small sequence divergence (4–7%) between notothenioid AFGP and trypsinogen genes indicates that the transformation of the proteinase gene into the novel ice-binding protein gene occurred quite recently, about 5–14 million years ago (mya), which is highly consistent with the estimated times of the freezing of the Antarctic Ocean at 10–14 mya, and of the main phyletic divergence of the AFGP-bearing notothenioid families at 7–15 mya. The notothenioid trypsinogen to AFGP conversion is the first clear example of how an old protein gene spawned a new gene for an entirely new protein with a new function. It also represents a rare instance in which protein evolution, organismal adaptation, and environmental conditions can be linked directly.
Polymorphism of murine Fas ligand that affects the biological activity
Authors: Kayagaki Nobuhiko , Yamaguchi Noriko , Nagao Fumiko , Matsuo Seishi , Maeda Hiroaki , Okumura Ko , Yagita Hideo
Published: July 2002

Fas ligand (FasL) is a member of the tumor necrosis factor family and induces apoptosis in Fas (CD95)-bearing target cells. In this study, we generated several mAbs that react with mouse FasL (mFasL) and characterized their functional properties. One of these mAbs, K10, specifically reacted with mFasL derived from C57BL/6 (B6) mice, but not that from BALB/c mice as estimated by surface staining and blocking of cytotoxic activities of mFasL transfectants, suggesting a polymorphism of mFasL. Sequence analysis of mFasL cDNA from several strains revealed that BALB/c and DBA/2 mice have three nucleotide differences from the known B6 and C3H sequences, which result in two amino acid substitutions (Thr-184 → Ala-184 and Glu-218 → Gly-218) in the extracellular region. Analysis of the K10 reactivity and genotyping by PCR-restriction fragment length polymorphism revealed that inbred mice segregate into the following two allotypes: mFasL.1 (B6, C3H, MRL, SJL, NOD, NZB, NZW) and mFasL.2 (BALB/c, DBA/1, DBA/2). Interestingly, COS7 cells expressing BALB/c FasL lysed Fas-bearing target cells more efficiently than those expressing B6 FasL. Furthermore, BALB/c-derived CD8-FasL fusion protein, which is composed of the extracellular domains of human CD8α and mFasL, exhibited 9-fold higher specific activity than did B6-derived CD8-FasL. These results suggest that in mFasL.2 mice the Fas/FasL system works more effectively than in mFasL.1 mice.
Motor-driven gene expression
Authors: Jarvis Erich D. , Nottebohm Fernando
Published: July 2002

There is increased neuronal firing in the high vocal center (a motor nucleus) and other song nuclei of canaries,
Serinus canaria
, and zebra finches,
Taeniopygia guttata
, whenever these songbirds sing or hear song. These observations suggested that song perception involved sensory and motor pathways. We now show that the act of singing, but not hearing song, induces a rapid and striking increase (up to 60-fold) in expression of the transcriptional regulator ZENK in the high vocal center and other song nuclei. This motor-driven gene expression is independent of auditory feedback, since it occurs in deafened birds when they sing and in muted birds when they produce silent song. Conversely, hearing song, but not the act of singing, induces ZENK expression in parts of the auditory forebrain. Our observations show that even though the same auditory stimulus activates sensory and motor pathways, perception and production of song are accompanied by anatomically distinct patterns of gene expression.
Modulation of murine systemic lupus erythematosus with peptides based on complementarity determining regions of a pathogenic anti-DNA monoclonal antibody
Authors: Waisman Ari , Ruiz Pedro J. , Israeli Eran , Eilat Eran , Könen-Waisman Stephanie , Zinger Heidy , Dayan Molly , Mozes Edna
Published: July 2002

Experimental systemic lupus erythematosus (SLE) can be induced in naive mice by immunization with a murine monoclonal anti-DNA antibody (mAb), 5G12, that bears a major idiotype designated 16/6 Id. Strain-dependent differences were observed in the proliferative responses of lymph node cells of mice immunized with two peptides based on the sequences of the complementarity determining region (CDR) 1 and 3 of mAb 5G12. The capacity of the peptides to bind to major histocompatibility complex class II molecules correlated with the proliferative responses. Immunization of high responder strains with the CDR-based peptides led to production of autoantibodies and clinical manifestations characteristic to experimental SLE. The CDR-based peptides could prevent autoantibody production in neonatal mice that were immunized later either with the peptide or with the pathogenic autoantibody. Furthermore, the peptides inhibited specific proliferation of lymph node cells of mice immunized with the same peptide, with mAb 5G12 or with the human mAb anti-DNA, 16/6 Id. Thus, the CDR-based peptides are potential candidates for therapy of SLE.
The antibipolar drug valproate mimics lithium in stimulating glutamate release and inositol 1,4,5-trisphosphate accumulation in brain cortex slices but not accumulation of inositol monophosphates and bisphosphates
Authors: Dixon John F. , Hokin Lowell E.
Published: July 2002

Valproic acid and lithium are effective antibipolar drugs. We recently showed that lithium stimulated the release of glutamate in monkey and mouse cerebral cortex slices, which, through activation of the
N
-methyl-
d
-aspartate receptor, increased accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P
3
]. We show here that valproate behaves similarly to lithium in that at therapeutic concentrations it stimulates glutamate release and Ins(1,4,5)P
3
accumulation in mouse cerebral cortex slices. The fact that these two effects are a common denominator for two structurally unrelated antibipolar drugs suggests that these effects are important in their antibipolar action. The effects of maximal concentrations of lithium and valproate on glutamate release are additive, suggesting different mechanisms for release, which are discussed. The additivity of the two drugs on glutamate release is consistent with the clinical benefit of combining the two drugs in the treatment of subsets of bipolar patients, e.g., in rapid cycling manic-depression. Unlike lithium, valproate does not increase accumulation of inositol monophosphates, inositol bisphosphates, or inositol 1,3,4-trisphosphate. This is additional evidence against the “inositol depletion” hypothesis, which states that, by trapping inositol in the form of inositol monophosphates and certain inositol polyphosphates, lithium exerts its antimanic action by inhibiting resynthesis of phosphoinositides with resultant blunting of Ins(1,4,5)P
3
signaling.
Second gene for gonadotropin-releasing hormone in humans
Authors: White Richard B. , Eisen Jonathan A. , Kasten Thomas L. , Fernald Russell D.
Published: July 2002

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. In addition to hypothalamic GnRH (GnRH-I), a second GnRH form (pGln-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly; GnRH-II) with unknown function has been localized to the midbrain of many vertebrates. We show here that a gene encoding GnRH-II is expressed in humans and is located on chromosome 20p13, distinct from the GnRH-I gene that is on 8p21-p11.2. The GnRH-II genomic and mRNA structures parallel those of GnRH-I. However, in contrast to GnRH-I, GnRH-II is expressed at significantly higher levels outside the brain (up to 30×), particularly in the kidney, bone marrow, and prostate. The widespread expression of GnRH-II suggests it may have multiple functions. Molecular phylogenetic analysis shows that this second gene is likely the result of a duplication before the appearance of vertebrates, and predicts the existence of a third GnRH form in humans and other vertebrates.
Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules
Authors: Avrameas Alexandre , Ternynck Thérèse , Nato Faridabano , Buttin Gérard , Avrameas Stratis
Published: July 2002

Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB × NZW)F1mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab′)2 and Fab′ fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes.
A bacterial two-hybrid system based on a reconstituted signal transduction pathway
Authors: Karimova Gouzel , Pidoux Josette , Ullmann Agnes , Ladant Daniel
Published: July 2002

We describe a bacterial two-hybrid system that allows an easy
in vivo
screening and selection of functional interactions between two proteins. This genetic test is based on the reconstitution, in an
Escherichia coli cya
strain, of a signal transduction pathway that takes advantage of the positive control exerted by cAMP. Two putative interacting proteins are genetically fused to two complementary fragments, T25 and T18, that constitute the catalytic domain of
Bordetella pertussis
adenylate cyclase. Association of the two-hybrid proteins results in functional complementation between T25 and T18 fragments and leads to cAMP synthesis. Cyclic AMP then triggers transcriptional activation of catabolic operons, such as lactose or maltose, that yield a characteristic phenotype. In this genetic test, the involvement of a signaling cascade offers the unique property that association between the hybrid proteins can be spatially separated from the transcriptional activation readout. This permits a versatile design of screening procedures either for ligands that bind to a given “bait,” as in the classical yeast two-hybrid system, or for molecules or mutations that block a given interaction between two proteins of interest.