bluuebunny/crossref_metadata_2025 huggingface dataset:
Trp zipper folding kinetics by molecular dynamics and temperature-jump spectroscopy
Authors: Snow Christopher D. , Qiu Linlin , Du Deguo , Gai Feng , Hagen Stephen J. , Pande Vijay S.
Published: March 2004

We studied the microsecond folding dynamics of three β hairpins (Trp zippers 1–3, TZ1–TZ3) by using temperature-jump fluorescence and atomistic molecular dynamics in implicit solvent. In addition, we studied TZ2 by using time-resolved IR spectroscopy. By using distributed computing, we obtained an aggregate simulation time of 22 ms. The simulations included 150, 212, and 48 folding events at room temperature for TZ1, TZ2, and TZ3, respectively. The all-atom optimized potentials for liquid simulations (OPLS
aa
) potential set predicted TZ1 and TZ2 properties well; the estimated folding rates agreed with the experimentally determined folding rates and native conformations were the global potential-energy minimum. The simulations also predicted reasonable unfolding activation enthalpies. This work, directly comparing large simulated folding ensembles with multiple spectroscopic probes, revealed both the surprising predictive ability of current models as well as their shortcomings. Specifically, for TZ1–TZ3, OPLS for united atom models had a nonnative free-energy minimum, and the folding rate for OPLS
aa
TZ3 was sensitive to the initial conformation. Finally, we characterized the transition state; all TZs fold by means of similar, native-like transition-state conformations.
WIN1, a transcriptional activator of epidermal wax accumulation inArabidopsis
Authors: Broun Pierre , Poindexter Patricia , Osborne Erin , Jiang Cai-Zhong , Riechmann José Luis
Published: March 2004

Epicuticular wax forms a layer of hydrophobic material on plant aerial organs, which constitutes a protective barrier between the plant and its environment. We report here the identification of WIN1, anArabidopsis thalianaethylene response factor-type transcription factor, which can activate wax deposition in overexpressing plants. We constitutively expressedWIN1in transgenicArabidopsisplants, and found that leaf epidermal wax accumulation was up to 4.5-fold higher in these plants than in control plants. A significant increase was also found in stems. Interestingly, ≈50% of the additional wax could only be released by complete lipid extractions, suggesting that not all of the wax is superficial. Gene expression analysis indicated that a number of genes, such asCER1, KCS1, andCER2, which are known to be involved in wax biosynthesis, were induced inWIN1overexpressors. This observation indicates that induction of wax accumulation in transgenic plants is probably mediated through an increase in the expression of genes encoding enzymes of the wax biosynthesis pathway.
Synaptic activity-independent persistent plasticity in endogenously active mammalian motoneurons
Authors: Bocchiaro Christopher M. , Feldman Jack L.
Published: March 2004

Potentiation and depression of glutamate receptor function in hippocampal, cerebellar, and cortical neurons are examples of persistent changes in synaptic function that underlie important behavioral adaptations such as learning and memory. Persistent changes in synaptic function relevant for motor behaviors have not been demonstrated in mammalian motoneurons. We demonstrate that adaptive changes in (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) receptor function at endogenously active synapses occur in motoneurons in neonatal rodents. We found a form of serotonin (5-HT)-dependent synaptic plasticity in hypoglossal (XII) motoneurons, which control tongue muscles affecting upper airway function, that is metamodulated by metabotropic glutamate receptors. Episodic, but not continuous, activation of postsynaptic 5-HT type 2 (5-HT
2
) receptors on hypoglossal (XII) motoneurons leads to long-lasting increases in their AMPA receptor-mediated respiratory drive currents and associated XII nerve motor output. Antagonism of group-I metabotropic glutamate receptors blocks induction of the 5-HT-induced increase in excitability. We propose that this activity-independent postsynaptic 5-HT-mediated plasticity represents the cellular mechanism underlying long-term facilitation, i.e., persistent increases in respiratory motor output and ventilation seen in humans and rodents in response to episodic hypoxia. Loss of activity in XII motoneurons is common during sleep causing snoring and, in serious cases, airway obstruction that interrupts breathing, a condition known as obstructive sleep apnea. These results may provide the basis for rationale development of therapeutics for obstructive sleep apnea in humans.
The yeast cell-cycle network is robustly designed
Authors: Li Fangting , Long Tao , Lu Ying , Ouyang Qi , Tang Chao
Published: March 2004

The interactions between proteins, DNA, and RNA in living cells constitute molecular networks that govern various cellular functions. To investigate the global dynamical properties and stabilities of such networks, we studied the cell-cycle regulatory network of the budding yeast. With the use of a simple dynamical model, it was demonstrated that the cell-cycle network is extremely stable and robust for its function. The biological stationary state, the G1state, is a global attractor of the dynamics. The biological pathway, the cell-cycle sequence of protein states, is a globally attracting trajectory of the dynamics. These properties are largely preserved with respect to small perturbations to the network. These results suggest that cellular regulatory networks are robustly designed for their functions.
Chaos may enhance information transmission in the inferior olive
Authors: Schweighofer Nicolas , Doya Kenji , Fukai Hidekazu , Chiron Jean Vianney , Furukawa Tetsuya , Kawato Mitsuo
Published: March 2004

Despite unique well characterized neuronal properties, such as extensive electrical coupling and low firing rates, the role of the inferior olive (IO), which is the source of the climbing fiber inputs to cerebellar Purkinje cells, is still controversial. We propose that the IO stochastically recodes the high-frequency information carried by its synaptic inputs into stochastic, low-rate spikes in its climbing fiber output. Computer simulations of realistic IO networks showed that moderate electrical coupling produced chaotic firing, which maximized the input-output mutual information. This “chaotic resonance” may allow rich error signals to reach individual Purkinje cells, even at low firing rates, allowing efficient cerebellar learning.
Disruption of communication between peripheral and central trigeminovascular neurons mediates the antimigraine action of 5HT
1B/1D
receptor agonists
Authors: Levy Dan , Jakubowski Moshe , Burstein Rami
Published: March 2004

Triptans are 5HT
1B/1D
receptor agonists commonly prescribed for migraine headache. Although originally designed to constrict dilated intracranial blood vessels, the mechanism and site of action by which triptans abort the migraine pain remain unknown. We showed recently that sensitization of peripheral and central trigeminovascular neurons plays an important role in the pathophysiology of migraine pain. Here we examined whether the drug sumatriptan can prevent and/or suppress peripheral and central sensitization by using single-unit recording in our animal model of intracranial pain. We found that sumatriptan effectively prevented the induction of sensitization (i.e., increased spontaneous firing; increased neuronal sensitivity to intracranial mechanical stimuli) in central trigeminovascular neurons (recorded in the dorsal horn), but not in peripheral trigeminovascular neurons (recorded in the trigeminal ganglion). After sensitization was established in both types of neuron, sumatriptan effectively normalized intracranial mechanical sensitivity of central neurons, but failed to reverse such hypersensitivity in peripheral neurons. In both the peripheral and central neurons, the drug failed to attenuate the increased spontaneous activity established during sensitization. These results suggest that neither peripheral nor central trigeminovascular neurons are directly inhibited by sumatriptan. Rather, triptan action appears to be exerted through presynaptic 5HT
1B/1D
receptors in the dorsal horn to block synaptic transmission between axon terminals of the peripheral trigeminovascular neurons and cell bodies of their central counterparts. We therefore suggest that the analgesic action of triptan can be attained specifically in the absence, but not in the presence, of central sensitization.
Bacterial biota in the human distal esophagus
Authors: Pei Zhiheng , Bini Edmund J. , Yang Liying , Zhou Meisheng , Francois Fritz , Blaser Martin J.
Published: March 2004

The esophagus, like other luminal organs of the digestive system, provides a potential environment for bacterial colonization, but little is known about the presence of a bacterial biota or its nature. By using broad-range 16S rDNA PCR, biopsies were examined from the normal esophagus of four human adults. The 900 PCR products cloned represented 833 unique sequences belonging to 41 genera, or 95 species-level operational taxonomic units (SLOTU); 59 SLOTU were homologous with culture-defined bacterial species, 34 with 16S rDNA clones, and two were not homologous with any known bacterial 16S rDNA. Members of six phyla,
Firmicutes
,
Bacteroides
,
Actinobacteria
,
Proteobacteria
,
Fusobacteria
, and
TM7
, were represented. A large majority of clones belong to 13 of the 41 genera (783/900, 87%), or 14 SLOTU (574/900, 64%) that were shared by all four persons.
Streptococcus
(39%),
Prevotella
(17%), and
Veilonella
(14%) were most prevalent. The present study identified ≈56–79% of SLOTU in this bacterial ecosystem. Most SLOTU of esophageal biota are similar or identical to residents of the upstream oral biota, but the major distinction is that a large majority (82%) of the esophageal bacteria are known and cultivable. These findings provide evidence for a complex but conserved bacterial population in the normal distal esophagus.
Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry
Authors: Simmons Graham , Reeves Jacqueline D. , Rennekamp Andrew J. , Amberg Sean M. , Piefer Andrew J. , Bates Paul
Published: March 2004

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell–cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell–cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell–cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.
Impact of isotype-selective estrogen receptor agonists on ovarian function
Authors: Hegele-Hartung Christa , Siebel Philip , Peters Olaf , Kosemund Dirk , Müller Gerd , Hillisch Alexander , Walter Alexander , Kraetzschmar Jörn , Fritzemeier Karl-Heinrich
Published: March 2004

Other isotype-selective estrogen receptor (ER) agonists, the selective ERα agonist 3,17-dihydroxy-19-nor-17α-pregna-1,3,5 (10)triene-21,16α-lactone and the selective ERβ agonist 8-vinylestra-1,3,5 (10)-triene-3,17β-diol, were used in hypophysectomized rats, gonadotropin-releasing hormone antagonist-treated mice, as well as intact rats to elucidate the effects of isotype-selective estrogens on the physiology of folliculogenesis and ovulation. In hypophysectomized rats and gonadotropin-releasing hormone antagonist-treated mice, the ERβ agonist caused stimulation of early folliculogenesis, a decrease in follicular atresia, induction of ovarian gene expression, and stimulation of late follicular growth, accompanied by an increase in the number of ovulated oocytes similar to 17β-estradiol (E2). In contrast, the ERα agonist had little or no effect on these parameters, implying that direct estrogen effects on ovarian follicular development are mediated by ERβ. In intact rats, E2 and the ERα agonist dose-dependently inhibited ovulation, in contrast to the ERβ agonist. On the other hand, the ERβ agonist did not stimulate uterine weight in intact rats, in contrast to E2 and the ERα agonist. This finding is in line with the assumption that estrogen mediated ovulation inhibition and stimulation of uterine growth are mediated by ERα but not by ERβ
Isolation of an endotoxin–MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations
Authors: Gioannini Theresa L. , Teghanemt Athmane , Zhang DeSheng , Coussens Nathan P. , Dockstader Wendie , Ramaswamy S. , Weiss Jerrold P.
Published: March 2004

Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin–protein and protein–protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin–sCD14 complexes. Subsequent interactions of endotoxin–sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin–MD-2 complex generated by treatment of endotoxin–sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin–CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin–MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin–MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.
CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata
Authors: Helvig C. , Koener J. F. , Unnithan G. C. , Feyereisen R.
Published: March 2004

The molecular analysis of insect hormone biosynthesis has long been hampered by the minute size of the endocrine glands producing them. Expressed sequence tags from the corpora allata of the cockroach
Diploptera punctata
yielded a new cytochrome P450, CYP15A1. Its full-length cDNA encoded a 493-aa protein that has only 34% amino acid identity with CYP4C7, a terpenoid ω-hydroxylase previously cloned from this tissue. Heterologous expression of the cDNA in
Escherichia coli
produced >300 nmol of CYP15A1 per liter of culture. After purification, its catalytic activity was reconstituted by using phospholipids and house fly P450 reductase. CYP15A1 metabolizes methyl (2
E
,6
E
)-3,7,11-trimethyl-2,6-dodecatrienoate (methyl farnesoate) to methyl (2
E
,6
E
)-(10
R
)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate [juvenile hormone III, JH III] with a turnover of 3–5 nmol/min/nmol P450. The enzyme produces JH III with a ratio of ≈98:2 in favor of the natural (10
R
)-epoxide enantiomer. This result is in contrast to other insect P450s, such as CYP6A1, that epoxidize methyl farnesoate with lower regio- and stereoselectivity. RT-PCR experiments show that the CYP15A1 gene is expressed selectively in the corpora allata of
D. punctata
, at the time of maximal JH production by the glands. We thus report the cloning and functional expression of a gene involved in an insect-specific step of juvenile hormone biosynthesis. Heterologously expressed CYP15A1 from
D. punctata
or its ortholog from economically important species may be useful in the design and screening of selective insect control agents.
Promoter unwinding and promoter clearance by RNA polymerase: Detection by single-molecule DNA nanomanipulation
Authors: Revyakin Andrey , Ebright Richard H. , Strick Terence R.
Published: March 2004

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with ≈1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with ≈5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.
Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol
Authors: Mitra Kakoli , Ubarretxena-Belandia Iban , Taguchi Tomohiko , Warren Graham , Engelman Donald M.
Published: March 2004

A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness
in vivo
because measurements
in vitro
have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ± 0.4 Å, 39.5 ± 0.4 Å, 35.6 ± 0.6 Å, and 42.5 ± 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (
i
) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (
ii
) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of
Escherichia coli
cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.
Down-regulation of DENN/MADD, a TNF receptor binding protein, correlates with neuronal cell death in Alzheimer's disease brain and hippocampal neurons
Authors: Del Villar Keith , Miller Carol A.
Published: March 2004

Tumor necrosis factor (TNF) α and mitogen-activated protein kinase/c-Jun N-terminal kinase (MAPK/JNK) pathways are both implicated in Alzheimer's disease (AD) pathogenesis. Increased expression of several members of the TNF pathway and JNK activation of c-Jun ultimately result in neuronal apoptosis. DENN/MADD, a multifunctional domain protein expressed in neurons, interacts with both the p55 TNF receptor (TNFR) type 1 and JNK3, placing it at a critical juncture in regulating signaling of neurodegeneration. We examined expression and interactions of the TNFR1 binding proteins, DENN/MADD, and TNFR-associated death domain (TRADD) protein in AD-affected tissues and cell cultures. We found reduced DENN/MADD and increased TRADD expression immunohistochemically in the hippocampus in areas of AD pathology compared to normal controls but little intraneuronal colocalization. In brain homogenates, DENN/MADD protein and mRNA expression was significantly reduced in AD compared to controls. Conversely, TRADD, TNFR1, and activated JNK were increased. Murine neuroblastoma and rat hippocampal cultures stressed with Aβ1–42 and the cortices of AD transgenic mice (Tg2576Swe) each showed decreased DENN/MADD expression and TRADD up-regulation in the mice, compared to controls. DENN/MADD antisense treatment of cultured rat hippocampal neurons reduced endogenous DENN/MADD and promoted neuronal cell death. DENN/MADD and TRADD competitively bound to TNFR1 when overexpressed in N2A cells, with DENN/MADD abrogating TNFR1 binding to TRADD. DENN/MADD may therefore be protective by inhibiting TRADD-induced apoptotic cell death. Reduction of DENN/MADD may affect long-term neuronal viability in AD by allowing TRADD mediation of TNFR1 signaling in response to oxidative or cytokine-promoted stresses.
Nitric oxide, oxidants, and protein tyrosine nitration
Authors: Radi Rafael
Published: March 2004

The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the signal transducing physiological actions of•NO to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite anion (ONOO–) and nitrogen dioxide (•NO2), formed as secondary products of•NO metabolism in the presence of oxidants including superoxide radicals (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document}), hydrogen peroxide (H2O2), and transition metal centers. The precise interplay between•NO and oxidants and the identification of the proximal intermediate(s) responsible for nitrationin vivohave been under controversy. Despite the capacity of peroxynitrite to mediate tyrosine nitrationin vitro, its role on nitrationin vivohas been questioned, and alternative pathways, including the nitrite/H2O2/hemeperoxidase and transition metal-dependent mechanisms, have been proposed. A balanced analysis of existing evidence indicates that (i) different nitration pathways can contribute to tyrosine nitrationin vivo, and (ii) most, if not all, nitration pathways involve free radical biochemistry with carbonate radicals (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{CO}}_{3}^{{\bullet}-}\end{equation*}\end{document}) and/or oxo–metal complexes oxidizing tyrosine to tyrosyl radical followed by the diffusion-controlled reaction with•NO2to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield processin vivo, 3-nitrotyrosine has been revealed as a relevant biomarker of•NO-dependent oxidative stress; additionally, site-specific nitration focused on particular protein tyrosines may result in modification of function and promote a biological effect. Tissue distribution and quantitation of protein 3-nitrotyrosine, recognition of the predominant nitration pathways and individual identification of nitrated proteins in disease states open new avenues for the understanding and treatment of human pathologies.
Both chemotaxis and net motility greatly influence the infectivity of
Vibrio cholerae
Authors: Butler Susan M. , Camilli Andrew
Published: March 2004

The role of chemotaxis in the virulence of gastrointestinal pathogens is ill defined. Counterintuitively, nonchemotactic mutants of the polarly flagellated pathogen
Vibrio cholerae
greatly out-compete the wild-type strain during infection of the small intestine. We show that the out-competition phenotype is dependent on the direction of flagellar rotation and independent of Toxin Co-regulated Pilus function. Specifically, the out-competition associated with the loss of chemotaxis required the presence of counterclockwise-biased flagellar rotation and smooth straight runs by the bacteria. In contrast, a nonchemotactic strain with clockwise-biased flagellar rotation was confined to small-scale net movement and was attenuated for infection. The significance of the out-competition phenotype was examined and was shown to correlate with a true increase in infectivity. Counterclockwise-biased mutants are aberrantly distributed throughout the infant mouse small intestine and we find that the expression of virulence factors occurs normally in all segments. Thus, alteration of the chemotactic properties of
V. cholerae
allows it to exploit additional niches in the host intestine.
Identification of the bacterial alarmone guanosine 5′-diphosphate 3′-diphosphate (ppGpp) in plants
Authors: Takahashi Kosaku , Kasai Koji , Ochi Kozo
Published: March 2004

Stringent control mediated by the bacterial alarmone guanosine 5′-diphosphate 3′-diphosphate (ppGpp) is a key regulatory process governing bacterial gene expression. By devising a system to measure ppGpp in plants, we have been able to identify ppGpp in the chloroplasts of plant cells. Levels of ppGpp increased markedly when plants were subjected to such biotic and abiotic stresses as wounding, heat shock, high salinity, acidity, heavy metal, drought, and UV irradiation. Abrupt changes from light to dark also caused a substantial elevation in ppGpp levels.
In vitro
, chloroplast RNA polymerase activity was inhibited in the presence of ppGpp, demonstrating the existence of a bacteria-type stringent response in plants. Elevation of ppGpp levels was elicited also by treatment with plant hormones jasmonic acid, abscisic acid, and ethylene, but these effects were blocked completely by another plant hormone, indole-3-acetic acid. On the basis of these findings, we propose that ppGpp plays a critical role in systemic plant signaling in response to environmental stresses, contributing to the adaptation of plants to environmental changes.
Histone H1 and the origin of protamines
Authors: Lewis John D. , Saperas Núria , Song Yue , Zamora Maria Jose , Chiva Manel , Ausió Juan
Published: March 2004

We present evidence that chordate protamines have evolved from histone H1. During the final stages of spermatogenesis, the compaction of DNA in many organisms is accomplished by the replacement of histones with a class of arginine-rich proteins called protamines. In other organisms, however, condensation of sperm DNA can occur with comparable efficiency in the presence of somatic-type histones or, alternatively, an intermediate class of proteins called protamine-like proteins. The idea that the highly specialized sperm chromosomal proteins (protamines) and somatic chromosomal proteins (histones) could be related dates back almost to the discovery of these proteins. Although this notion has frequently been revisited since that time, there has been a complete lack of supporting experimental evidence. Here we show that the emergence of protamines in chordates occurred very quickly, as a result of the conversion of a lysine-rich histone H1 to an arginine-rich protamine. We have characterized the sperm nuclear basic proteins of the tunicateStyela montereyensis, which we show consists of both a protamine and a sperm-specific histone H1 with a protamine tail. Comparison of the genes encoding these proteins to that of a sister protochordate,Ciona intestinalis, has indicated this rapid and dramatic change is most likely the result of frameshift mutations in the tail of the sperm-specific histone H1. By establishing an evolutionary link between the chromatin-condensing histone H1s of somatic tissues and the chromatin-condensing proteins of the sperm, these results provide unequivocal support to the notion that vertebrate protamines evolved from histones.
Epitope-specific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis
Authors: Prakken Berent J. , Samodal Rodrigo , Le Tho D. , Giannoni Francesca , Yung Gisella Puga , Scavulli John , Amox Diane , Roord Sarah , de Kleer Isme , Bonnin Dustan , Lanza Paola , Berry Charles , Massa Margherita , Billetta Rosario , Albani Salvatore
Published: March 2004

Modulation of epitope-specific immune responses would represent a major addition to available therapeutic options for many autoimmune diseases. The objective of this work was to induce immune deviation by mucosal peptide-specific immunotherapy in rheumatoid arthritis (RA) patients, and to dissect the related immunological mechanisms by using a technology for the detection of low-affinity class II-restricted peptide-specific T cells. A group of patients with early RA was treated for 6 months orally with dnaJP1, a peptide that induces proinflammatory T cell responses in naive RA patients. Immunological analysis at initial, intermediate and end treatment points showed an intriguing change from proinflammatory to regulatory T cell function. In fact, dnaJP1-induced T cell production of IL-4 and IL-10 increased significantly when initial and end treatment points were compared, whereas dnaJP1-induced T cell proliferation and production of IL-2, IFN-γ, and tumor necrosis factor-α decreased significantly. The total number of dnaJP1-specific cells did not change over time, whereas expression of foxP3 by CD4+CD25brightcells increased, suggesting that the treatment affected regulatory T cell function. Thus, rather than clonal deletion, the observed change in immune reactivity to dnaJP1 was the outcome of treatment-induced emergence of T cells with a different functional phenotype. This study contributes to our knowledge of mechanisms and tools needed for antigen-specific immune modulation in humans, thus laying the foundation for exploitation of this approach for therapeutic purposes.
Inducible costimulator-dependent IL-10 production by regulatory T cells specific for self-antigen
Authors: Kohyama Masako , Sugahara Daisuke , Sugiyama Shigeru , Yagita Hideo , Okumura Ko , Hozumi Nobumichi
Published: March 2004

In this study, we investigated the relationship between the expression levels of self-antigen and the function of self-reactive T cells in the periphery. To this end, we used two rat insulin promoter-ovalbumin (RIP-OVA) transgenic mice (RIP-OVAhigh, RIP-OVAlow) in which was produced only in pancreatic β-islet cells. The OVA-producing transgenic mice were crossed to DO.11.10 (DO) mice expressing a T cell antigen receptor specific for OVA323–339. The responsiveness of peripheral CD4+T cells in the double transgenic mice was examined. We demonstrated that hyporesponsive but highly IL-10-producing T cells were developed in DO × OVAhighmice only, not in DO × OVAlowmice. These IL-10-producing T cells exhibited regulatory activity both inin vitroandin vivoexperiments. Moreover, these IL-10-producing regulatory T (Tr) cells expressed high levels of inducible costimulator (ICOS) beforein vitrostimulation. Blockade of ICOS-signaling inhibited the production of IL-10 and abrogated the inhibitory function of these Tr cells. Thus, these results suggested that the development of IL-10-producing Tr cells depends on the expression levels of self-antigenin vivoand that ICOS signal plays a critical role in immune regulation by IL-10-producing Tr cells in self-tolerance.
Elastic coupling of integral membrane protein stability to lipid bilayer forces
Authors: Hong Heedeok , Tamm Lukas K.
Published: March 2004

It has been traditionally difficult to measure the thermodynamic stability of membrane proteins because fully reversible protocols for complete folding these proteins were not available. Knowledge of the thermodynamic stability of membrane proteins is desirable not only from a fundamental theoretical standpoint, but is also of enormous practical interest for the rational design of membrane proteins and for optimizing conditions for their structure determination by crystallography or NMR. Here, we describe the design of a fully reversible system to study equilibrium folding of the outer membrane protein A from
Escherichia coli
in lipid bilayers. Folding is shown to be two-state under appropriate conditions permitting data analysis with a classical folding model developed for soluble proteins. The resulting free energy and
m
value, i.e., a measure of cooperativity, of unfolding are

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\begin{equation*}{\Delta}G_{u,{\mathrm{H}}_{2}{\mathrm{O}}}^{{\mathrm{o}}}=3.4~{\mathrm{kcal/mol}}\end{equation*}\end{document}

and
m
= 1.1 kcal/mol M
–1
, respectively, in a reference bilayer composed of palmitoyl-oleoyl-phosphatidylcholine (C
16:0
C
18:1
PC) and palmitoyloleoyl-phosphatidylglycerol (C
16:0
C
18:1
PG). These values are strong functions of the lipid bilayer environment. By systematic variation of lipid headgroup and chain composition, we show that elastic bilayer forces such as curvature stress and hydrophobic mismatch modulate the free energy and cooperativity of folding of this and perhaps many other membrane proteins.
The structure of zetekitoxin AB, a saxitoxin analog from the Panamanian golden frog
Atelopus zeteki
: A potent sodium-channel blocker
Authors: Yotsu-Yamashita Mari , Kim Yong H. , Dudley Samuel C. , Choudhary Gaurav , Pfahnl Arnold , Oshima Yasukatsu , Daly John W.
Published: March 2004

Bufonid anurans of the genus
Atelopus
contain both steroidal bufadienolides and various guanidinium alkaloids of the tetrodotoxin class. The former inhibit sodium-potassium ATPases, whereas the latter block voltage-dependent sodium channels. The structure of one guanidinium alkaloid, zetekitoxin AB, has remained a mystery for over 30 years. The structure of this alkaloid now has been investigated with a sample of ≈0.3 mg, purified from extracts obtained decades ago from the Panamanian golden frog
Atelopus zeteki
. Detailed NMR and mass spectral analyses have provided the structure and relative stereochemistry of zetekitoxin AB and have revealed that it is an analog of saxitoxin. The proposed structure is characterized by richness of heteroatoms (C
16
H
25
N
8
O
12
S) and contains a unique 1,2-oxazolidine ring-fused lactam, a sulfate ester, and an
N
-hydroxycarbamate moiety. Zetekitoxin AB proved to be an extremely potent blocker of voltage-dependent sodium channels expressed in
Xenopus
oocytes. The IC
50
values were 280 pM for human heart channels, 6.1 pM for rat brain IIa channels, and 65 pM for rat skeletal muscle channels, thus being roughly 580-, 160-, and 63-fold more potent at these channels than saxitoxin.
Nuclear protein-coding genes support lungfish and not the coelacanth as the closest living relatives of land vertebrates
Authors: Brinkmann Henner , Venkatesh Byrappa , Brenner Sydney , Meyer Axel
Published: March 2004

The colonization of land by tetrapod ancestors is one of the major questions in the evolution of vertebrates. Despite intense molecular phylogenetic research on this problem during the last 15 years, there is, until now, no statistically supported answer to the question of whether coelacanths or lungfish are the closest living relatives of tetrapods. We determined DNA sequences of the nuclear-encoded recombination activating genes (
Rag1
and
Rag2
) from all three major lungfish groups, the Australian
Neoceratodis forsteri
, the South American
Lepidosiren paradoxa
and the African lungfish
Protopterus dolloi
, and the Indonesian coelacanth
Latimeria menadoensis
. Phylogenetic analyses of both the single gene and the concatenated data sets of RAG1 and RAG2 found that the lungfishes are the closest living relatives of the land vertebrates. These results are supported by high bootstrap values, Bayesian posterior probabilities, and likelihood ratio tests.
Coupling morphogenesis to mitotic entry
Authors: Sakchaisri Krisada , Asano Satoshi , Yu Li-Rong , Shulewitz Mark J. , Park Chong J. , Park Jung-Eun , Cho Young-Wook , Veenstra Timothy D. , Thorner Jeremy , Lee Kyung S.
Published: March 2004

In eukaryotes, cyclin B-bound cyclin-dependent protein kinase 1 promotes mitotic entry but is held in check, in part, by Wee1 protein kinase. Timely mitotic entry in budding yeast requires inactivation of Swe1 (Wee1 ortholog). Perturbations of the septin collar at the bud neck lead to Swe1 stabilization, delaying the G
2
/M transition. Swe1 is recruited to the neck and hyperphosphorylated before ubiquitin-mediated degradation. Hsl1 kinase (Nim1 ortholog), a negative regulator of Wee1, is required for efficient Swe1 localization at the neck but seems not to phosphorylate Swe1. Here, we show that two other kinases targeted sequentially to the neck, Cla4/PAK and Cdc5/Polo, are responsible for stepwise phosphorylation and down-regulation of Swe1. This mechanism links assembly of a cellular structure to passage into mitosis.
S
-adenosylhomocysteine hydrolase deficiency in a human: A genetic disorder of methionine metabolism
Authors: Barić Ivo , Fumić Ksenija , Glenn Byron , Ćuk Mario , Schulze Andreas , Finkelstein James D. , James S. Jill , Mejaški-Bošnjak Vlatka , Pažanin Leo , Pogribny Igor P. , Radoš Marko , Sarnavka Vladimir , Šćukanec-Špoljar Mira , Allen Robert H. , Stabler Sally , Uzelac Lidija , Vugrek Oliver , Wagner Conrad , Zeisel Steven , Mudd S. Harvey
Published: March 2004

We report studies of a Croatian boy, a proven case of human
S
-adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 μM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5–15.9 μM. In plasma,
S
-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was ≈3% of control in liver and was 5–10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.
The plant hormone indoleacetic acid induces invasive growth in
Saccharomyces cerevisiae
Authors: Prusty Reeta , Grisafi Paula , Fink Gerald R.
Published: March 2004

Fungi must recognize plant-specific signals to initiate subsequent morphogenetic events such as filamentation that lead to infection. Here we show that the plant hormone indoleacetic acid (IAA) induces adhesion and filamentation of
Saccharomyces cerevisiae
. Genome expression profiling of cells treated with IAA identified Yap1, a fungal specific transcription factor, as a key mediator of this response. Strains lacking
YAP1
(
yap1-1
) are hypersensitive to growth on IAA because they accumulate more IAA than can wild type. Members of a family of transporters the amino acid/auxin:proton symport permeases with homology to
AUX1
, a putative IAA transporter from plants, are up-regulated in the
yap1-1
mutant. Deletion of any one of these transporters makes
yap1-1
mutants more resistant to IAA by decreasing its uptake. The permease mutants are defective in IAA perception and filamentation. The ability of a fungus to perceive a plant hormone that causes it to differentiate into an invasive form has important implications for plant–pathogen interactions.
Synaptic organization in the adult honey bee brain is influenced by brood-temperature control during pupal development
Authors: Groh Claudia , Tautz Jürgen , Rössler Wolfgang
Published: March 2004

Recent studies have shown that the behavioral performance of adult honey bees is influenced by the temperature experienced during pupal development. Here we explore whether there are temperature-mediated effects on the brain. We raised pupae at different constant temperatures between 29 and 37°C and performed neuroanatomical analyses of the adult brains. Analyses focused on sensory-input regions in the mushroom bodies, brain areas associated with higher-order processing such as learning and memory. Distinct synaptic complexes [microglomeruli (MG)] within the mushroom body calyces were visualized by using fluorophore-conjugated phalloidin and an antibody to synapsin. The numbers of MG were different in bees that had been raised at different temperatures, and these differences persisted after the first week of adult life. In the olfactory-input region (lip), MG numbers were highest in bees raised at the temperature normally maintained in brood cells (34.5°C) and significantly decreased in bees raised at 1°C below and above this norm. Interestingly, in the neighboring visual-input region (collar), MG numbers were less affected by temperature. We conclude that thermoregulatory control of brood rearing can generate area- and modality-specific effects on synaptic neuropils in the adult brain. We propose that resulting differences in the synaptic circuitry may affect neuronal plasticity and may underlie temperature-mediated effects on multimodal communication and learning.
Substance P in the medial amygdala: Emotional stress-sensitive release and modulation of anxiety-related behavior in rats
Authors: Ebner Karl , Rupniak Nadia M. , Saria Alois , Singewald Nicolas
Published: March 2004

Increasing evidence implicates the substance P (SP)/neurokinin-1 receptor system in anxiety and depression. However, it is not known whether emotional stimulation alters endogenous extracellular SP levels in brain areas important for processing of anxiety and mood, a prerequisite for a contribution of this neuropeptide system in modulating these behaviors. Therefore, we examined in rats whether the release of SP is sensitive to emotional stressors in distinct subregions of the amygdala, a key area in processing of emotions. By using
in vivo
micropush–pull superfusion and microdialysis techniques, we found a pronounced and long-lasting increase (150%) in SP release in the medial nucleus of the amygdala (MeA), but not in the central nucleus of the amygdala, in response to immobilization stress. SP release in the MeA was transiently enhanced (40%) in response to elevated platform exposure, which is regarded as a mild emotional stressor. Immobilization enhanced the anxiety-related behavior evaluated in the subsequently performed elevated plus-maze test. Bilateral microinjections of the neurokinin-1 receptor antagonist [2-cyclopropoxy-5-(5-(trifluoromethyl)tetrazol-1-yl)benzyl]-(2-phenylpiperidin-3-yl)amine into the MeA blocked the stress-induced anxiogenic-like effect, supporting a functional significance of enhanced SP release. In unstressed rats, the neurokinin-1 receptor antagonist displayed no significant anxiolytic effect but reversed the anxiogenic effect of SP microinjected into the MeA. Our findings identify the MeA as a critical brain area for the involvement of SP transmission in anxiety responses and as a putative site of action for the recently discovered therapeutic effects of SP antagonists in the treatment of stress-related disorders.
A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme
Authors: Gregory B. D. , Nickels B. E. , Garrity S. J. , Severinova E. , Minakhin L. , Bieber Urbauer R. J. , Urbauer J. L. , Heyduk T. , Severinov K. , Hochschild A.
Published: March 2004

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the β subunit and conserved region 4 of the σ subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the σ region 4/β-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the β-flap for binding to σ region 4, thereby inhibiting transcription initiation by disrupting the σ region 4/β-flap interaction.
Conserved T cell receptor usage in primary and recall responses to an immunodominant influenza virus nucleoprotein epitope
Authors: Kedzierska Katherine , Turner Stephen J. , Doherty Peter C.
Published: March 2004

The CD8+T cell response to the immunodominant DbNP366epitope has been analyzed sequentially to determine the prevalence and persistence of different T cell antigen receptor (TCR)Vβ8.3 clonotypes after primary and secondary influenza virus challenge. Based on the length and amino acid sequences of the complementarity-determining region 3 of TCRβ (CDR3β) loop and associated Jβ usage, the same dominant TCRβ signatures were found in the blood, the spleen, and the site of virus-induced pathology in the infected respiratory tract. Longitudinal analysis demonstrated that TCRβ prominent in the antigen-driven phase of response persisted into memory and were again expanded after secondary challenge. A proportion of these high-frequency TCRβ expressed “public” CDR3β sequences that were detected in every mouse sampled, whereas others were found more than once but were not invariably present. Analysis of N-region nucleotide diversity established that as many as 10 different nucleic acid sequences (maximum of four “nucleotypes” in any one mouse) could encode a single public TCRβ amino acid sequence. Conversely, whereas some of the unique, “private” TCRβ achieved a substantial clone size, they were always specified by a single nucleotype. Although there is a strong stochastic element in this response, the public TCRβ seem to represent a “best fit” for this immunodominant epitope, are selected preferentially from the naive TCR repertoire, and assume even greater prominence after secondary challenge.