bluuebunny/crossref_metadata_2025 huggingface dataset:
Variable character of O—O and M—O bonding in side-on (η
2
) 1:1 metal complexes of O
2
Authors: Cramer Christopher J. , Tolman William B. , Theopold Klaus H. , Rheingold Arnold L.
Published: April 2003

The structures and the O—O and M—O bonding characters of a series of reported side-on (η
2
) 1:1 metal complexes of O
2
are analyzed by using density functional theory calculations. Comparison of the calculated and experimental systems with respect to O—O bond distance, O—O stretching frequency, and O—O and M—O bond orders provides new insights into subtle influences relevant to O
2
activation processes in biology and catalysis. The degree of charge transfer from the generally electron-rich metals to the dioxygen fragment is found to be variable, such that there are species well described as superoxides, others well described as peroxides, and several cases having intermediate character. Increased charge transfer to dioxygen takes place via overlap of the metal d

xy

orbital with the in-plane π* orbital of O
2
and results in increased M—O bond orders and decreased O—O bond orders. Comparison of theory and experiment over the full range of compounds studied suggests that reevaluation of the O—O bond lengths determined from certain x-ray crystal structures is warranted; in one instance, an x-ray crystal structure redetermination was performed at low temperature, confirming the theoretical prediction. Librational motion of the coordinated O
2
is identified as a basis for significant underestimation of the O—O distance at high temperature.
Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis
Authors: da Fonseca Paula C. A. , Morris Stephen A. , Nerou Edmund P. , Taylor Colin W. , Morris Edward P.
Published: April 2003

The inositol 1,4,5-trisphosphate receptor (IP
3
R) is a tetrameric intracellular Ca
2+
channel, which mediates the release of Ca
2+
from the endoplasmic reticulum in response to many different extracellular stimuli. We present a 3D structure of the type 1 IP
3
R obtained by electron microscopy and single-particle analysis that reveals its domain organization. The IP
3
R has a flower-like appearance with fourfold symmetry and is made up of three distinct domains connected by slender links. By relating the organization of the structural domains to secondary-structure predictions and biochemical data we develop a model in which structural domains are mapped onto the amino acid sequence to deduce the location of the channel region and the cytoplasmic inositol 1,4,5-trisphosphate-binding and modulatory subdomains. The structure of the IP
3
R is compared with that of other tetrameric cation channels. The channel domain is similar in size and shape to its counterparts in the ryanodine receptor and the Shaker voltage-gated K
+
channel.
Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease
Authors: Welch Joel T. , Kearney William R. , Franklin Sonya J.
Published: April 2003

A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in α-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 ± 0.3 μM) and for Eu(III) (6.2 ± 0.3 μM) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helix-turn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metal-binding loop but also retains the α−α corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence.
Electrochemical and NMR spectroscopic studies of distal pocket mutants of nitrophorin 2: Stability, structure, and dynamics of axial ligand complexes
Authors: Shokhireva Tatjana Kh. , Berry Robert E. , Uno Elizabeth , Balfour Celia A. , Zhang Hongjun , Walker F. Ann
Published: April 2003

WT and leucine → valine distal pocket mutants of nitrophorin 2 (NP2) and their NO complexes have been investigated by spectroelectrochemistry. NO complexes of two of the mutants exhibit more positive reduction potential shifts than does the WT protein, thus indicating stabilization of the Fe(II)–NO state. This more positive reduction potential for NP2-L132V and the double mutant is consistent with the hypothesis that smaller valine residues may allow the heme to regain planarity instead of being significantly ruffled, as in WT NP2. Thus, ruffling may stabilize the Fe(III)–NO state, which is required for facile NO dissociation. NMR spectroscopic investigations show that the sterically demanding 2-methylimidazole ligand readily binds to all three distal pocket mutants to create low-spin Fe(III) complexes having axial ligands in nearly perpendicular planes; it also binds to the WT protein in the presence of higher concentrations of 2-methylimidazole, but yields a different ligand plane orientation than is present in any of the three distal pocket mutants. NOESY spectra of NP2–ImH mutants exhibit chemical exchange cross peaks, whereas WT NP2–ImH shows no chemical exchange. Chemical exchange in the case of the distal leucine → valine mutants is caused by ImH ligand orientational dynamics. The two angular orientations of the ImH ligand could be determined from the
1
H chemical shifts of the heme methyls, and the rate of interconversion of the two forms could be estimated from the NOESY diagonal and cross peak intensities.
K
eq
is 100 or larger and favors an orientation similar to that found for the WT NP2–ImH complex.
Purified particulate methane monooxygenase from
Methylococcus capsulatus
(Bath) is a dimer with both mononuclear copper and a copper-containing cluster
Authors: Lieberman Raquel L. , Shrestha Deepak B. , Doan Peter E. , Hoffman Brian M. , Stemmler Timothy L. , Rosenzweig Amy C.
Published: April 2003

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from
Methylococcus capsulatus
(Bath) and detected activity. The purified enzyme has a molecular mass of ≈200 kDa, probably corresponding to an α
2
β
2
γ
2
polypeptide arrangement. Each 200-kDa pMMO complex contains 4.8 ± 0.8 copper ions and 1.5 ± 0.7 iron ions. Electron paramagnetic resonance spectroscopic parameters corresponding to 40–60% of the total copper are consistent with the presence of a mononuclear type 2 copper site. X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states. Finally, extended x-ray absorption fine structure data are best fit with oxygen/nitrogen ligands and a 2.57-Å Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO.
The influence of the synergistic anion on iron chelation by ferric binding protein, a bacterial transferrin
Authors: Dhungana Suraj , Taboy Céline H. , Anderson Damon S. , Vaughan Kevin G. , Aisen Philip , Mietzner Timothy A. , Crumbliss Alvin L.
Published: April 2003

Although the presence of an exogenous anion is a requirement for tight Fe
3+
binding by the bacterial (
Neisseria
) transferrin
n
Fbp, the identity of the exogenous anion is not specific
in vitro. n
Fbp was reconstituted as a stable iron containing protein by using a number of different exogenous anions [arsenate, citrate, nitrilotriacetate, pyrophosphate, and oxalate (symbolized by X)] in addition to phosphate, predominantly present in the recombinant form of the protein. Spectroscopic characterization of the Fe
3+
/anion interaction in the reconstituted protein was accomplished by UV-visible and EPR spectroscopies. The affinity of the protein for Fe
3+
is anion dependent, as evidenced by the effective Fe
3+
binding constants (
K
′
eff
) observed, which range from 1 × 10
17
M
−1
to 4 × 10
18
M
−1
at pH 6.5 and 20°C. The redox potentials for Fe
3+
n
FbpX/Fe
2+
n
FbpX reduction are also found to depend on the identity of the synergistic anion required for Fe
3+
sequestration. Facile exchange of exogenous anions (Fe
3+
n
FbpX + X′ → Fe
3+
n
FbpX′ + X) is established and provides a pathway for environmental modulation of the iron chelation and redox characteristics of
n
Fbp. The affinity of the iron loaded protein for exogenous anion binding at pH 6.5 was found to decrease in the order phosphate > arsenate ∼ pyrophosphate > nitrilotriacetate > citrate ∼ oxalate ≫ carbonate. Anion influence on the iron primary coordination sphere through iron binding and redox potential modulation may have
in vivo
application as a mechanism for periplasmic control of iron delivery to the cytosol.
The organometallic active site of [Fe]hydrogenase: Models and entatic states
Authors: Darensbourg Marcetta Y. , Lyon Erica J. , Zhao Xuan , Georgakaki Irene P.
Published: April 2003

The simple organometallic, (μ-S
2
)Fe
2
(CO)
6
, serves as a precursor to synthetic analogues of the chemically rudimentary iron-only hydrogenase enzyme active site. The fundamental properties of the (μ-SCH
2
CH
2
CH
2
S)[Fe(CO)
3
]
2
compound, including structural mobility and regioselectivity in cyanide/carbon monoxide substitution reactions, relate to the enzyme active site in the form of transition-state structures along reaction paths rather than ground-state structures. Even in the absence of protein-based active-site organization, the ground-state structural model complexes are shown to serve as hydrogenase enzyme reaction models, H
2
uptake and H
2
production, with the input of photo- or electrochemical energy, respectively.
An engineered two-iron superoxide reductase lacking the [Fe(SCys)
4
] site retains its catalytic properties
in vitro
and
in
vivo
Authors: Emerson Joseph P. , Cabelli Diane E. , Kurtz Donald M.
Published: April 2003

Superoxide reductases (SORs) contain a characteristic square-pyramidal [Fe(NHis)
4
(SCys)] active site that catalyzes reduction of superoxide to hydrogen peroxide in several anaerobic bacteria and archaea. Some SORs, referred to as two-iron SORs (2Fe-SORs), also contain a lower-potential [Fe(SCys)
4
] site that is presumed to have an electron transfer function. However, the intra- and inter-subunit distances between [Fe(SCys)
4
] and [Fe(NHis)
4
(SCys)] iron centers within the 2Fe-SOR homodimer seem too long for efficient electron transfer between these sites. The possible role of the [Fe(SCys)
4
] site in 2Fe-SORs was addressed in this work by examination of an engineered
Desulfovibrio vulgaris
2Fe-SOR variant, C13S, in which one ligand residue of the [Fe(SCys)
4
] site, cysteine 13, was changed to serine. This single amino acid residue change destroyed the native [Fe(SCys)
4
] site with complete loss of its iron, but left the [Fe(NHis)
4
(SCys)] site and the protein homodimer intact. The spectroscopic, redox and superoxide reactivity properties of the [Fe(NHis)
4
(SCys)] site in the C13S variant were nearly indistinguishable from those of the wild-type 2Fe-SOR. Aerobic growth complementation of a superoxide dismutase (SOD)-deficient
Escherichia coli
strain showed that the presence of the [Fe(NHis)
4
(SCys)] site in C13S 2Fe-SOR was apparently sufficient to catalyze reduction of the intracellular superoxide to nonlethal levels. As is the case for the wild-type protein, C13S 2Fe-SOR did not show any detectable SOD activity, i.e., destruction of the [Fe(SCys)
4
] site did not unmask latent SOD activity of the [Fe(NHis)
4
(SCys)] site. Possible alternative roles for the [Fe(SCys)
4
] site in 2Fe-SORs are considered.
A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition
Authors: Junicke Henrik , Hart Jonathan R. , Kisko Jennifer , Glebov Oleg , Kirsch Ilan R. , Barton Jacqueline K.
Published: April 2003

A rhodium(III) complex,
rac
-[Rh(bpy)
2
phzi]
3+
(bpy, 2,2′-bipyridine; phzi, benzo[
a
]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)
2
(chrysi)]
3+
[chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 10
7
M
−1
, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design.
Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation
Authors: Nguyen Houbi , Jäger Marcus , Moretto Alessandro , Gruebele Martin , Kelly Jeffery W.
Published: April 2003

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded β-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28–37 residues), the availability of a quantitative reaction coordinate (φ
T
), the fast folding time scale (10s of μs), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory.
Enterobactin: An archetype for microbial iron transport
Authors: Raymond Kenneth N. , Dertz Emily A. , Kim Sanggoo S.
Published: April 2003

Bacteria have aggressive acquisition processes for iron, an essential nutrient. Siderophores are small iron chelators that facilitate cellular iron transport. The siderophore enterobactin is a triscatechol derivative of a cyclic triserine lactone. Studies of the chemistry, regulation, synthesis, recognition, and transport of enterobactin make it perhaps the best understood of the siderophore-mediated iron uptake systems, displaying a lot of function packed into this small molecule. However, recent surprises include the isolation of corynebactin, a closely related trithreonine triscatechol derivative lactone first found in Gram-positive bacteria, and the crystal structure of a ferric enterobactin complex of a protein identified as an antibacterial component of the human innate immune system.
Speculative synthetic chemistry and the nitrogenase problem
Authors: Lee Sonny C. , Holm Richard H.
Published: April 2003

There exist a limited but growing number of biological metal centers whose properties lie conspicuously outside the realm of known inorganic chemistry. The synthetic analogue approach, broadly directed, offers a powerful exploratory tool that can define intrinsic chemical possibilities for these sites while simultaneously expanding the frontiers of fundamental inorganic chemistry. This speculative application of analogue study is exemplified here in the evolution of synthetic efforts inspired by the cluster chemistry of biological nitrogen fixation.
Genetic characterization of glucose transporter function inLeishmania mexicana
Authors: Burchmore Richard J. S. , Rodriguez-Contreras Dayana , McBride Kathleen , Barrett Michael P. , Modi Govind , Sacks David , Landfear Scott M.
Published: April 2003

Both insect and mammalian life cycle stages ofLeishmania mexicanatake up glucose and express all three isoforms encoded by theLmGTglucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of theLmGTlocus that encompasses theLmGT1,LmGT2, andLmGT3genes. This Δlmgtnull mutant exhibited no detectable glucose transport activity. The growth rate of the Δlmgtknockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. Δlmgtcells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WTL. mexicanawere not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of eitherLmGT2orLmGT3, but not ofLmGT1, in Δlmgtnull mutants significantly restored growth as promastigotes, but onlyLmGT3expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in Δlmgtcells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.
Transcriptome dynamics of
Deinococcus radiodurans
recovering from ionizing radiation
Authors: Liu Yongqing , Zhou Jizhong , Omelchenko Marina V. , Beliaev Alex S. , Venkateswaran Amudhan , Stair Julia , Wu Liyou , Thompson Dorothea K. , Xu Dong , Rogozin Igor B. , Gaidamakova Elena K. , Zhai Min , Makarova Kira S. , Koonin Eugene V. , Daly Michael J.
Published: April 2003

Deinococcus radiodurans
R1 (DEIRA) is a bacterium best known for its extreme resistance to the lethal effects of ionizing radiation, but the molecular mechanisms underlying this phenotype remain poorly understood. To define the repertoire of DEIRA genes responding to acute irradiation (15 kGy), transcriptome dynamics were examined in cells representing early, middle, and late phases of recovery by using DNA microarrays covering ≈94% of its predicted genes. At least at one time point during DEIRA recovery, 832 genes (28% of the genome) were induced and 451 genes (15%) were repressed 2-fold or more. The expression patterns of the majority of the induced genes resemble the previously characterized expression profile of
recA
after irradiation. DEIRA
recA
, which is central to genomic restoration after irradiation, is substantially up-regulated on DNA damage (early phase) and down-regulated before the onset of exponential growth (late phase). Many other genes were expressed later in recovery, displaying a growth-related pattern of induction. Genes induced in the early phase of recovery included those involved in DNA replication, repair, and recombination, cell wall metabolism, cellular transport, and many encoding uncharacterized proteins. Collectively, the microarray data suggest that DEIRA cells efficiently coordinate their recovery by a complex network, within which both DNA repair and metabolic functions play critical roles. Components of this network include a predicted distinct ATP-dependent DNA ligase and metabolic pathway switching that could prevent additional genomic damage elicited by metabolism-induced free radicals.
A duodenally absorbable CXC chemokine receptor 4 antagonist, KRH-1636, exhibits a potent and selective anti-HIV-1 activity
Authors: Ichiyama Kozi , Yokoyama-Kumakura Sei , Tanaka Yuetsu , Tanaka Reiko , Hirose Kunitaka , Bannai Kenji , Edamatsu Takeo , Yanaka Mikiro , Niitani Yoshiaki , Miyano-Kurosaki Naoko , Takaku Hiroshi , Koyanagi Yoshio , Yamamoto Naoki
Published: April 2003

A low molecular weight nonpeptide compound, KRH-1636, efficiently blocked replication of various T cell line-tropic (X4) HIV type 1 (HIV-1) in MT-4 cells and peripheral blood mononuclear cells through the inhibition of viral entry and membrane fusion via the CXC chemokine receptor (CXCR)4 coreceptor but not via CC chemokine receptor 5. It also inhibited binding of the CXC chemokine, stromal cell-derived factor 1α, to CXCR4 specifically and subsequent signal transduction. KRH-1636 prevented monoclonal antibodies from binding to CXCR4 without down-modulation of the coreceptor. The inhibitory effect against X4 viral replication by KRH-1636 was clearly reproduced in the human peripheral blood lymphocyte/severe combined immunodeficiency mouse system. Furthermore, this compound was absorbed into the blood after intraduodenal administration as judged by anti-HIV-1 activity and liquid chromatography MS in the plasma. Thus, KRH-1636 seems to be a promising agent for the treatment of HIV-1 infection.
The role of disulfide bonds in the assembly and function of MD-2
Authors: Mullen Gregory E. D. , Kennedy Margaret N. , Visintin Alberto , Mazzoni Alessandra , Leifer Cynthia A. , Davies David R. , Segal David M.
Published: April 2003

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDS/PAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-κB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.
Neuregulin 1–erbB2 signaling is required for the establishment of radial glia and their transformation into astrocytes in cerebral cortex
Authors: Schmid Ralf S. , McGrath Barbara , Berechid Bridget E. , Boyles Becky , Marchionni Mark , Šestan Nenad , Anton Eva S.
Published: April 2003

Radial glial cells and astrocytes function to support the construction and maintenance, respectively, of the cerebral cortex. However, the mechanisms that determine how radial glial cells are established, maintained, and transformed into astrocytes in the cerebral cortex are not well understood. Here, we show that neuregulin-1 (NRG-1) exerts a critical role in the establishment of radial glial cells. Radial glial cell generation is significantly impaired in NRG mutants, and this defect can be rescued by exogenous NRG-1. Down-regulation of expression and activity of erbB2, a member of the NRG-1 receptor complex, leads to the transformation of radial glial cells into astrocytes. Reintroduction of erbB2 transforms astrocytes into radial glia. The activated form of the Notch1 receptor, which promotes the radial glial phenotype, activates the erbB2 promoter in radial glial cells. These results suggest that developmental changes in NRG-1–erbB2 interactions modulate the establishment of radial glia and contribute to their appropriate transformation into astrocytes.
Rapid evolution of the neutralizing antibody response to HIV type 1 infection
Authors: Richman Douglas D. , Wrin Terri , Little Susan J. , Petropoulos Christos J.
Published: April 2003

A recombinant virus assay was used to characterize in detail neutralizing antibody responses directed at circulating autologous HIV in plasma. Examining serial plasma specimens in a matrix format, most patients with primary HIV infection rapidly generated significant neutralizing antibody responses to early (0–39 months) autologous viruses, whereas responses to laboratory and heterologous primary strains were often lower and delayed. Plasma virus continually and rapidly evolved to escape neutralization, indicating that neutralizing antibody exerts a level of selective pressure that has been underappreciated based on earlier, less comprehensive characterizations. These data argue that neutralizing antibody responses account for the extensive variation in the envelope gene that is observed in the early months after primary HIV infection.
Convergent evolution of perenniality in rice and sorghum
Authors: Hu F. Y. , Tao D. Y. , Sacks E. , Fu B. Y. , Xu P. , Li J. , Yang Y. , McNally K. , Khush G. S. , Paterson A. H. , Li Z.-K.
Published: April 2003

Annual and perennial habit are two major strategies by which grasses adapt to seasonal environmental change, and these distinguish cultivated cereals from their wild relatives. Rhizomatousness, a key trait contributing to perenniality, was investigated by using an F
2
population from a cross between cultivated rice (
Oryza sativa
) and its wild relative,
Oryza longistaminata
. Molecular mapping based on a complete simple sequence-repeat map revealed two dominant-complementary genes controlling rhizomatousness.
Rhz3
was mapped to the interval between markers OSR16 [1.3 centimorgans (cM)] and OSR13 (8.1 cM) on rice chromosome 4 and
Rhz2
located between RM119 (2.2 cM) and RM273 (7.4 cM) on chromosome 3. Comparative mapping indicated that each gene closely corresponds to major quantitative trait loci (QTLs) controlling rhizomatousness in
Sorghum propinquum
, a wild relative of cultivated sorghum. Correspondence of these genes in rice and sorghum, which diverged from a common ancestor ≈50 million years ago, suggests that the two genes may be key regulators of rhizome development in many Poaceae. Many additional QTLs affecting abundance of rhizomes in
O. longistaminata
were identified, most of which also corresponded to the locations of
S. propinquum
QTLs. Convergent evolution of independent mutations at, in some cases, corresponding genes may have been responsible for the evolution of annual cereals from perennial wild grasses. DNA markers closely linked to
Rhz2
and
Rhz3
will facilitate cloning of the genes, which may contribute significantly to our understanding of grass evolution, advance opportunities to develop perennial cereals, and offer insights into environmentally benign weed-control strategies.
Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations
Authors: Johnson Sean J. , Taylor Jeffrey S. , Beese Lorena S.
Published: April 2003

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable
Bacillus
DNA polymerase I. The 6-bp extension involves a 20-Å translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a “closed” conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an “open” conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.
Analysis of the eye developmental pathway in
Drosophila
using DNA microarrays
Authors: Michaut Lydia , Flister Susanne , Neeb Martin , White Kevin P. , Certa Ulrich , Gehring Walter J.
Published: April 2003

Pax
-
6
genes encode evolutionarily conserved transcription factors capable of activating the gene-expression program required to build an eye. When ectopically expressed in
Drosophila
imaginal discs,
Pax
-
6
genes induce the eye formation on the corresponding appendages of the adult fly. We used two different
Drosophila
full-genome DNA microarrays to compare gene expression in wild-type leg discs versus leg discs where
eyeless
, one of the two
Drosophila Pax
-
6
genes, was ectopically expressed. We validated these data by analyzing the endogenous expression of selected genes in eye discs and identified 371 genes that are expressed in the eye imaginal discs and up-regulated when an eye morphogenetic field is ectopically induced in the leg discs. These genes mainly encode transcription factors involved in photoreceptor specification, signal transducers, cell adhesion molecules, and proteins involved in cell division. As expected, genes already known to act downstream of
eyeless
during eye development were identified, together with a group of genes that were not yet associated with eye formation.
Identification and functional characterization of a novel binding site on TNF-α promoter
Authors: Tang Xiaoren , Fenton Matthew J. , Amar Salomon
Published: April 2003

Transcription of the tumor necrosis factor (TNF) gene is rapidly and transiently induced by lipopolysaccharide in cells of monocyte/macrophage lineage. Previous studies have suggested that in the mouse, multiple NF-κB/Rel-binding sites contribute to the TNF transcriptional response to LPS. But the role of these regulatory elements in transcriptional activation of the TNF-α gene in human monocytes remains unclear. Previously, a transcription factor, termed lipopolysaccharide-induced TNF-α factor (LITAF), was found to regulate TNF-α gene expression. However, the specific protein domain(s) of human (h)LITAF that interact with the hTNF-α promoter had not been identified. In this study, we identify by footprinting a sequence motif, CTCCC (−515 to −511), within the TNF-α promoter that binds to hLITAF. We also identify the region of hLITAF (amino acids 165–180) that was named peptide B and specifically mediates binding to the hTNF-α promoter. When THP-1 cells were stimulated with this peptide B, it was sufficient to induce TNF-α secretion. Induction of TNF-α transcription by LPS or peptide B depended on the presence of the −515 to −511 promoter region, which was found to be essential for hLITAF binding. Together, these findings help to clarify the mechanism of hLITAF/hTNF-α interaction and the manner by which hLITAF contributes to hTNF-α regulation in an attempt to design new pharmacological interventions to address TNF-related diseases.
Induction of G
1
cycle arrest in T lymphocytes results in increased extracellular levels of β-chemokines: A strategy to inhibit R5 HIV-1
Authors: Heredia Alonso , Davis Charles , Amoroso Anthony , Dominique Joyelle K. , Le Nhut , Klingebiel Erin , Reardon Elise , Zella Davide , Redfield Robert R.
Published: April 2003

The β-chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1α (MIP-1α), and MIP-1β are the natural ligands of the HIV-1 coreceptor CCR5 and compete with the virus for receptor binding. We show that secretion of the β-chemokines by activated lymphocytes starts before cellular DNA synthesis is detected and demonstrate that transient prolongation of the G
1
phase of the cell cycle by treatment with cytostatic drugs results in increased levels of the three chemokines in culture supernatants. Supernatants collected from peripheral blood mononuclear cells exposed to hydroxyurea, which arrests the cell cycle in late G
1
, contained high levels of β-chemokines. These supernatants were able to inhibit HIV-1 replication when added to cultures of infected lymphocytes. The observed antiviral effect likely was due to the increased levels of β-chemokines RANTES, MIP-1α, and MIP-1β because (
i
) supernatants greatly inhibited the replication of HIV-1 BaL, whereas they affected HIV-1 IIIb replication only slightly; (
ii
) neutralizing antibodies against the chemokines abrogated the antiviral effect of the supernatants; and (
iii
) the hydroxyurea concentrations shown to up-regulate chemokine levels were not sufficient to inhibit virus replication by depletion of intracellular nucleotide pools. Although antiviral properties have been reported previously for the cytostatic agents shown here to up-regulate β-chemokine levels, our results provide an additional mechanism by which these drugs may exert antiviral activity. In summary, increased extracellular levels of anti-HIV-1 β-chemokines resulting from transient prolongation of the G
1
phase of the lymphocyte cell cycle by treatment with cytostatic drugs may help to control the replication of CCR5-using strains of HIV-1.
Induction of bilirubin clearance by the constitutive androstane receptor (CAR)
Authors: Huang Wendong , Zhang Jun , Chua Steven S. , Qatanani Mohammed , Han Yunqing , Granata Riccarda , Moore David D.
Published: April 2003

Bilirubin clearance is one of the numerous important functions of the liver. Defects in this process result in jaundice, which is particularly common in neonates. Elevated bilirubin levels can be decreased by treatment with phenobarbital. Because the nuclear hormone receptor constitutive androstane receptor (CAR) mediates hepatic effects of this xenobiotic inducer, we hypothesized that CAR could be a regulator of bilirubin clearance. Activation of the nuclear hormone receptor CAR increases hepatic expression of each of five components of the bilirubin-clearance pathway. This induction is absent in homozygous CAR null mice but is observed in mice expressing human CAR instead of mouse CAR. Pretreatment with xenobiotic inducers markedly increases the rate of clearance of an exogenous bilirubin load in wild-type but not CAR knockout animals. Bilirubin itself can also activate CAR, and mice lacking CAR are defective in clearing chronically elevated bilirubin levels. Unexpectedly, CAR expression is very low in livers of neonatal mice and humans. We conclude that CAR directs a protective response to elevated bilirubin levels and suggest that a functional deficit of CAR activity may contribute to neonatal jaundice.
Dynamic behavior of paired claudin strands within apposing plasma membranes
Authors: Sasaki Hiroyuki , Matsui Chiyuki , Furuse Kyoko , Mimori-Kiyosue Yuko , Furuse Mikio , Tsukita Shoichiro
Published: April 2003

The tight junction (TJ) strand is a linear proteinaceous polymer spanning plasma membranes, and each TJ strand associates laterally with another TJ strand in the apposing membranes of adjacent cells to form “paired” TJ strands. Claudins have been identified as the major constituents of TJ strands, and when exogenously expressed in L fibroblasts, they polymerize into paired strands, which are morphologically similar to paired TJ strands in epithelia. Here, we show that a fusion protein of GFP with claudin-1 can also form similar paired strands in L fibroblasts, allowing us to directly observe individual paired claudin strands in live cells in real time. These paired strands showed more dynamic behavior than expected; they were occasionally broken and annealed, and dynamically associated with each other in both an end-to-side and side-to-side manner. Through this behavior of individual paired claudin strands, the network of strands was reorganized dynamically. Furthermore, fluorescence recovery after photobleaching analyses revealed that claudin molecules were not mobile within paired strands. Although these observations are not necessarily representative of TJ strands
per se
in epithelial cells, they provide important information on the structural and kinetic properties of TJ strands
in situ
with significant implications for barrier function of TJs.
A model of the glycine receptor deduced from Brownian dynamics studies
Authors: O'Mara Megan , Barry Peter H. , Chung Shin-Ho
Published: April 2003

We have developed a three-dimensional model of the α1 homomeric glycine receptor by using Brownian dynamics simulations to account for its observed physiological properties. The model channel contains a large external vestibule and a shallow internal vestibule, connected by a narrow, cylindrical selectivity filter. Three rings of charged residues from the pore-lining M2 domain are modeled as point charges in the protein. Our simulations reproduce many of the key features of the channel, such as the current–voltage profiles, permeability ratios, and ion selectivity. When we replace the ring of alanine residues lining the selectivity filter with glutamates, the mutant model channel becomes permeable to cations, as observed experimentally. In this mutation, anions act as chaperones for sodium ions in the extracellular vestibule, and together they penetrate deep inside the channel against a steep energy barrier encountered by unaccompanied ions. Two subsequent amino acid mutations increase the cation permeability, enabling monovalent cations to permeate through the channel unaided and divalent cations to permeate when chaperoned by anions. These results illustrate the key structural features and underlying mechanism for charge selectivity in the glycine receptor.
Impaired adrenocorticotropic hormone response to bacterial endotoxin in mice deficient in prostaglandin E receptor EP1 and EP3 subtypes
Authors: Matsuoka Yoko , Furuyashiki Tomoyuki , Bito Haruhiko , Ushikubi Fumitaka , Tanaka Yasuhiro , Kobayashi Takuya , Muro Seiji , Satoh Noriko , Kayahara Tetsuro , Higashi Mikito , Mizoguchi Akira , Shichi Hitoshi , Fukuda Yoshihiro , Nakao Kazuwa , Narumiya Shuh
Published: April 2003

Sickness evokes various neural responses, one of which is activation of the hypothalamo–pituitary–adrenal (HPA) axis. This response can be induced experimentally by injection of bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1. Although prostaglandins (PGs) long have been implicated in LPS-induced HPA axis activation, the mechanism downstream of PGs remained unsettled. By using mice lacking each of the four PGE receptors (EP1–EP4) and an EP1-selective antagonist, ONO-8713, we showed that both EP1 and EP3 are required for adrenocorticotropic hormone release in response to LPS. Analysis of c-Fos expression as a marker for neuronal activity indicated that both EP1 and EP3 contribute to activation of neurons in the paraventricular nucleus of the hypothalamus (PVN). This analysis also revealed that EP1, but not EP3, is involved in LPS-induced activation of the central nucleus of the amygdala. EP1 immunostaining in the PVN revealed its localization at synapses on corticotropin-releasing hormone-containing neurons. These findings suggest that EP1- and EP3-mediated neuronal pathways converge at corticotropin-releasing hormone-containing neurons in the PVN to induce HPA axis activation upon sickness.
Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulatesSMN2gene expression in patient fibroblasts
Authors: Skordis Leigh A. , Dunckley Matthew G. , Yue Baigong , Eperon Ian C. , Muntoni Francesco
Published: April 2003

The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of theSurvival Motor Neuron(SMN) gene. Mutations ofSMN1cause SMA, whereasSMN2acts as a modifying gene. The two genes undergo alternative splicing withSMN1, producing an abundance of full-length mRNA transcripts, whereasSMN2predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference inSMN2exon 7, which disrupts an exonic splicing enhancer containing an SF2/ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increasedSMN2exon 7 splicingin vitroand rescued the incorporation ofSMN2exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered.
A gain-of-function mutation in
ftsA
bypasses the requirement for the essential cell division gene
zipA
in
Escherichia coli
Authors: Geissler Brett , Elraheb Dany , Margolin William
Published: April 2003

ZipA and FtsA are recruited independently to the FtsZ cytokinetic ring (Z ring) and are essential for cell division of
Escherichia coli
. The molecular role of FtsA in cell division is unknown; however, ZipA is thought to stabilize the Z ring, anchor it to the membrane, and recruit downstream cell division proteins. Here we demonstrate that the requirement for ZipA can be bypassed completely by a single alteration in a conserved residue of FtsA (FtsA*). Cells with
ftsA
* in single copy in place of WT
ftsA
or with
ftsA
* alone on a multicopy plasmid divide mostly normally, whether they are
zipA
+ or
zipA
−. Experiments with
ftsQAZ
and
ftsQA
*
Z
on multicopy plasmids indicate that
ftsQAZ
/
zipA
+ and
ftsQA
*
Z
/
zipA
− cells divide fairly normally, whereas
ftsQAZ
/
zipA
− cells divide poorly and
ftsQA
*
Z
/
zipA
+ cells display a phenotype that suggests their septa are unusually stable. In support of the idea that
ftsA
* stabilizes Z rings, single-copy
ftsA
* confers resistance to excess MinC, which destabilizes Z rings. The inhibitory effect of excess ZipA on division is also suppressed by
ftsA
*. These results suggest that the molecular mechanism of the FtsA* bypass is to stabilize FtsZ assembly via a parallel pathway and that FtsA* can replace the multiple functions of ZipA. This is an example of a complete functional replacement of an essential prokaryotic cell division protein by another and may explain why most bacteria can divide without an obvious ZipA homolog.
Context sensitivity of activity-dependent increases in cerebral blood flow
Authors: Caesar Kirsten , Gold Lorenz , Lauritzen Martin
Published: April 2003

Functional neuroimaging in humans is used widely to study brain function in relation to human disease and cognition. The neural basis of neuroimaging signals is probably synaptic activity, but the effect of context, defined as the interaction between synaptic inhibition, excitation, and the electroresponsive properties of the targeted neurons, is not well understood. We examined here the effect of interaction of synaptic excitation and net inhibition on the relationship between electrical activity and vascular signals in the cerebellar cortex. We show that stimulation of the net inhibitory parallel fibers simultaneously with stimulation of the excitatory climbing fibers leads to a further rise in total local field potentials (LFP) and cerebral blood flow (CBF) amplitudes, not a decrease, as predicted from theoretical studies. However, the combined stimulation of the parallel and climbing fiber systems produced changes in CBF and LFP that were smaller than their algebraic sum evoked by separate stimulation of either system. This finding was independent of the starting condition, i.e., whether inhibition was superimposed on a state of excitation or vice versa. The attenuation of the increases in LFP and CBF amplitudes was similar, suggesting that synaptic activity and CBF were coupled under these conditions. The result might be explained by a relative neuronal refractoriness that relates to the intrinsic membrane properties of Purkinje cells, which determine the recovery time of these cells. Our work implies that neuronal and vascular signals are context-sensitive and that their amplitudes are modulated by the electroresponsive properties of the targeted neurons.