bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4
Authors: Arita Kyouhei , Shimizu Toshiyuki , Hashimoto Hiroshi , Hidaka Yuji , Yamada Michiyuki , Sato Mamoru
Published: March 2006

Histone arginine methylation is a posttranslational modification linked to the regulation of gene transcription. Unlike other posttranslational modifications, methylation has generally been regarded as stable, and enzymes that demethylate histone arginine residues have not been identified. However, it has recently been shown that human peptidylarginine deiminase 4 (PAD4), a Ca
2+
-dependent enzyme previously known to convert arginine residues to citrulline in histones, can also convert monomethylated arginine residues to citrulline both
in vivo
and
in vitro
. Citrullination of histone arginine residues by the enzyme antagonizes methylation by histone arginine methyltransferases and is thus a novel posttranslational modification that regulates the level of histone arginine methylation and gene activity. Here we present the crystal structures of a Ca
2+
-bound PAD4 mutant in complex with three histone N-terminal peptides, each consisting of 10 amino acid residues that include one target arginine residue for the enzyme (H3/Arg-8, H3/Arg-17, and H4/Arg-3). To each histone N-terminal peptide, the enzyme induces a β-turn-like bent conformation composed of five successive residues at the molecular surface near the active site cleft. The remaining five residues are highly disordered. The enzyme recognizes each peptide through backbone atoms of the peptide with a possible consensus recognition motif. The sequence specificity of the peptide recognized by this enzyme is thought to be fairly broad. These observations provide structural insights into target protein recognition by histone modification enzymes and illustrate how PAD4 can target multiple arginine sites in the histone N-terminal tails.
Folding-based molecular simulations reveal mechanisms of the rotary motor F
1
–ATPase
Authors: Koga Nobuyasu , Takada Shoji
Published: March 2006

Biomolecular machines fulfill their function through large conformational changes that typically occur on the millisecond time scale or longer. Conventional atomistic simulations can only reach microseconds at the moment. Here, extending the minimalist model developed for protein folding, we propose the “switching Gō model” and use it to simulate the rotary motion of ATP-driven molecular motor F
1
–ATPase. The simulation recovers the unidirectional 120° rotation of the γ-subunit, the rotor. The rotation was induced solely by steric repulsion from the α
3
β
3
subunits, the stator, which undergoes conformation changes during ATP hydrolysis.
In silico
alanine mutagenesis further elucidated which residues play specific roles in the rotation. Finally, regarding the mechanochemical coupling scheme, we found that the tri-site model does not lead to successful rotation but that the always-bi-site model produces ≈30° and ≈90° substeps, perfectly in accord with experiments. In the always-bi-site model, the number of sites occupied by nucleotides is always two during the hydrolysis cycle. This study opens up an avenue of simulating functional dynamics of huge biomolecules that occur on the millisecond time scales involving large-amplitude conformational change.
The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease
Authors: Shillingford Jonathan M. , Murcia Noel S. , Larson Claire H. , Low Seng Hui , Hedgepeth Ryan , Brown Nicole , Flask Chris A. , Novick Andrew C. , Goldfarb David A. , Kramer-Zucker Albrecht , Walz Gerd , Piontek Klaus B. , Germino Gregory G. , Weimbs Thomas
Published: March 2006

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently leads to renal failure. Mutations in polycystin-1 (PC1) underlie most cases of ADPKD, but the function of PC1 has remained poorly understood. No preventive treatment for this disease is available. Here, we show that the cytoplasmic tail of PC1 interacts with tuberin, and the mTOR pathway is inappropriately activated in cyst-lining epithelial cells in human ADPKD patients and mouse models. Rapamycin, an inhibitor of mTOR, is highly effective in reducing renal cystogenesis in two independent mouse models of PKD. Treatment of human ADPKD transplant-recipient patients with rapamycin results in a significant reduction in native polycystic kidney size. These results indicate that PC1 has an important function in the regulation of the mTOR pathway and that this pathway provides a target for medical therapy of ADPKD.
Hyperaccumulation of arsenic in the shoots of
Arabidopsis
silenced for arsenate reductase (ACR2)
Authors: Dhankher Om Parkash , Rosen Barry P. , McKinney Elizabeth C. , Meagher Richard B.
Published: March 2006

Endogenous plant arsenate reductase (ACR) activity converts arsenate to arsenite in roots, immobilizing arsenic below ground. By blocking this activity, we hoped to construct plants that would mobilize more arsenate aboveground. We have identified a single gene in the
Arabidopsis thaliana
genome,
ACR2
, with moderate sequence homology to yeast arsenate reductase. Expression of
ACR2
cDNA in
Escherichia coli
complemented the arsenate-resistant and arsenate-sensitive phenotypes of various bacterial
ars
operon mutants. RNA interference reduced ACR2 protein expression in
Arabidopsis
to as low as 2% of wild-type levels. The various knockdown plant lines were more sensitive to high concentrations of arsenate, but not arsenite, than wild type. The knockdown lines accumulated 10- to 16-fold more arsenic in shoots (350–500 ppm) and retained less arsenic in roots than wild type, when grown on arsenate medium with <8 ppm arsenic. Reducing expression of
ACR2
homologs in tree, shrub, and grass species should play a vital role in the phytoremediation of environmental arsenic contamination.
Structural basis of docking interactions between ERK2 and MAP kinase phosphatase 3
Authors: Liu Sijiu , Sun Jin-Peng , Zhou Bo , Zhang Zhong-Yin
Published: March 2006

Mitogen-activated protein (MAP) kinases are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. Signaling efficiency and specificity are modulated in large part by docking interactions between individual MAP kinase and the kinase interaction motif (KIM), (R/K)
2–3
-X
1–6
-Φ
A
-X-Φ
B
, in its cognate kinases, phosphatases, scaffolding proteins, and substrates. We have determined the crystal structure of extracellular signal-regulated protein kinase 2 bound to the KIM peptide from MAP kinase phosphatase 3, an extracellular signal-regulated protein kinase 2-specific phosphatase. The structure reveals that the KIM docking site, situated in a noncatalytic region opposite of the kinase catalytic pocket, is comprised of a highly acidic patch and a hydrophobic groove, which engage the basic and Φ
A
-X-Φ
B
residues, respectively, in the KIM sequence. The specific docking interactions observed in the structure consolidate all known biochemical data. In addition, structural comparison indicates that the KIM docking site is conserved in all MAP kinases. The results establish a structural model for understanding how MAP kinases interact with their regulators and substrates and provide new insights into how MAP kinase docking specificity can be achieved.
Simulation of polymer translocation through protein channels
Authors: Muthukumar M. , Kong C. Y.
Published: March 2006

A modeling algorithm is presented to compute simultaneously polymer conformations and ionic current, as single polymer molecules undergo translocation through protein channels. The method is based on a combination of Langevin dynamics for coarse-grained models of polymers and the Poisson–Nernst–Planck formalism for ionic current. For the illustrative example of ssDNA passing through the α-hemolysin pore, vivid details of conformational fluctuations of the polymer inside the vestibule and β-barrel compartments of the protein pore, and their consequent effects on the translocation time and extent of blocked ionic current are presented. In addition to yielding insights into several experimentally reported puzzles, our simulations offer experimental strategies to sequence polymers more efficiently.
l-Tartaric acid synthesis from vitamin C in higher plants
Authors: DeBolt Seth , Cook Douglas R. , Ford Christopher M.
Published: March 2006

The biosynthetic pathway ofl-tartaric acid, the form most commonly encountered in nature, and its catabolic ties to vitamin C, remain a challenge to plant scientists. Vitamin C andl-tartaric acid are plant-derived metabolites with intrinsic human value. In contrast to most fruits during development, grapes accumulatel-tartaric acid, which remains within the berry throughout ripening. Berry taste and the organoleptic properties and aging potential of wines are intimately linked to levels ofl-tartaric acid present in the fruit, and those added during vinification. Elucidation of the reactions relatingl-tartaric acid to vitamin C catabolism in the Vitaceae showed that they proceed via the oxidation ofl-idonic acid, the proposed rate-limiting step in the pathway. Here we report the use of transcript and metabolite profiling to identify candidate cDNAs from genes expressed at developmental times and in tissues appropriate forl-tartaric acid biosynthesis in grape berries. Enzymological analyses of one candidate confirmed its activity in the proposed rate-limiting step of the direct pathway from vitamin C to tartaric acid in higher plants. Surveying organic acid content inVitisand related genera, we have identified a non-tartrate-forming species in which this gene is deleted. This species accumulates in excess of three times the levels of vitamin C than comparably ripe berries of tartrate-accumulating species, suggesting that modulation of tartaric acid biosynthesis may provide a rational basis for the production of grapes rich in vitamin C.
The inherent structure landscape of a protein
Authors: Nakagawa Naoko , Peyrard Michel
Published: March 2006

Using the Gō model of a real protein, we explore the landscape of its metastable structures. First, we show how the inherent structure energy density can be obtained from the probability density determined by sampling molecular dynamics trajectories and quenching. The analysis of the inherent structure landscape can characterize the folding transition. Then we show how thermodynamics of the inherent states can be established to study the equilibrium properties of proteins. Our work brings some elements into the current discussion about the protein dynamical transition. The study uses a simplified model to illustrate the ideas, but, as the inherent structure landscape is much simpler than the free energy surface of the protein, it appears to be accessible for an all-atom model of a small protein, at the expense of much longer calculations.
Suppression of prion protein in livestock by RNA interference
Authors: Golding Michael C. , Long Charles R. , Carmell Michelle A. , Hannon Gregory J. , Westhusin Mark E.
Published: March 2006

Given the difficulty of applying gene knockout technology to species other than mice, we decided to explore the utility of RNA interference (RNAi) in silencing the expression of genes in livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability to suppress the expression of caprine and bovine prion protein (PrP). Lentiviral vectors were used to deliver a transgene expressing GFP and an shRNA targeting PrP into goat fibroblasts. These cells were then used for nuclear transplantation to produce a cloned goat fetus, which was surgically recovered at 81 days of gestation and compared with an age-matched control derived by natural mating. All tissues examined in the cloned fetus expressed GFP, and PCR analysis confirmed the presence of the transgene encoding the PrP shRNA. Most relevant, Western blot analysis performed on brain tissues comparing the transgenic fetus with control demonstrated a significant (>90%) decrease in PrP expression levels. To confirm that similar methodologies could be applied to the bovine, recombinant virus was injected into the perivitelline space of bovine ova. After
in vitro
fertilization and culture, 76% of the blastocysts exhibited GFP expression, indicative that they expressed shRNAs targeting PrP. Our results provide strong evidence that the approach described here will be useful in producing transgenic livestock conferring potential disease resistance and provide an effective strategy for suppressing gene expression in a variety of large-animal models.
Green primaries: Environmentally friendly energetic complexes
Authors: Huynh My Hang V. , Hiskey Michael A. , Meyer Thomas J. , Wetzler Modi
Published: March 2006

Primary explosives are used in small quantities to generate a detonation wave when subjected to a flame, heat, impact, electric spark, or friction. Detonation of the primary explosive initiates the secondary booster or main-charge explosive or propellant. Long-term use of lead azide and lead styphnate as primary explosives has resulted in lead contamination at artillery and firing ranges and become a major health hazard and environmental problem for both military and civilian personnel. Devices using lead primary explosives are manufactured by the tens of millions every year in the United States from primers for bullets to detonators for mining. Although substantial synthetic efforts have long been focused on the search for greener primary explosives, this unresolved problem has become a “holy grail” of energetic materials research. Existing candidates suffer from instability or excessive sensitivity, or they possess toxic metals or perchlorate. We report here four previously undescribed green primary explosives based on complex metal dianions and environmentally benign cations, (cat)
2
[M
II
(NT)
4
(H
2
O)
2
] (where cat is NH
4
+
or Na
+
, M is Fe
2+
or Cu
2+
, and NT
−
is 5-nitrotetrazolato-
N
2
). They are safer to prepare, handle, and transport than lead compounds, have comparable initiation efficiencies to lead azide, and offer rapid reliable detonation comparable with lead styphnate. Remarkably, they possess all current requirements for green primary explosives and are suitable to replace lead primary explosives in detonators. More importantly, they can be synthesized more safely, do not pose health risks to personnel, and cause much less pollution to the environment.
A protein interaction surface in nonribosomal peptide synthesis mapped by combinatorial mutagenesis and selection
Authors: Lai Jonathan R. , Fischbach Michael A. , Liu David R. , Walsh Christopher T.
Published: March 2006

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with
in vivo
selection for the production of the
Escherichia coli
NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine in a structural model, and two of these positions are in the predicted helix 3 region. Subsequent
in vitro
studies are consistent with the hypothesis that these residues form a surface on EntB required for interaction with EntF. These results suggest that helix 3 is a major recognition element in EntB-ArCP and demonstrate the utility of selection-based approaches for studying NRPS biosynthesis.
NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression
Authors: Rosendorff Adam , Sakakibara Shuhei , Lu Sixin , Kieff Elliott , Xuan Yan , DiBacco Alessandra , Shi Yujiang , Shi Yang , Gill Grace
Published: March 2006

Small ubiquitin-like modifier (SUMO) modification of transcription factors is generally associated with repression. Reverse genetic analysis of SUMO-1, and -2 conserved residues emphasized the importance of dual charge reversals in abrogating the critical role of SUMO-2 K33, K35, and K42 in repression. GST-SUMO-2-affinity chromatography followed by liquid chromatography (LC)-MS analysis identified proteins that appeared to bind preferentially to WT SUMO-2 versus SUMO-2 K33E and K35E. LSD1, NXP-2, KIAA0809 (ARIP4), SAE2, RanGAP1, PELP1, and SETDB1 bound to SUMO-2 and not to SUMO-2 K33E, K42E, or K35E and K42E. Although LSD1 is a histone lysine demethylase, and histone H3K4 was demethylated at a SUMO-2-repressed promoter, neither overexpression of a dominant-negative LSD1 nor LSD1 depletion with RNA interference affected SUMO-2-mediated repression, indicating that LSD1 is not essential for repression, in this context. When tethered to a promoter by fusion to Gal4, NXP-2 repressed transcription, consistent with a role for NXP-2 in SUMO-mediated repression. SUMO-2-associated proteins identified in this study may contribute to SUMO-dependent regulation of transcription or other processes.
Transfer characteristics of the hair cell’s afferent synapse
Authors: Keen Erica C. , Hudspeth A. J.
Published: March 2006

The sense of hearing depends on fast, finely graded neurotransmission at the ribbon synapses connecting hair cells to afferent nerve fibers. The processing that occurs at this first chemical synapse in the auditory pathway determines the quality and extent of the information conveyed to the central nervous system. Knowledge of the synapse’s input–output function is therefore essential for understanding how auditory stimuli are encoded. To investigate the transfer function at the hair cell’s synapse, we developed a preparation of the bullfrog’s amphibian papilla. In the portion of this receptor organ representing stimuli of 400–800 Hz, each afferent nerve fiber forms several synaptic terminals onto one to three hair cells. By performing simultaneous voltage-clamp recordings from presynaptic hair cells and postsynaptic afferent fibers, we established that the rate of evoked vesicle release, as determined from the average postsynaptic current, depends linearly on the amplitude of the presynaptic Ca
2+
current. This result implies that, for receptor potentials in the physiological range, the hair cell’s synapse transmits information with high fidelity.
Songbirds tune their vocal tract to the fundamental frequency of their song
Authors: Riede Tobias , Suthers Roderick A. , Fletcher Neville H. , Blevins William E.
Published: March 2006

In human speech, the sound generated by the larynx is modified by articulatory movements of the upper vocal tract, which acts as a variable resonant filter concentrating energy near particular frequencies, or formants, essential in speech recognition. Despite its potential importance in vocal communication, little is known about the presence of tunable vocal tract filters in other vertebrates. The tonal quality of much birdsong, in which upper harmonics have relatively little energy, depends on filtering of the vocal source, but the nature of this filter is controversial. Current hypotheses treat the songbird vocal tract as a rigid tube with a resonance that is modulated by the end-correction of a variable beak opening. Through x-ray cinematography of singing birds, we show that birdsong is accompanied by cyclical movements of the hyoid skeleton and changes in the diameter of the cranial end of the esophagus that maintain an inverse relationship between the volume of the oropharyngeal cavity and esophagus and the song’s fundamental frequency. A computational acoustic model indicates that this song-related motor pattern tunes the major resonance of the oropharyngeal–esophageal cavity to actively track the song’s fundamental frequency.
Reelin expression and glycosylation patterns are altered in Alzheimer’s disease
Authors: Botella-López Arancha , Burgaya Ferran , Gavín Rosalina , García-Ayllón M. Salud , Gómez-Tortosa Estrella , Peña-Casanova Jordi , Ureña Jesús M. , Del Río José A. , Blesa Rafael , Soriano Eduardo , Sáez-Valero Javier
Published: March 2006

Reelin is a glycoprotein that is essential for the correct cytoarchitectonic organization of the developing CNS. Its function in the adult brain is less understood, although it has been proposed that Reelin is involved in signaling pathways linked to neurodegeneration. Here we analyzed Reelin expression in brains and cerebrospinal fluid (CSF) from Alzheimer’s disease (AD) patients and nondemented controls. We found a 40% increase in the Reelin protein levels in the cortex of AD patients compared with controls. Similar increases were detected at the Reelin mRNA transcriptional level. This expression correlates with parallel increases in CSF but not in plasma samples. Next, we examined whether CSF Reelin levels were also altered in neurological diseases, including frontotemporal dementia, progressive supranuclear palsy, and Parkinson’s disease. The Reelin 180-kDa band increased in all of the neurodegenerative disorders analyzed. Moreover, the 180-kDa Reelin levels correlated positively with Tau protein in CSF. Finally, we studied the pattern of Reelin glycosylation by using several lectins and the anti-HNK-1 antibody. Glycosylation differed in plasma and CSF. Furthermore, the pattern of Reelin lectin binding differed between the CSF of controls and in AD. Our results show that Reelin is up-regulated in the brain and CSF in several neurodegenerative diseases and that CSF and plasma Reelin have distinct cellular origins, thereby supporting that Reelin is involved in the pathogenesis of a number of neurodegenerative diseases.
A functional AMPA receptor–calcium channel complex in the postsynaptic membrane
Authors: Kang Myoung-Goo , Chen Chien-Chang , Wakamori Minoru , Hara Yuji , Mori Yasuo , Campbell Kevin P.
Published: March 2006

Ca
2+
channels play critical roles in the regulation of synaptic activity. In contrast to the well established function of voltage-activated Ca
2+
channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the Ca
2+
channels in the postsynaptic membrane. In this study, we demonstrated the functional association of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal Ca
2+
channels. A series of biochemical studies showed the specific association of Ca
v
2.1 (α
1A
-class) and Ca
v
2.2 (α
1B
-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and Ca
2+
imaging analyses of recombinant Ca
v
2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of Ca
2+
([Ca
2+
]
i
) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of Ca
2+
channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity.
Complex haplotypes derived from noncoding polymorphisms of the intronless α
2A
-adrenergic gene diversify receptor expression
Authors: Small Kersten M. , Brown Kari M. , Seman Carrie A. , Theiss Cheryl T. , Liggett Stephen B.
Published: March 2006

α
2A
-adrenergic receptors (α
2A
AR) regulate multiple central nervous system, cardiovascular, and metabolic processes including neurotransmitter release, platelet aggregation, blood pressure, insulin secretion, and lipolysis. Complex diseases associated with α
2A
AR dysfunction display familial clustering, phenotypic heterogeneity, and interindividual variability in response to therapy targeted to α
2A
ARs, suggesting common, functional polymorphisms. In a multiethnic discovery cohort we identified 16 single-nucleotide polymorphisms (SNPs) in the α
2A
AR gene organized into 17 haplotypes of two major phylogenetic clades. In contrast to other adrenergic genes, variability of the α
2A
AR was primarily due to SNPs in the promoter, 5′ UTR and 3′ UTR, as opposed to the coding block. Marked ethnic variability in the frequency of SNPs and haplotypes was observed: one haplotype represented 70% of Caucasians, whereas Africans and Asians had a wide distribution of less common haplotypes, with the highest haplotype frequencies being 16% and 35%, respectively. Despite the compact nature of this intronless gene, local linkage disequilibrium between a number of SNPs was low and ethnic-dependent. Whole-gene transfections into BE(
2
)-C human neuronal cells using vectors containing the entire ≈5.3-kb gene without exogenous promoters were used to ascertain the effects of haplotypes on α
2A
AR expression. Substantial differences (
P
< 0.001) in transcript and cell-surface protein expression, by as much as ≈5-fold, was observed between haplotypes, including those with common frequencies. Thus, signaling by this virtually ubiquitous receptor is under major genetic influence, which may be the basis for highly divergent phenotypes in complex diseases such as systemic and pulmonary hypertension, heart failure, diabetes, and obesity.
CXCL1 induced by prostaglandin E2 promotes angiogenesis in colorectal cancer
Authors: Wang Dingzhi , Wang Haibin , Brown Joanne , Daikoku Takiko , Ning Wei , Shi Qiong , Richmond Ann , Strieter Robert , Dey Sudhansu K. , DuBois Raymond N.
Published: March 2006

Chronic inflammation is a well-known risk factor for cancer. Proinflammatory mediators such as prostaglandin E2 (PGE2) promote colorectal tumor growth by stimulating angiogenesis, cell invasion, and cell growth, and inhibiting apoptosis. Molecules that regulate tumor-associated angiogenesis provide promising therapeutic targets for treatment of colorectal cancer (CRC) as indicated by the recent development of the novel anti-angiogenic agent bevacizumab (Avastin). However, use of this drug only prolongs survival by several months, highlighting the importance of finding more effective treatment regimens. We report here that PGE2 induces expression of CXCL1 (growth-regulated oncogene α), a pro-angiogenic chemokine, in human CRC cells. More importantly, CXCL1 released from carcinoma cells induces microvascular endothelial cell migration and tube formation in vitro. Furthermore, PGE2 promotes tumor growth in vivo by induction of CXCL1 expression, which results in increased tumor microvessel formation. These results have potential clinical significance because we found that CXCL1 expression correlates with PGE2 levels in human CRCs. Collectively, our findings show for the first time that CXCL1 is regulated by PGE2 and indicate that CXCL1 inhibitors should be evaluated further as potential anti-angiogenic agents for treatment of CRC.
Regulation of phosphatidylinositol 3–kinase by polyisoprenyl phosphates in neutrophil-mediated tissue injury
Authors: Bonnans Caroline , Fukunaga Koichi , Keledjian Raquel , Petasis Nicos A. , Levy Bruce D.
Published: March 2006

Neutrophils play a central role in host defense, inflammation, and tissue injury. Recent findings indicate a novel role for polyisoprenyl phosphates (PIPPs) as natural down-regulatory signals in neutrophils. The relationship between PIPPs and neutrophil early activating signals, such as phosphoinositides, has not been previously determined. Here, we establish presqualene diphosphate (PSDP) as an endogenous PIPP regulator of phosphatidylinositol 3–kinase (PI3K). In human neutrophils, leukotriene B4 (LTB4) triggered rapid decreases in PSDP and reciprocal increases in PI3K activity. In addition, PSDP was identified by gas chromatography/mass spectrometry in p110γ–PI3K immunoprecipitates obtained 30 s after LTB4, indicating a physical interaction between PSDP and PI3K in activated neutrophils. Moreover, PSDP (0.4–800 pmol) directly inhibited recombinant human p110γ-PI3K activity. During an experimental model of lung injury and inflammation, a reciprocal relationship was also present in vivo for lung PSDP and PI3K activity. To investigate its therapeutic potential, we developed a new PSDP structural mimetic that blocked human neutrophil activation and mouse lung PI3K activity and inflammation. Together, our findings indicate that PSDP is an endogenous PI3K inhibitor, and suggest that in inflammatory diseases characterized by excessive neutrophil activation, PIPPs can serve as structural templates in a novel antineutrophil therapeutic strategy to limit tissue injury.
Secondary memory CD8+ T cells are more protective but slower to acquire a central–memory phenotype
Authors: Jabbari Ali , Harty John T.
Published: March 2006

The formation of memory CD8 T cells is an important goal of vaccination. However, although widespread use of booster immunizations in humans generates secondary and tertiary CD8 T cell memory, experimental data are limited to primary CD8 T cell memory. Here, we show that, compared with primary memory CD8 T cells, secondary memory CD8 T cells exhibit substantially delayed conversion to a central–memory phenotype, as determined by CD62L expression and interleukin (IL)-2 production. This delayed conversion to a central–memory phenotype correlates with reduced basal proliferation and responsiveness to IL-15, although in vitro coculture with a high concentration of IL-15 is capable of inducing proliferation and CD62L upregulation. Functionally, secondary memory CD8 T cells are more protective in vivo on a per cell basis, and this may be explained by sustained lytic ability. Additionally, secondary memory CD8 T cells are more permissive than primary memory CD8 T cells for new T cell priming in lymph nodes, possibly suggesting a mechanism of replacement for memory T cells. Thus, primary and secondary memory CD8 T cells are functionally distinct, and the number of encounters with antigen influences memory CD8 T cell function.
A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis
Authors: Kim Nancy D. , Chou Richard C. , Seung Edward , Tager Andrew M. , Luster Andrew D.
Published: March 2006

Neutrophil recruitment into tissue plays an important role in host defense and disease pathogenesis, including the inflammatory arthritides. A multitude of diverse chemoattractants have been implicated in neutrophil recruitment, suggesting that they have overlapping functions in mediating this critical biological response. However, here we demonstrate a unique, non-redundant role for the leukotriene B4 receptor BLT1 in mediating neutrophil recruitment into the joint in the K/BxN mouse model of inflammatory arthritis. We demonstrate that neutrophil expression of BLT1 was absolutely required for arthritis generation and chemokine production in this model, and that specific BLT1 inhibition reversed established disease. Adoptive transfer of wild-type (WT) neutrophils restored arthritis and chemokine production in BLT1−/− mice. Surprisingly, the primary effect of the transferred WT neutrophils into BLT1−/− mice was to promote the entry of endogenous BLT1−/− neutrophils into the joints of these mice. However, continued joint inflammation was dependent on the presence of WT neutrophils, indicating an ongoing specific requirement for BLT1-activated neutrophils in mediating BLT1−/− neutrophil recruitment by other chemoattractants. These experiments demonstrate that neutrophil BLT1 functions in a novel and essential non–cell-autonomous manner to enable the recruitment of additional neutrophils not expressing this receptor, thereby amplifying the inflammatory response in autoantibody-induced arthritis.
Neutrophil-derived leukotriene B4 is required for inflammatory arthritis
Authors: Chen Mei , Lam Bing K. , Kanaoka Yoshihide , Nigrovic Peter A. , Audoly Laurent P. , Austen K. Frank , Lee David M.
Published: March 2006

Neutrophils serve as a vanguard of the acute innate immune response to invading pathogens. Neutrophils are also abundant at sites of autoimmune inflammation, such as the rheumatoid joint, although their pathophysiologic role is incompletely defined and relevant effector functions remain obscure. Using genetic and pharmacologic approaches in the K/BxN serum transfer model of arthritis, we find that autoantibody-driven erosive synovitis is critically reliant on the generation of leukotrienes, and more specifically on leukotriene B4 (LTB4), for disease induction as well as perpetuation. Pursuing the cellular source for this mediator, we find via reconstitution experiments that mast cells are a dispensable source of leukotrienes, whereas arthritis susceptibility can be restored to leukotriene-deficient mice by intravenous administration of wild-type neutrophils. These experiments demonstrate a nonredundant role for LTB4 in inflammatory arthritis and define a neutrophil mediator involved in orchestrating the synovial eruption.
Critical role of endothelial P-selectin glycoprotein ligand 1 in chronic murine ileitis
Authors: Rivera-Nieves Jesús , Burcin Tracy L. , Olson Timothy S. , Morris Margaret A. , McDuffie Marcia , Cominelli Fabio , Ley Klaus
Published: March 2006

L-selectin ligands might be relevant for inflammatory cell trafficking into the small intestine in a spontaneous model of chronic ileitis (i.e., SAMP1/YitFc mice). Immunoblockade of peripheral node addressin or mucosal addressin cell adhesion molecule 1 failed to ameliorate ileitis, whereas P-selectin glycoprotein ligand 1 (PSGL-1) neutralization attenuated both the adoptively transferred and spontaneous disease. PSGL-1 was detected in venules of mesenteric lymph node and small intestine by immunohistochemistry and confirmed by real-time reverse transcription polymerase chain reaction and flow cytometry. In addition, reconstitution of wild-type mice with PSGL-1−/− bone marrow demonstrated that PSGL-1 messenger RNA and PSGL-1 protein expression remained on endothelium, localized within mesenteric lymph node and small intestine. Endothelial PSGL-1 bound P-selectin–IgG and its blockade or genetic deletion altered the recruitment of lymphocytes to the small intestine, as revealed by intravital microscopy and homing studies. Endothelial expression of PSGL-1 adds a new dimension to the various cellular interactions involved in small intestinal recruitment. Thus, the multiple roles of PSGL-1 may explain why targeting this single adhesion molecule results in attenuation of chronic murine ileitis, a disease previously resistant to antiadhesion molecule strategies.
Finding regions of significance in SELDI measurements for identifying protein biomarkers
Authors: Tan Chuen Seng , Ploner Alexander , Quandt Andreas , Lehtiö Janne , Pawitan Yudi
Published: March 2006

Motivation: There is a well-recognized potential of protein expression profiling using the surface-enhanced laser desorption and ionization technology for discovering biomarkers that can be applied in clinical diagnosis, prognosis and therapy prediction. The pre-processing of the raw data, however, is still problematic.

Methods: We focus on the peak detection step, where the standard method is marked by poor specificity. Currently, scientists need to inspect individual spectra visually and laboriously in order to verify that spectral peaks identified by the standard method are real. Motivated by this multi-spectral process, we investigate an analytical approach—called RS for ‘regions of significance’—that reduces the data to a single spectrum of F-statistics capturing significant variability between spectra. To account for multiple testing, we use a false discovery rate criterion for identifying potentially interesting proteins.

Results: We show that RS has better operating characteristics than several existing methods and demonstrate routine applications on a number of large datasets.

Availability: RS is implemented in an R package called ProSpect which is available at

Contact:  yudi.pawitan@ki.se

Supplementary information: Supplementary data are available at Bioinformatics online.
GBPM: GRID-based pharmacophore model: concept and application studies to protein–protein recognition
Authors: Ortuso Francesco , Langer Thierry , Alcaro Stefano
Published: March 2006

Motivation: Automatic procedures to obtain pharmacophore models from experimentally determined macromolecular complexes can help in the drug discovery process, especially when protein–protein recognition plays an important biological role.

Results: The GRID-based pharmacophore model (GBPM) is a fully objective method for defining most relevant interaction areas in complexes deriving pharmacophore models from three-dimensional (3D) molecular structure information. It is based on logical and clustering operations with 3D maps computed by the GRID program on structurally known molecular complexes. In this manuscript we describe the concept and discuss application examples regarding protein–protein recognition. In particular two complexes selected in the Protein Data Bank have been tested to evaluate the GBPM capability to identify interaction areas. The results obtained show the capabilities of this new bioinformatic method.

Availability: The GBPM method has not been developed as a new computational code. It is based on the combination of existing scientific programs.

Contact:  alcaro@unicz.it
Heart Rate Variability–guided Prophylactic Treatment of Severe Hypotension after Subarachnoid Block for Elective Cesarean Delivery
Authors: Hanss Robert , Bein Berthold , Francksen Helga , Scherkl Wiebke , Bauer Martin , Doerges Volker , Steinfath Markus , Scholz Jens , Tonner Peter H.
Published: March 2006

Background

Baseline low-to-high frequency ratio (LF/HF) of heart rate variability predicted hypotension after subarachnoid block (SAB). LF/HF-guided treatment of hypotension with vasopressors or colloids was investigated.

Methods

In 80 women scheduled to undergo cesarean delivery during SAB, LF/HF and systolic blood pressure (SBP) were analyzed. Patients were randomly assigned to a control group (n = 40) or a treatment group (n = 40). Control patients were assigned by their baseline LF/HF to one of two subgroups: LF/HF less than 2.5 or LF/HF greater than 2.5. Treatment patients with baseline LF/HF greater than 2.5 were treated with vasopressor infusion right after SAB (n = 20) or colloid prehydration until LF/HF decreased below 2.5 (n = 20). The incidences of hypotension (SBP < 80 mmHg) and hypertension (SBP > 140 mmHg) were investigated. LF/HF is presented as median and range, and SBP is presented as mean +/- SD.

Results

Three of 17 control patients with low baseline LF/HF (1.7 [1.3/1.8]) demonstrated hypotension, and mean SBP remained stable (lowest SBP = 105 +/- 14 mmHg). In contrast, 20 of 23 control patients with high baseline LF/HF (3.8 [3.3/4.8]; P < 0.0001 vs. low baseline LF/HF) demonstrated hypotension after SAB: lowest SBP = 78 +/- 15 mmHg (P < 0.0001 vs. lowest SBP of control group with low baseline LF/HF). LF/HF-guided vasopressor therapy prevented hypotension in 19 of 20 patients: baseline SBP = 123 +/- 15 mmHg, lowest SBP = 116 +/- 17 mmHg. Mean prophylactic colloid infusion of 1,275 +/- 250 ml reduced elevated baseline LF/HF from 5.4 (4.1/7.5) to 1.3 (0.8/1.59) (P < 0.0001). Hypotension was prevented in 17 of 20 patients: baseline SBP = 115 +/- 13 mmHg, lowest SBP = 104 +/- 19 mmHg. No hypertensive episode was recognized.

Conclusions

LF/HF may be a tool to guide prophylactic therapy of patients at high risk for hypotension after SAB. Vasopressor therapy tended to be more effective compared with colloid prehydration.
Uterine Cervical Afferents in Thoracolumbar Dorsal Root Ganglia Express Transient Receptor Potential Vanilloid Type 1 Channel and Calcitonin Gene–related Peptide, but Not P2X3 Receptor and Somatostatin
Authors: Tong Chuanyao , Conklin Dawn , Clyne Brittany B. , Stanislaus Jennifer D. , Eisenach James C.
Published: March 2006

Background

Little is known regarding the phenotype of afferents that innervate the uterine cervix. Chronic estrogen sensitizes uterine cervical afferents to mechanical distension, but whether this reflects changes in afferent neurotransmitter or excitatory ion channel expression is unknown. The authors used immunocytochemistry to characterize uterine cervical afferents and the effects of estrogen on them.

Methods

Fluorogold was injected into the uterine cervix of intact rats (n = 7) and those with ovariectomy alone (n = 9) or with estrogen supplementation (n = 8). Bilateral dorsal root ganglia at T12-L2 were removed and immunostained for transient receptor potential vanilloid type 1 (TRPV1), P2X3 receptor, calcitonin gene-related peptide, and somatostatin. The proportion of fluorogold-traced dorsal root ganglion neurons expressing each of these markers was compared with untraced neurons.

Results

Most fluorogold-traced cells were found at L1 (> 55%) and were of small diameter (24 microm). TRPV1 expression was similar between traced and untraced cells, except the estrogen treatment increased TRPV1 expression in traced cells. Calcitonin gene-related peptide expression was greater in traced than in untraced cells, with no effect of experimental treatment. No traced cells expressed the P2X3 receptor or somatostatin, although each of these was present in untraced cells.

Conclusion

Uterine cervical afferents in the hypogastric nerve express TRPV1, an important nociceptive channel, which may play a role in estrogen-induced sensitization of cervical afferents. High expression of calcitonin gene-related peptide suggests a sensory and efferent role for this peptide. In contrast to other viscera, these afferents do not express somatostatin or P2X3 receptor, indicating a unique phenotype of these C fibers.
Prognosis Value of Plasma S100B Protein Levels after Subarachnoid Aneurysmal Hemorrhage
Authors: Weiss Nicolas , Sanchez-Peña Paola , Roche Sabine , Beaudeux Jean L. , Colonne Chantal , Coriat Pierre , Puybasset Louis
Published: March 2006

Background

S100B has been described as a biologic marker of neuronal damage. The purpose of this study was to assess its prognostic value in patients with subarachnoid aneurysmal hemorrhage.

Methods

Seventy-four patients (32 men and 42 women; age, 48 +/- 11 yr) admitted within 48 h after subarachnoid hemorrhage onset and treated by surgical clipping or coiling within 2 days after admission were included. World Federation of Neurological Surgeons, Fisher, and Glasgow outcome scores at intensive care unit discharge and at 6 months were evaluated. Blood concentrations of S100B were determined at admission and daily up to day 8.

Results

The time course of S100B was increased in patients with high World Federation of Neurological Surgeons and Fisher scores. Patients who underwent surgical clipping had an S100B time course longer than that of those who underwent coiling. This difference remained true after stratification for World Federation of Neurological Surgeons and Fisher scores. The threshold of mean daily value of S100B predicting a poor outcome at 6 months was 0.4 microg/l (sensitivity = 0.50 [95% confidence interval (CI), 0.29-0.71], specificity = 0.87[corrected] [95% CI, 0.76-0.95]). In multivariate analysis, high World Federation of Neurological Surgeons score (odds ratio = 9.5 [95% CI, 3.1-29.4]), mean daily S100B value above 0.4 microg/l (odds ratio = 7.3 [95% CI, 2.3-23.6]), and age (odds ratio = 1.08 per year [95% CI, 1.01-1.15]) were independent predictors of a poor 6-month outcome (Glasgow outcome score 1-3).

Conclusion

Mean daily value of S100B assessed during the first 8 days is a prognostic tool complementary to initial clinical evaluation in subarachnoid hemorrhage patients.
Reversal of Rocuronium-induced Neuromuscular Block by the Selective Relaxant Binding Agent Sugammadex
Authors: Sorgenfrei Iben F. , Norrild Kathrine , Larsen Per Bo , Stensballe Jakob , Østergaard Doris , Prins Martine E. , Viby-Mogensen Jørgen
Published: March 2006

Background

Sugammadex (Org 25969) forms a complex with steroidal neuromuscular blocking agents, thereby reversing neuromuscular block. This study investigated the dose-response relation, safety, and pharmacokinetics of sugammadex to reverse rocuronium-induced block.

Methods

Twenty-seven male surgical patients aged 18-64 yr were randomly assigned to receive placebo or sugammadex (0.5, 1.0, 2.0, 3.0, or 4.0 mg/kg) for reversal of 0.6 mg/kg rocuronium-induced neuromuscular block. Anesthesia was induced and maintained using intravenous fentanyl and propofol. Neuromuscular function was assessed using acceleromyography. Sugammadex or placebo was administered at reappearance of T2 of the train-of-four. The primary efficacy variable was the time required for recovery to a train-of-four ratio of 0.9.

Results

Sugammadex decreased median recovery time in a dose-dependent manner from 21.0 min in the placebo group to 1.1 min in the group receiving 4.0 mg/kg sugammadex. Doses of sugammadex of 2.0 mg/kg or greater reversed rocuronium-induced neuromuscular block within 3 min. A median of 59-77% of sugammadex was excreted unchanged in the urine within 16 h, mostly in the first 8 h. Sugammadex increased the proportion of the rocuronium dose excreted unchanged in the urine (from a median of 19% in the placebo group to 53% in the 4.0-mg/kg group within 16 h). Sugammadex was safe and well tolerated. No evidence of recurarization was observed in any patient.

Conclusion

At doses of 2.0 mg/kg or greater, sugammadex safely reversed 0.6 mg/kg rocuronium-induced neuromuscular block in a dose-dependent manner. Sugammadex enhanced renal excretion of rocuronium and was excreted unchanged by the kidneys.
Atlantoaxial Subluxation in Different Intraoperative Head Positions in Patients with Rheumatoid Arthritis
Authors: Tokunaga Daisaku , Hase Hitoshi , Mikami Yasuo , Hojo Tatsuya , Ikoma Kazuya , Hatta Yoichiro , Ishida Masashi , Sessler Daniel I. , Mizobe Toshiki , Kubo Toshikazu
Published: March 2006

Background

Disorders of the cervical spine are often observed in patients with rheumatoid arthritis (RA). However, the best head position for RA patients with atlantoaxial subluxation in the perioperative period is unknown. This study investigated head position during general anesthesia for the patients with RA and proven atlantoaxial subluxation.

Methods

During anesthesia of patients with RA and proven atlantoaxial subluxation, the authors used fluoroscopy to obtain a lateral view of the upper cervical spine in four different positions: the mask position, the intubation position, the flat pillow position, and the protrusion position. Copies of the still fluoroscopic images were used to determine the anterior atlantodental interval, the posterior atlantodental interval, and the angle of atlas and axis (C1-C2 angle).

Results

The anterior atlantodental interval was significantly smaller in the protrusion position (2.3 mm) than in the flat pillow position (5.1 mm) (P < 0.05). The posterior atlantodental interval was significantly greater in the protrusion position (18.9 mm) than in the flat pillow position (16.2 mm) (P < 0.05). The C1-C2 angle was, on average, 9.3 degrees greater in the protrusion position than in the flat pillow position (P < 0.05).

Conclusion

This study showed that the protrusion position using a flat pillow and a donut-shaped pillow during general anesthesia reduced the anterior atlantodental interval and increased the posterior atlantodental interval in RA patients with atlantoaxial subluxation. This suggests that the protrusion position, which involves support of the upper cervical spine and extension at the craniocervical junction, might be advantageous for these patients.