bluuebunny/crossref_metadata_2025 huggingface dataset:
The structure and precision of retinal spike trains
Authors: Berry Michael J. , Warland David K. , Meister Markus
Published: July 2002

Assessing the reliability of neuronal spike trains is fundamental to an understanding of the neural code. We measured the reproducibility of retinal responses to repeated visual stimuli. In both tiger salamander and rabbit, the retinal ganglion cells responded to random flicker with discrete, brief periods of firing. For any given cell, these firing events covered only a small fraction of the total stimulus time, often less than 5%. Firing events were very reproducible from trial to trial: the timing jitter of individual spikes was as low as 1 msec, and the standard deviation in spike count was often less than 0.5 spikes. Comparing the precision of spike timing to that of the spike count showed that the timing of a firing event conveyed several times more visual information than its spike count. This sparseness and precision were general characteristics of ganglion cell responses, maintained over the broad ensemble of stimulus waveforms produced by random flicker, and over a range of contrasts. Thus, the responses of retinal ganglion cells are not properly described by a firing probability that varies continuously with the stimulus. Instead, these neurons elicit discrete firing events that may be the fundamental coding symbols in retinal spike trains.
Hematopoiesis in the fetal liver is impaired by targeted mutagenesis of a gene encoding a non-DNA binding subunit of the transcription factor, polyomavirus enhancer binding protein 2/core binding factor
Authors: Niki Masaru , Okada Hitoshi , Takano Hiroshi , Kuno Junko , Tani Kenzaburo , Hibino Hitoshi , Asano Shigetaka , Ito Yoshiaki , Satake Masanobu , Noda Tetsuo
Published: July 2002

The
Pebpb2
gene encodes a non-DNA binding subunit of the heterodimeric transcription factor, polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF), and is rearranged in inversion of chromosome 16 associated with human acute myeloid leukemia. To investigate its physiological function,
Pebpb2
was mutated by a targeting strategy to generate a null mutant. The homozygous mutation in mice proved lethal in embryos around embryonic day 12.5, apparently due to massive hemorrhaging in the central nervous system. In addition, definitive hematopoiesis in the liver was severely impaired. The observed phenotype was indistinguishable from that reported for homozygous disruption of
AML1
, which encodes a DNA binding subunit of PEBP2/CBF. Thus, the results indicate that the two subunits function together as a heterodimeric PEBP2/CBF
in vivo
and that PEBP2/CBF plays an essential role in the development of definitive hematopoiesis.
Gene expression from plasmids containing the
araBAD
promoter at subsaturating inducer concentrations represents mixed populations
Authors: Siegele Deborah A. , Hu James C.
Published: July 2002

Gene expression from plasmids containing the
araBAD
promoter can be regulated by the concentration of arabinose in the growth medium. Guzman
et al.
[Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995)
J. Bacteriol.
177, 4121–4130] showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concentrations of arabinose. We constructed plasmids expressing a fast-folding mutant
Aequorea victoria
green fluorescent protein from the
araBAD
promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels. Microscopic examination of cells grown at low arabinose concentrations shows mixtures of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population average of induced and uninduced cells. The kinetics of green fluorescent protein induction suggest that this reflects an “autocatalytic” induction mechanism due to accumulation of the inducer by active transport. This mechanism, which is analogous to the induction of the
lac
operon at subsaturating inducer concentrations in
lacY
+
cells, was described 40 years ago by Novick and Weiner [Novick, A. & Weiner, M. (1957)
Proc. Natl. Acad. Sci. USA
43, 553–566].
Linkage of a neurophysiological deficit in schizophrenia to a chromosome 15 locus
Authors: Freedman Robert , Coon Hilary , Myles-Worsley Marina , Orr-Urtreger Avi , Olincy Ann , Davis Ashley , Polymeropoulos Mihael , Holik John , Hopkins Jan , Hoff Mark , Rosenthal Judy , Waldo Merilyne C. , Reimherr Fred , Wender Paul , Yaw Jeffrey , Young David A. , Breese Charles R. , Adams Catherine , Patterson David , Adler Lawrence E. , Kruglyak Leonid , Leonard Sherry , Byerley William
Published: July 2002

Inheritance of a defect in a neuronal mechanism that regulates response to auditory stimuli was studied in nine families with multiple cases of schizophrenia. The defect, a decrease in the normal inhibition of the P50 auditory-evoked response to the second of paired stimuli, is associated with attentional disturbances in schizophrenia. Decreased P50 inhibition occurs not only in most schizophrenics, but also in many of their nonschizophrenic relatives, in a distribution consistent with inherited vulnerability for the illness. Neurobiological investigations in both humans and animal models indicated that decreased function of the α7-nicotinic cholinergic receptor could underlie the physiological defect. In the present study, a genome-wide linkage analysis, assuming autosomal dominant transmission, showed that the defect is linked [maximum logarithm of the odds (lod) score = 5.3 with zero recombination] to a dinucleotide polymorphism at chromosome 15q13-14, the site of the α7-nicotinic receptor. Despite many schizophrenics’ extremely heavy nicotine use, nicotinic receptors were not previously thought to be involved in schizophrenia. The linkage data thus provide unique new evidence that the α7-nicotinic receptor gene may be responsible for the inheritance of a pathophysiological aspect of the illness.
Archaeal nucleosomes
Authors: Pereira Suzette L. , Grayling Rowan A. , Lurz Rudi , Reeve John N.
Published: July 2002

Archaeacontain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNAin vivointo structures similar to the structure formed by the histone (H3+H4)2tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linkingin vivo, these archaeal nucleosomes have been isolated fromMethanobacterium thermoautotrophicumandMethanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect ≈60 bp of DNA and multiples of ≈60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all,M. fervidusgenomic DNA sequences are associatedin vivowith archaeal histones.
Preferential activation of the p46 isoform of JNK/SAPK in mouse macrophages by TNFα
Authors: Chan Edward D. , Winston Brent W. , Jarpe Matthew B. , Wynes Murry W. , Riches David W. H.
Published: July 2002

A pleiotropic cytokine, tumor necrosis factor-α (TNFα), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. In this study, we have investigated the specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages in response to stimulation with TNFα. Exposure of macrophages to TNFα stimulated a preferential increase in catalytic activity of the p46 JNK/SAPK isoform compared with the p54 JNK/SAPK isoform as determined by: (i) separation of p46 and p54 JNK/SAPKs by anion exchange liquid chromatography and (ii) selective immunodepletion of the p46 JNK/SAPK from macrophage lysates. To investigate the level of regulation of p46 JNK/SAPK activation, we determined the ability of MKK4/SEK1/JNKK, an upstream regulator of JNK/SAPKs, to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 was able to transphosphorylate both isoforms. In addition, both the p46 and p54 JNK/SAPK isoforms were phosphorylated on their TPY motif in response to TNFα stimulation as reflected by immunoblotting with a phospho-specific antibody that recognizes both kinases. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNFα regulates macrophage phenotypic heterogeneity and differentiation.
Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway
Authors: Bond Chris J. , Wong Kam-Bo , Clarke Jane , Fersht Alan R. , Daggett Valerie
Published: July 2002

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 10
4
structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix α1 (residues 13–17) and in helix α2 as well as a turn and nonnative hydrophobic clustering between β3 and β4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in α1 and the C-terminal portion of the β-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from φ value analysis and NMR.
Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome
Authors: Messerle Martin , Crnkovic Irena , Hammerschmidt Wolfgang , Ziegler Heike , Koszinowski Ulrich H.
Published: July 2002

A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in
E. coli
. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.
Severe spatial navigation deficit in the Morris water maze after single high dose of neonatal x-ray irradiation in the rat
Authors: Czurkó András , Czéh Boldizsár , Seress László , Nadel Lynn , Bures Jan
Published: July 2002

Ambiguous spatial behavior deficits induced in adult rats by different types of dentate gyrus lesions were examined by subjecting neonatal rats to x-ray irradiation, which reduces the granule cell population in fascia dentata without affecting the number of hilar neurons and pyramidal cells of Ammon’s horn. Three- to six-month-old irradiated and intact male Long–Evans rats were tested in the Morris water maze. Four experiments were done. (
i
) Rats were trained to find an invisible escape platform, when started from any of four equidistant points at the circumference of the pool. (
ii
) The same rats then were trained to find a visible platform in the same pool. Poor performance of irradiated rats in both experiments suggested a visual deficit. (
iii
) Navigation in the absence of visual cues was studied in other rats trained in total darkness to find the escape platform under conditions of fixed start–fixed goal geometry. (
iv
) Contribution of nonvisual allocentric cues and egocentric path integration mechanisms to spatial performance of the above rats was tested in darkness after rotating both the start and goal positions by 90° clockwise. Impairment of irradiated rats in Exp. 3 and 4 and histological examination of their brains support the conclusion that 60–70% reduction of granule cells in the dorsal hippocampus causes significant deterioration in both allocentric and egocentric orientation.
The structure of a CAP–DNA complex having two cAMP molecules bound to each monomer
Authors: Passner J. M. , Steitz T. A.
Published: July 2002

The 2.2 Å resolution crystal structure of the
Escherichia coli
catabolite gene activator protein (CAP) complexed with cAMP and a 46-bp DNA fragment reveals a second cAMP molecule bound to each protein monomer. The second cAMP is in the
syn
conformation and is located on the DNA binding domain interacting with the helix-turn-helix, a β-hairpin from the regulatory domain and the DNA (via water molecules). The presence of this second cAMP site resolves the apparent discrepancy between the NMR and x-ray data on the conformation of cAMP, and explains the cAMP concentration-dependent behaviors of the protein. In addition, this site’s close proximity to mutations affecting transcriptional activation and its water-mediated interactions with a DNA recognition residue (E181) and DNA raise the possibility that this site has biological relevance.
Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B
Authors: Zhao Allan Z. , Zhao Hong , Teague Jeanette , Fujimoto Wilfred , Beavo Joseph A.
Published: July 2002

Both insulin and insulin-like growth factor 1 (IGF-1) are known to reduce glucose-dependent insulin secretion from the β cells of pancreatic islets. In this paper we show that the mechanism by which IGF-1 mediates this effect is in large part through activation of a specific cyclic nucleotide phosphodiesterase, phosphodiesterase 3B (PDE3B). More specifically, in both isolated pancreatic islets and insulin-secreting HIT-T15 cells, IGF-1 inhibits insulin secretion that has been increased by glucose and glucagonlike peptide 1 (GLP-1). Moreover, IGF-1 decreases cAMP levels in parallel to the reduction of insulin secretion. Insulin secretion stimulated by cAMP analogs that activate protein kinase A and also are substrates for PDE3B is also inhibited by IGF-1. However, IGF-1 does not inhibit insulin secretion stimulated by nonhydrolyzable cAMP analogs. In addition, selective inhibitors of PDE3B completely block the ability of IGF-1 to inhibit insulin secretion. Finally, PDE3B activity measured
in vitro
after immunoprecipitation from cells treated with IGF-1 is higher than the activity from control cells. Taken together with the fact that pancreatic β cells express little or no insulin receptor but large amounts of IGF-1 receptor, these data strongly suggest a new regulatory feedback loop model for the control of insulin secretion. In this model, increased insulin secretion
in vivo
will stimulate IGF-1 synthesis by the liver, and the secreted IGF-1 in turn feedback inhibits insulin secretion from the β cells through an IGF-1 receptor-mediated pathway. This pathway is likely to be particularly important when levels of both glucose and secretagogues such as GLP-1 are elevated.
Generation of Alzheimer β-amyloid protein in the trans-Golgi network in the apparent absence of vesicle formation
Authors: Xu Huaxi , Sweeney David , Wang Rong , Thinakaran Gopal , Lo Amy C. Y. , Sisodia Sangram S. , Greengard Paul , Gandy Sam
Published: July 2002

β-amyloid protein (Aβ) formation was reconstituted in permeabilized neuroblastoma cells expressing human Alzheimer β-amyloid precursor protein (βAPP) harboring the Swedish double mutation associated with familial early-onset Alzheimer disease. Permeabilized cells were prepared following metabolic labeling and incubation at 20°C, a temperature that allows βAPP to accumulate in the trans-Golgi network (TGN) without concomitant Aβ formation. Subsequent incubation at 37°C led to the generation of Aβ. Aβ production in the TGN persisted even under conditions in which formation of nascent post-TGN vesicles was inhibited by addition of guanosine 5′-
O
-(3-thiotriphosphate), a nonhydrolyzable GTP analogue, or by omission of cytosol. These and other results indicate that vesicle budding and trafficking may not be required for proteolytic metabolism of βAPP to Aβ, a process that includes “γ-secretase” cleavage within the βAPP transmembrane domain.
Intracellular and secreted Alzheimer β-amyloid species are generated by distinct mechanisms in cultured hippocampal neurons
Authors: Tienari Pentti J. , Ida Nobuo , Ikonen Elina , Simons Mikael , Weidemann Andreas , Multhaup Gerd , Masters Colin L. , Dotti Carlos G. , Beyreuther Konrad
Published: July 2002

Cerebral plaques containing β-amyloid (βA4) represent an invariant pathological feature of Alzheimer disease (AD). βA4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In nonneuronal cells βA4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of βA4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of βA4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular βA4. We also show that a common feature of two types of APP mutations (“Swedish” and “London”) implicated in early-onset AD is their increased production of C-terminally elongated βA4 (β
42
), both intra- and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular βA4 and also the main victims in AD, these findings may provide an important link between βA4 and neurodegeneration.
Sarcoplasmic reticulum Ca
2+
release flux underlying Ca
2+
sparks in cardiac muscle
Authors: Blatter Lothar A. , Hüser Jörg , Ríos Eduardo
Published: July 2002

Discrete events of Ca
2+
release from the sarcoplasmic
reticulum (SR) have been described in cardiac, skeletal, and smooth
muscle. In skeletal muscle these release events originate at individual
channels. In cardiac muscle, however, it remains a question of debate
whether localized Ca
2+
release transients, termed
Ca
2+
sparks, originate from single release channels or
multiple channels clustered in close vicinity. Generalizing methods
used earlier to describe cell-averaged Ca
2+
release, we
derived, as a function of space and time, the flux of Ca
2+
release that underlies Ca
2+
sparks. Using the method to
analyze spontaneous sparks recorded with confocal microscopy in
dissociated cat atrial cells, we obtained in most cases single sparks
of Ca
2+
release that appear to originate from approximately
1-μm-wide regions. In many cases, doublets, triplets, and greater
groups of release sparks were observed. This multiplicity, the
estimated release flux magnitude, and existing data on the structure of
junctions between SR and plasmalemma suggest that individual release
sparks result from the opening of multiple Ca
2+
release
channels clustered within discrete SR junctional regions.
Crystal structure of the channel-forming polypeptide antiamoebin in a membrane-mimetic environment
Authors: Karle Isabella L. , Perozzo Mary Ann , Mishra Vinod K. , Balaram Padmanabhan
Published: July 2002

Crystals of an ion-channel-forming peptaibol peptide in a partial membrane environment have been obtained by cocrystallizing antiamoebin with
n
-octanol. The antiamoebin molecule has a bent helical conformation very similar to that established for Leu-zervamicin, despite a significantly different sequence for residues 1–8. The bent helices assemble to form a polar channel in the shape of an hour glass that is quite comparable to that of Leu-zervamicin. The molecules of cocrystallized octanol are found in two different areas with respect to the assembly of peptide molecules. One octanol molecule mimics a membrane segment along the hydrophobic exterior of the channel assembly. The other octanol molecules fill the channel in such a way that their OH termini satisfy the C=O moieties directed into the interior of the channel. Structure parameters for C
82
H
27
N
17
O
20
⋅3C
8
H
18
O are space group
P
2
1
2
1
2
1
,
a
= 9.143(2) Å,
b
= 28.590(8) Å,
c
= 44.289(8) Å,
Z
= 4, agreement factor
R
1
= 11.95% for 4,113 observed reflections [>4σ(
F
)], resolution ∼1.0 Å.
In vivo
detection and imaging of phosphatidylserine expression during programmed cell death
Authors: Blankenberg Francis G. , Katsikis Peter D. , Tait Jonathan F. , Davis R. Eric , Naumovski Louis , Ohtsuki Katsuichi , Kopiwoda Susan , Abrams Michael J. , Darkes Marilyn , Robbins Robert C. , Maecker Holden T. , Strauss H.W.
Published: July 2002

One of the earliest events in programmed cell death is the externalization of phosphatidylserine, a membrane phospholipid normally restricted to the inner leaflet of the lipid bilayer. Annexin V, an endogenous human protein with a high affinity for membrane bound phosphatidylserine, can be used
in vitro
to detect apoptosis before other well described morphologic or nuclear changes associated with programmed cell death. We tested the ability of exogenously administered radiolabeled annexin V to concentrate at sites of apoptotic cell death
in vivo
. After derivatization with hydrazinonicotinamide, annexin V was radiolabeled with technetium 99m.
In vivo
localization of technetium 99m hydrazinonicotinamide-annexin V was tested in three models: fuminant hepatic apoptosis induced by anti-Fas antibody injection in BALB/c mice; acute rejection in ACI rats with transplanted heterotopic PVG cardiac allografts; and cyclophosphamide treatment of transplanted 38C13 murine B cell lymphomas. External radionuclide imaging showed a two- to sixfold increase in the uptake of radiolabeled annexin V at sites of apoptosis in all three models. Immunohistochemical staining of cardiac allografts for exogenously administered annexin V revealed intense staining of numerous myocytes at the periphery of mononuclear infiltrates of which only a few demonstrated positive apoptotic nuclei by the terminal deoxynucleotidyltransferase-mediated UTP end labeling method. These results suggest that radiolabeled annexin V can be used
in vivo
as a noninvasive means to detect and serially image tissues and organs undergoing programmed cell death.
Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in
Arabidopsis
Authors: Meyer Knut , Shirley Amber M. , Cusumano Joanne C. , Bell-Lelong Dolly A. , Chapple Clint
Published: July 2002

The phenylpropanoid pathway provides precursors for the biosynthesis of soluble secondary metabolites and lignin in plants. Ferulate-5-hydroxylase (F5H) catalyzes an irreversible hydroxylation step in this pathway that diverts ferulic acid away from guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin. This fact led us to postulate that F5H was a potential regulatory step in the determination of lignin monomer composition. To test this hypothesis, we have used
Arabidopsis
to examine the impact of F5H overexpression.
Arabidopsis
is a useful model system in which to study lignification because in wild-type plants, guaiacyl and syringyl lignins are deposited in a tissue-specific fashion, while the F5H-deficient
fah1
mutant accumulates only guaiacyl lignin. Here we show that ectopic overexpression of F5H in
Arabidopsis
abolishes tissue-specific lignin monomer accumulation. Surprisingly, overexpression of F5H under the control of the lignification-associated cinnamate-4-hydroxylase promoter, but not the commonly employed cauliflower mosaic virus 35S promoter, generates a lignin that is almost entirely comprised of syringylpropane units. These experiments demonstrate that modification of F5H expression may enable engineering of lignin monomer composition in agronomically important plant species.
Expressed protein ligation: A general method for protein engineering
Authors: Muir Tom W. , Sondhi Dolan , Cole Philip A.
Published: July 2002

A protein semisynthesis method—expressed protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.
Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice
Authors: Andersson Ellen Christina , Hansen Bjarke Endel , Jacobsen Helle , Madsen Lars S. , Andersen Claus B. , Engberg Jan , Rothbard Jonathan B. , McDevitt Grete Sønderstrup , Malmström Vivianne , Holmdahl Rikard , Svejgaard Arne , Fugger Lars
Published: July 2002

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.
Nature of PEVK-titin elasticity in skeletal muscle
Authors: Linke Wolfgang A. , Ivemeyer Marc , Mundel Peter , Stockmeier Marc R. , Kolmerer Bernhard
Published: July 2002

A unique sequence within the giant titin molecule, the PEVK domain, has been suggested to greatly contribute to passive force development of relaxed skeletal muscle during stretch. To explore the nature of PEVK elasticity, we used titin-specific antibodies to stain both ends of the PEVK region in rat psoas myofibrils and determined the region’s force-extension relation by combining immunofluorescence and immunoelectron microscopy with isolated myofibril mechanics. We then tried to fit the results with recent models of polymer elasticity. The PEVK segment elongated substantially at sarcomere lengths above 2.4 μm and reached its estimated contour length at ≈3.5 μm. In immunofluorescently labeled sarcomeres stretched and released repeatedly above 3 μm, reversible PEVK lengthening could be readily visualized. At extensions near the contour length, the average force per titin molecule was calculated to be ≈45 pN. Attempts to fit the force-extension curve of the PEVK segment with a standard wormlike chain model of entropic elasticity were successful only for low to moderate extensions. In contrast, the experimental data also could be correctly fitted at high extensions with a modified wormlike chain model that incorporates enthalpic elasticity. Enthalpic contributions are likely to arise from electrostatic stiffening, as evidenced by the ionic-strength dependency of titin-based myofibril stiffness; at high stretch, hydrophobic effects also might become relevant. Thus, at physiological muscle lengths, the PEVK region does not function as a pure entropic spring. Rather, PEVK elasticity may have both entropic and enthalpic origins characterizable by a polymer persistence length and a stretch modulus.
Interleukin 6 plays a key role in the development of antigen-induced arthritis
Authors: Ohshima Shiro , Saeki Yukihiko , Mima Toru , Sasai Mitsuko , Nishioka Katsuhiro , Nomura Shintaro , Kopf Manfred , Katada Yoshinori , Tanaka Toshio , Suemura Masaki , Kishimoto Tadamitsu
Published: July 2002

To investigate the direct role of interleukin (IL) 6 in the development of rheumatoid arthritis, IL-6-deficient (IL-6 −/−) mice were backcrossed for eight generations into C57BL/6 mice, a strain of mice with a genetic background of susceptibility for antigen-induced arthritis (AIA). Both histological and immunological comparisons were made between IL-6-deficient (IL-6 −/−) mice and wild-type (IL-6 +/+) littermates after the induction of AIA. Although all IL-6 +/+ mice developed severe arthritis, only mild arthritis was observed in IL-6 −/− mice. Safranin O staining demonstrated that articular cartilage was well preserved in IL-6 −/− mice, whereas it was destroyed completely in IL-6 +/+ mice. In addition, comparable mRNA expression for both IL-1β and tumor necrosis factor α, but not for IL-6, was detected in the inflamed joints of IL-6 −/− mice, suggesting that IL-6 may play a more crucial role in cartilage destruction than either IL-1β or tumor necrosis factor α. In immunological comparisons, both antigen-specific
in vitro
proliferative response in lymph node cells and
in vivo
antibody production were elicited in IL-6 −/− mice, but they were reduced to less than half of that found in IL-6 +/+ mice. Lymph node cells of IL-6 −/− mice produced many more Th2 cytokines than did IL-6 +/+ mice with either antigen-specific or nonspecific stimulation in
in vitro
culture. Taken together, these results indicate that IL-6 may play a key role in the development of AIA at the inductive as well as the effector phase, and the blockade of IL-6 is possibly beneficial in the treatment of rheumatoid arthritis.
The early stage of folding of villin headpiece subdomain observed in a 200-nanosecond fully solvated molecular dynamics simulation
Authors: Duan Yong , Wang Lu , Kollman Peter A.
Published: July 2002

A new approach in implementing classical molecular dynamics simulation for parallel computers has enabled a simulation to be carried out on a protein with explicit representation of water an order of magnitude longer than previously reported and will soon enable such simulations to be carried into the microsecond time range. We have used this approach to study the folding of the villin headpiece subdomain, a 36-residue small protein consisting of three helices, from an unfolded structure to a molten globule state, which has a number of features of the native structure. The time development of the solvation free energy, the radius of gyration, and the mainchain rms difference from the native NMR structure showed that the process can be seen as a 60-nsec “burst” phase followed by a slow “conformational readjustment” phase. We found that the burial of the hydrophobic surface dominated the early phase of the folding process and appeared to be the primary driving force of the reduction in the radius of gyration in that phase.
The negative feedback actions of progesterone on gonadotropinreleasing hormone secretion are transduced by the classical progesterone receptor
Authors: Skinner Donal C. , Evans Neil P. , Delaleu Bernadette , Goodman Robert L. , Bouchard Philippe , Caraty Alain
Published: July 2002

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0–12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12–24 hr, P implant removed; 24–36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3α-hydroxy-5α-pregnan-20-one, which operates through the type A γ-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.
Differential display of peptides induced during the immune response of
Drosophila
: A matrix-assisted laser desorption ionization time-of-flight mass spectrometry study
Authors: Uttenweiler-Joseph Sandrine , Moniatte Marc , Lagueux Marie , Van Dorsselaer Alain , Hoffmann Jules A. , Bulet Philippe
Published: July 2002

We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of
Drosophila
, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5–11 kDa, of 24
Drosophila
immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-flight MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.
Evidence for posttranscriptional activation of γ-glutamylcysteine synthetase during plant stress responses
Authors: May Mike J. , Vernoux Teva , Sánchez-Fernández Rocío , Van Montagu Marc , Inzé Dirk
Published: July 2002

Glutathione (GSH) is a key component of plant antioxidant defenses. We have sought to determine how the rate-limiting step in GSH biosynthesis, catalyzed by γ-glutamylcysteine synthase (γECS) is regulated inArabidopsis. Functional complementation of a yeast mutant deficient in this enzyme with anArabidopsisexpression library yielded two cDNAs with sequence identical to the previously describedAtγECS. Nevertheless, the cellular concentration of GSH in these transformants was only 10% of wild-type concentrations and this was not a result of Cys availability. To explore the possibility thatArabidopsisγECS requires additional factors for full catalytic activity, we analyzed the GSH levels and the enzyme activities and transcript levels of both enzymes of the GSH biosynthetic pathway inArabidopsissuspension cultures subjected to a variety of stresses that raise GSH levels. Our results demonstrate rapid posttranscriptional activation ofArabidopsisγECS. The implications of these findings for the mechanisms by which GSH concentrations are regulated during plant-stress responses are discussed.
A conventional myosin motor drives neurite outgrowth
Authors: Wylie Steven R. , Wu Pei-jun , Patel Hitesh , Chantler Peter D.
Published: July 2002

Neuritic outgrowth is a striking example of directed motility, powered through the actions of molecular motors. Members of the myosin superfamily of actin-associated motors have been implicated in this complex process. Although conventional myosin II is known to be present in neurons, where it is localized at the leading edge of growth cones and in the cell cortex close to the plasma membrane, its functional involvement in growth cone motility has remained unproven. Here, we show that antisense oligodeoxyribonucleotides, complementary to a specific isoform of conventional myosin (myosin IIB), attenuate filopodial extension whereas sense and scrambled control oligodeoxyribonucleotides have no effect. Attenuation is shown to be reversible, neurite outgrowth being restored after cessation of the antisense regimen. Myosin IIB mRNA was present during active neurite extension, but levels were minimal in phenotypically rounded cells before neurite outgrowth and message levels decreased during antisense treatment. By contrast, the myosin IIA isoform is shown to be expressed constitutively both before and during neurite outgrowth and throughout exposure to myosin IIB antisense oligodeoxyribonucleotides. These results provide direct evidence that a conventional two-headed myosin is required for growth cone motility and is responsible, at least in part, for driving neuritic process outgrowth.
Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ
Authors: Suh Won-Chul , Burkholder William F. , Lu Chi Zen , Zhao Xun , Gottesman Max E. , Gross Carol A.
Published: July 2002

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the
Escherichia coli
Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (
i
) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (
ii
) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.
Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone
Authors: Gässler Claudia S. , Buchberger Alexander , Laufen Thomas , Mayer Matthias P. , Schröder Hartwig , Valencia Alfonso , Bukau Bernd
Published: July 2002

Hsp70 chaperones assist protein folding by ATP-controlled cycles of substrate binding and release. ATP hydrolysis is the rate-limiting step of the ATPase cycle that causes locking in of substrates into the substrate-binding cavity of Hsp70. This key step is strongly stimulated by DnaJ cochaperones. We show for the
Escherichia coli
Hsp70 homolog, DnaK, that stimulation by DnaJ requires the linked ATPase and substrate-binding domains of DnaK. Functional interaction with DnaJ is affected by mutations in an exposed channel located in the ATPase domain of DnaK. It is proposed that binding to this channel, possibly involving the J-domain, allows DnaJ to couple substrate binding with ATP hydrolysis by DnaK. Evolutionary conservation of the channel and the J-domain suggests conservation of the mechanism of action of DnaJ proteins.
RNA as a target of double-stranded RNA-mediated genetic interference in
Caenorhabditis elegans
Authors: Montgomery Mary K. , Xu SiQun , Fire Andrew
Published: July 2002

Introduction of exogenous double-stranded RNA (dsRNA) into
Caenorhabditis elegans
has been shown to specifically and potently disrupt the activity of genes containing homologous sequences. In this study we present evidence that the primary interference effects of dsRNA are post-transcriptional. First, we examined the primary DNA sequence after dsRNA-mediated interference and found no evidence for alterations. Second, we found that dsRNA-mediated interference with the upstream gene in a polar operon had no effect on the activity of the downstream gene; this finding argues against an effect on initiation or elongation of transcription. Third, we observed by
in situ
hybridization that dsRNA-mediated interference produced a substantial, although not complete, reduction in accumulation of nascent transcripts in the nucleus, while cytoplasmic accumulation of transcripts was virtually eliminated. These results indicate that the endogenous mRNA is the target for interference and suggest a mechanism that degrades the targeted RNA before translation can occur. This mechanism is not dependent on the SMG system, an mRNA surveillance system in
C. elegans
responsible for targeting and destroying aberrant messages. We suggest a model of how dsRNA might function in a catalytic mechanism to target homologous mRNAs for degradation.
Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice
Authors: Kushi Atsuko , Sasai Hitoshi , Koizumi Haruko , Takeda Noriko , Yokoyama Masahiro , Nakamura Motonao
Published: July 2002

To elucidate the role of neuropeptide Y (NPY)-Y1 receptor (Y1-R) in food intake, energy expenditure, and other possible functions, we have generated Y1-R-deficient mice (Y1-R
−/−
) by gene targeting. Contrary to our hypothesis that the lack of NPY signaling via Y1-R would result in impaired feeding and weight loss, Y1-R
−/−
mice showed a moderate obesity and mild hyperinsulinemia without hyperphagia. Although there was some variation between males and females, typical characteristics of Y1-R
−/−
mice include: greater body weight (females more than males), an increase in the weight of white adipose tissue (WAT) (approximately 4-fold in females), an elevated basal level of plasma insulin (approximately 2-fold), impaired insulin secretion in response to glucose administration, and a significant changes in mitochondrial uncoupling protein (UCP) gene expression (up-regulation of UCP1 in brown adipose tissue and down-regulation of UCP2 in WAT). These results suggest either that the Y1-R in the hypothalamus is not a key molecule in the leptin/NPY pathway, which controls feeding behavior, or that its deficiency is compensated by other receptors, such as NPY-Y5 receptor. We believe that the mild obesity found in Y1-R
−/−
mice (especially females) was caused by the impaired control of insulin secretion and/or low energy expenditure, including the lowered expression of UCP2 in WAT. This model will be useful for studying the mechanism of mild obesity and abnormal insulin metabolism in noninsulin-dependent diabetes mellitus.
Structural basis for methylesterase CheB regulation by a phosphorylation-activated domain
Authors: Djordjevic Snezana , Goudreau Paul N. , Xu Qingping , Stock Ann M. , West Ann H.
Published: July 2002

We report the x-ray crystal structure of the methylesterase CheB, a phosphorylation-activated response regulator involved in reversible modification of bacterial chemotaxis receptors. Methylesterase CheB and methyltransferase CheR modulate signaling output of the chemotaxis receptors by controlling the level of receptor methylation. The structure of CheB, which consists of an N-terminal regulatory domain and a C-terminal catalytic domain joined by a linker, was solved by molecular replacement methods using independent search models for the two domains. In unphosphorylated CheB, the N-terminal domain packs against the active site of the C-terminal domain and thus inhibits methylesterase activity by directly restricting access to the active site. We propose that phosphorylation of CheB induces a conformational change in the regulatory domain that disrupts the domain interface, resulting in a repositioning of the domains and allowing access to the active site. Structural similarity between the two companion receptor modification enzymes, CheB and CheR, suggests an evolutionary and/or functional relationship. Specifically, the phosphorylated N-terminal domain of CheB may facilitate interaction with the receptors, similar to the postulated role of the N-terminal domain of CheR. Examination of surfaces in the N-terminal regulatory domain of CheB suggests that despite a common fold throughout the response regulator family, surfaces used for protein–protein interactions differ significantly. Comparison between CheB and other response regulators indicates that analogous surfaces are used for different functions and conversely, similar functions are mediated by different molecular surfaces.
Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy
Authors: Eggeling C. , Fries J. R. , Brand L. , Günther R. , Seidel C. A. M.
Published: July 2002

A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (≈10 fl) of a confocal epi-illuminated set-up. In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis. The two-dimensional BIFL data allow the identification and detection of different temporally resolved conformational states. A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity. The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR–DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.