bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome-wide analysis of mRNA translation profiles inSaccharomycescerevisiae
Authors: Arava Yoav , Wang Yulei , Storey John D. , Liu Chih Long , Brown Patrick O. , Herschlag Daniel
Published: April 2003

We have analyzed the translational status of each mRNA in rapidly growingSaccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, includingGCN4,CPA1, andICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.
The CsrR/CsrS two-component system of group A
Streptococcus
responds to environmental Mg
2+
Authors: Gryllos Ioannis , Levin James C. , Wessels Michael R.
Published: April 2003

Group A streptococci control expression of key virulence determinants via the two-component sensor/regulator system CsrR/CsrS. The membrane-bound sensor CsrS is thought to respond to previously unknown environmental signal(s) by controlling phosphorylation of its cognate regulator component CsrR. Phosphorylation of CsrR increases its affinity for binding to the promoter regions of Csr-regulated genes to repress transcription. Here we show that environmental Mg
2+
concentration is a potent and specific stimulus for CsrR/CsrS-mediated regulation. We studied the effect of divalent cations on expression of the Csr-regulated hyaluronic acid capsule genes (
hasABC
) by measuring chloramphenicol acetyltransferase (CAT) activity in a reporter strain of group A
Streptococcus
carrying a
has
operon promoter-
cat
fusion. Addition of Mg
2+
, but not of Ca
2+
, Mn
2+
, or Zn
2+
, repressed capsule gene expression by up to 80% in a dose-dependent fashion. The decrease in capsule gene transcription was associated with a marked reduction in cell-associated capsular polysaccharide. RNA hybridization analysis demonstrated reduced expression of the Csr-regulated
hasABC
operon, streptokinase (
ska
), and streptolysin S (
sagA
) during growth in the presence of 15 mM Mg
2+
for the wild-type strain 003CAT but not for an isogenic
csrS
mutant. We propose that Mg
2+
binds to CsrS to induce phosphorylation of CsrR and subsequent repression of virulence gene expression. The low concentration of Mg
2+
in extracellular body fluids predicts that the CsrR/CsrS system is maintained in the inactive state during infection, thereby allowing maximal expression of critical virulence determinants in the human host.
Efficient active transport of gene nanocarriers to the cell nucleus
Authors: Suh Junghae , Wirtz Denis , Hanes Justin
Published: April 2003

The intracellular transport of therapeutic gene carriers is poorly understood, limiting the rational design of efficient new vectors. We used live-cell real-time multiple particle tracking to quantify the intracellular transport of hundreds of individual nonviral DNA nanocarriers with 5-nm and 33-ms resolution. Unexpected parallels between several of nature's most efficient DNA viruses and nonviral polyethylenimine/DNA nanocomplexes were revealed to include motor protein-driven transport through the cytoplasm toward the nucleus on microtubules. Active gene carrier transport led to efficient perinuclear accumulation within minutes. The results provide direct evidence to dispute the common belief that the efficiency of nonviral gene carriers is dramatically reduced because of the need for their relatively slow random diffusion through the cell cytoplasm to the nucleus and, instead, focuses the attention of rational carrier design on overcoming barriers downstream of perinuclear accumulation.
Gigantic cannibals driving a whole-lake trophic cascade
Authors: Persson Lennart , De Roos André M. , Claessen David , Byström Pär , Lövgren Johan , Sjögren Stefan , Svanbäck Richard , Wahlström Eva , Westman Erika
Published: April 2003

Trophic cascades have been a central paradigm in explaining the structure of ecological communities but have been demonstrated mainly through comparative studies or experimental manipulations. In contrast, evidence for shifts in trophic cascades caused by intrinsically driven population dynamics is meager. By using empirical data of a cannibalistic fish population covering a 10-year period and a size-structured population model, we show the occurrence of a dynamic trophic cascade in a lake ecosystem, in which the community over time alternates between two different configurations. The intrinsically driven change in the size structure of the fish population from a dominance of stunted individuals to a dominance of gigantic cannibals among adult individuals is the driving force behind distinct abundance switches observed in zooplankton and phytoplankton. The presence of the phase with gigantic cannibals depends critically on the energy they extract from their victims, allowing strong reproduction for a number of years.
Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry
Authors: Hoffman Brian M.
Published: April 2003

This perspective discusses the ways that advanced paramagnetic resonance techniques, namely electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies, can help us understand how metal ions function in biological systems.
Calpain regulates neutrophil chemotaxis
Authors: Lokuta M. A. , Nuzzi P. A. , Huttenlocher A.
Published: April 2003

Cell polarization is required for directed cell migration. We investigated the role of the calcium-dependent protease calpain during neutrophil chemotaxis and found that calpain inhibition induced neutrophil adhesion, polarization, and rapid chemokinesis in the absence of exogenous activators. Resting neutrophils display constitutive calpain activity with μ-calpain being the predominant active isoform. Our findings suggest that constitutive calpain activity in resting neutrophils may function as a negative regulator of protrusion and migration. Specific inhibition of μ-calpain, but not
m
-calpain, induced neutrophil polarization and chemokinesis. In contrast to IL-8-induced chemokinesis, the chemokinesis induced by calpain inhibition was not reduced in the presence of pertussis toxin, suggesting that calpain functions downstream of G protein-coupled receptors. Further, both calpain inhibition and stimulation with IL-8 and formyl-Met-Leu-Phe (fMLP) induced an increase in Cdc42 and Rac activation. These findings are consistent with the involvement of calpain in chemotaxis pathways. Accordingly, calpain inhibition decreased neutrophil chemotaxis and directional persistence in a gradient of IL-8 and fMLP. Together, these data reveal a previously uncharacterized function for calpain in neutrophils and suggest that localized modulation of calpain activity may regulate neutrophil chemotaxis downstream of G-protein-coupled receptors.
Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and
p-
xylene
Authors: Mitchell Kevin H. , Rogge Corina E. , Gierahn Todd , Fox Brian G.
Published: April 2003

The present studies address the mechanism of aromatic hydroxylation used by the natural and G103L isoforms of the diiron enzyme toluene 4-monooxygenase. These isoforms have comparable catalytic parameters but distinct regiospecificities for toluene hydroxylation. Hydroxylation of ring-deuterated
p
-xylene by the natural isoform revealed a substantial inverse isotope effect of 0.735, indicating a change in hybridization from sp
2
to sp
3
for hydroxylation at a carbon atom bearing the deuteron. During the hydroxylation of 4-
2
H
1
- and 3,5-
2
H
2
-toluene, similar magnitudes of intramolecular isotope effects and patterns of deuterium retention were observed from both isoforms studied, indicating that the active-site mutation affected substrate orientation but did not influence the mechanism of hydroxylation. The results with deuterated toluenes show inverse intramolecular isotope effects for hydroxylation at the position of deuteration, normal secondary isotope effects for hydroxylation adjacent to the position of deuteration, near-quantitative deuterium retention in
m
-cresol obtained from 4-
2
H
1
-toluene, and partial loss of deuterium from all phenolic products obtained from 3,5-
2
H
2
-toluene. This combination of results suggests that an active site-directed opening of position-specific transient epoxide intermediates may contribute to the chemical mechanism and the high degree of regiospecificity observed for aromatic hydroxylation in this evolutionarily specialized diiron enzyme.
Nutritional supplement chromium picolinate causes sterility and lethal mutations inDrosophilamelanogaster
Authors: Hepburn Dion D. D. , Xiao Jiarong , Bindom Sharell , Vincent John B. , O'Donnell Janis
Published: April 2003

The nutritional dietary supplement chromium picolinate, [Cr(pic)3], has gained much notoriety as a safe supplement that supposedly promotes fat loss and muscle enhancement in humans. Thus, a significant industry has materialized around the incorporation of [Cr(pic)3] in many sports foods and drinks and a variety of weight loss products. However,in vitrostudies have suggested that low levels of [Cr(pic)3] in the presence of biological reducing agents can catalytically generate reactive oxygen species, and recentin vivostudies have detected oxidative damage in rats receiving the supplement. The potential deleteriousin vivoeffects of this activity were examined by usingDrosophila melanogaster. [Cr(pic)3], but not CrCl3, at levels of 260 μg Cr/kg food or less were found to lower the success rate of pupation and eclosion and to arrest development of pupae in a concentration dependent fashion. X-linked lethal analysis indicates that the supplement greatly enhances the rate of appearance of lethal mutations and dominant female sterility.
Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin
Authors: Hunter Christie L. , Maurus Robert , Mauk Marcia R. , Lee Hung , Raven Emma L. , Tong Harry , Nguyen Nham , Smith Michael , Brayer Gary D. , Mauk A. Grant
Published: April 2003

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional
1
H NMR spectroscopy indicates that Mn
2+
binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn
2+
is 1.28(4) × 10
4
M
−1
(pH 6.96, ionic strength
I
= 17.2 μM, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK
a
for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn
2+
-peroxidase activity of the variant are both increased ≈3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn
2+
. The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn
2+
and Cd
2+
. Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.
Interconversion of two oxidized forms of taurine/α-ketoglutarate dioxygenase, a non-heme iron hydroxylase: Evidence for bicarbonate binding
Authors: Ryle Matthew J. , Koehntop Kevin D. , Liu Aimin , Que Lawrence , Hausinger Robert P.
Published: April 2003

Taurine/α-ketoglutarate (αKG) dioxygenase, or TauD, is a mononuclear non-heme iron hydroxylase that couples the oxidative decarboxylation of αKG to the decomposition of taurine, forming sulfite and aminoacetaldehyde. Prior studies revealed that taurine-free TauD catalyzes an O
2
- and αKG-dependent self-hydroxylation reaction involving Tyr-73, yielding an Fe(III)-catecholate chromophore with a λ
max
of 550 nm. Here, a chromophore (λ
max
720 nm) is described and shown to arise from O
2
-dependent self-hydroxylation of TauD in the
absence
of αKG, but requiring the product succinate. A similar chromophore rapidly develops with the alternative oxidant H
2
O
2
. Resonance Raman spectra indicate that the ≈700-nm chromophore also arises from an Fe(III)-catecholate species, and site-directed mutagenesis studies again demonstrate Tyr-73 involvement. The ≈700-nm and 550-nm species are shown to interconvert by the addition or removal of bicarbonate, consistent with the αKG-derived CO
2
remaining tightly bound to the oxidized metal site as bicarbonate. The relevance of the metal-bound bicarbonate in TauD to reactions of other members of this enzyme family is discussed.
A soluble protein is immobile in dormant spores of
Bacillus subtilis
but is mobile in germinated spores: Implications for spore dormancy
Authors: Cowan Ann E. , Koppel Dennis E. , Setlow Barbara , Setlow Peter
Published: April 2003

Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of
Bacillus subtilis
but becomes freely mobile in germinated spores in which cytoplasmic water content has increased ≈2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated
cwlD
spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other
Bacillus
species. The immobility of a normally mobile protein in dormant wild-type spores and germinated
cwlD
spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of
Bacillus
species.
An Fe
IV
O complex of a tetradentate tripodal nonheme ligand
Authors: Lim Mi Hee , Rohde Jan-Uwe , Stubna Audria , Bukowski Michael R. , Costas Miquel , Ho Raymond Y. N. , Münck Eckard , Nam Wonwoo , Que Lawrence
Published: April 2003

The reaction of [Fe
II
(tris(2-pyridylmethyl)amine, TPA)(NCCH
3
)
2
]
2+
with 1 equiv. peracetic acid in CH
3
CN at −40°C results in the nearly quantitative formation of a pale green intermediate with λ
max
at 724 nm (ɛ ≈ 300 M
−1
⋅cm
−1
) formulated as [Fe
IV
(O)(TPA)]
2+
by a combination of spectroscopic techniques. Its electrospray mass spectrum shows a prominent feature at
m
/
z
461, corresponding to the [Fe
IV
(O)(TPA)(ClO
4
)]
+
ion. The Mössbauer spectra recorded in zero field reveal a doublet with Δ
E

Q

= 0.92(2) mm/s and δ = 0.01(2) mm/s; analysis of spectra obtained in strong magnetic fields yields parameters characteristic of
S
= 1 Fe
IV

O complexes. The presence of an Fe
IV

O unit is also indicated in its Fe K-edge x-ray absorption spectrum by an intense 1-s → 3-d transition and the requirement for an O/N scatterer at 1.67 Å to fit the extended x-ray absorption fine structure region. The [Fe
IV
(O)(TPA)]
2+
intermediate is stable at −40°C for several days but decays quantitatively on warming to [Fe
2
(μ
-
O)(μ
-
OAc)(TPA)
2
]
3+
. Addition of thioanisole or cyclooctene at −40°C results in the formation of thioanisole oxide (100% yield) or cyclooctene oxide (30% yield), respectively; thus [Fe
IV
(O)(TPA)]
2+
is an effective oxygen-atom transfer agent. It is proposed that the Fe
IV

O species derives from O—O bond heterolysis of an unobserved Fe
II
(TPA)-acyl peroxide complex. The characterization of [Fe
IV
(O)(TPA)]
2+
as having a reactive terminal Fe
IV

O unit in a nonheme ligand environment lends credence to the proposed participation of analogous species in the oxygen activation mechanisms of many mononuclear nonheme iron enzymes.
Investigation of metal–dithiolate fold angle effects: Implications for molybdenum and tungsten enzymes
Authors: Joshi Hemant K. , Cooney J. Jon A. , Inscore Frank E. , Gruhn Nadine E. , Lichtenberger Dennis L. , Enemark John H.
Published: April 2003

Gas-phase photoelectron spectroscopy and density functional theory have been used to investigate the interactions between the sulfur π-orbitals of arene dithiolates and high-valent transition metals as minimum molecular models of the active site features of pyranopterin Mo/W enzymes. The compounds (Tp*)MoO(bdt) (compound 1), Cp2Mo(bdt) (compound 2), and Cp2Ti(bdt) (compound 3) [where Tp* is hydrotris(3,5-dimethyl-1-pyrazolyl)borate, bdt is 1,2-benzenedithiolate, and Cp is η5- cyclopentadienyl] provide access to three different electronic configurations of the metal, formally d1, d2, and d0, respectively. The gas-phase photoelectron spectra show that ionizations from occupied metal and sulfur based valence orbitals are more clearly observed in compounds 2 and 3 than in compound 1. The observed ionization energies and characters compare very well with those calculated by density functional theory. A “dithiolate-folding-effect” involving an interaction of the metal in-plane and sulfur-π orbitals is proposed to be a factor in the electron transfer reactions that regenerate the active sites of molybdenum and tungsten enzymes.
Nitric oxide binding at the mononuclear active site of reduced
Pyrococcus furiosus
superoxide reductase
Authors: Clay Michael D. , Cosper Christopher A. , Jenney Francis E. , Adams Michael W. W. , Johnson Michael K.
Published: April 2003

Nitric oxide (NO) has been used as a substrate analog to explore the structural and electronic determinants of enzymatic superoxide reduction at the mononuclear iron active site of
Pyrococcus furiosus
superoxide reductase (SOR) through the use of EPR, resonance Raman, Fourier transform IR, UV-visible absorption, and variable-temperature variable-field magnetic CD spectroscopies. The NO adduct of reduced SOR is shown to have a near-axial
S
= 3/2 ground state with
E/D
= 0.06 and
D
= 12 ± 2 cm
−1
(where
D
and
E
are the axial and rhombic zero-field splitting parameters, respectively) and the UV-visible absorption and magnetic CD spectra are dominated by an out-of-plane NO
−
(π*)-to-Fe
3+
(dπ) charge–transfer transition, polarized along the zero-field splitting axis. Resonance Raman studies indicate that the NO adduct is six-coordinate with NO ligated in a bent conformation trans to the cysteinyl
S
, as evidenced by the identification of ν(N–O) at 1,721 cm
−1
, ν(Fe–NO) at 475 cm
−1
, and ν(Fe–S(Cys), at 291 cm
−1
, via
34
S and
15
NO isotope shifts. The electronic and vibrational properties of the
S
= 3/2 {FeNO}
7
unit are rationalized in terms of a limiting formulation involving a high-spin (
S
= 5/2) Fe
3+
center antiferromagnetically coupled to a (
S
= 1) NO
−
anion, with a highly covalent Fe
3+
−NO
−
interaction. The results support a catalytic mechanism for SOR, with the first step involving oxidative addition of superoxide to form a ferric-peroxo intermediate, and indicate the important roles that the Fe spin state and the trans cysteinate ligand play in effecting superoxide reduction and peroxide release.
Emergency response to an anthrax attack
Authors: Wein Lawrence M. , Craft David L. , Kaplan Edward H.
Published: April 2003

We developed a mathematical model to compare various emergency responses in the event of an airborne anthrax attack. The system consists of an atmospheric dispersion model, an age-dependent dose–response model, a disease progression model, and a set of spatially distributed two-stage queueing systems consisting of antibiotic distribution and hospital care. Our results underscore the need for the extremely aggressive and timely use of oral antibiotics by all asymptomatics in the exposure region, distributed either preattack or by nonprofessionals postattack, and the creation of surge capacity for supportive hospital care via expanded training of nonemergency care workers at the local level and the use of federal and military resources and nationwide medical volunteers. The use of prioritization (based on disease stage and/or age) at both queues, and the development and deployment of modestly rapid and sensitive biosensors, while helpful, produce only second-order improvements.
Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain
Authors: Wang Huimin , Shimizu Eiji , Tang Ya-Ping , Cho Min , Kyin Maureen , Zuo Wenqi , Robinson Daphne A. , Alaimo Peter J. , Zhang Chao , Morimoto Hiromi , Zhuo Min , Feng Ruiben , Shokat Kevan M. , Tsien Joe Z.
Published: April 2003

By integrating convergent protein engineering and rational inhibitor design, we have developed an
in vivo
conditional protein knockout and/or manipulation technology. This method is based on the creation of a specific interaction interface between a modified protein domain and sensitized inhibitors. By introducing this system into genetically modified mice, we can readily manipulate the activity of a targeted protein, such as α-Ca
2+
/calmodulin-dependent protein kinase II (αCAMKII), on the time scale of minutes in specific brain subregions of freely behaving mice. With this inducible and region-specific protein knockout technique, we analyzed the temporal stages of memory consolidation process and revealed the first postlearning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.
A redox switch in CopC: An intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites
Authors: Arnesano Fabio , Banci Lucia , Bertini Ivano , Mangani Stefano , Thompsett Andrew R.
Published: April 2003

The protein CopC from
Pseudomonas syringae
has been found capable of binding copper(I) and copper(II) at two different sites, occupied either one at a time or simultaneously. The protein, consisting of 102 amino acids, is known to bind copper(II) in a position that is now found consistent with a coordination arrangement including His-1, Glu-27, Asp-89, and His-91. A full solution structure analysis is reported here for Cu(I)-CopC. The copper(I) site is constituted by His-48 and three of the four Met residues (40, 43, 46, 51), which are clustered in a Met-rich region. Both copper binding sites have been characterized through extended x-ray absorption fine structure studies. They represent novel coordination environments for copper in proteins. The two sites are ≈30 Å far apart and have little affinity for the ion in the other oxidation state. Oxidation of Cu(I)-CopC or reduction of Cu(II)-CopC causes migration of copper from one site to the other. This behavior is observed both in NMR and EXAFS studies and indicates that CopC can exchange copper between two sites activated by a redox switch. CopC resides in the periplasm of Gram-negative bacteria where there is a multicopper oxidase, CopA, which may modulate the redox state of copper. CopC and CopA are coded in the same operon, responsible for copper resistance. These peculiar and novel properties of CopC are discussed with respect to their relevance for copper homeostasis.
Opening protein pores with chaotropes enhances Fe reduction and chelation of Fe from the ferritin biomineral
Authors: Liu Xiaofeng , Jin Weili , Theil Elizabeth C.
Published: April 2003

Iron is concentrated in ferritin, a spherical protein with a capacious cavity for ferric nanominerals of <4,500 Fe atoms. Global ferritin structure is very stable, resisting 6 M urea and heat (85°C) at neutral pH. Eight pores, each formed by six helices from 3 of the 24 polypeptide subunits, restrict mineral access to reductant, protons, or chelators. Protein-directed transport of Fe and aqueous Fe
3+
chemistry (solubility ≈10
−18
M) drive mineralization. Ferritin pores are “gated” based on protein crystals and Fe chelation rates of wild-type (WT) and engineered proteins. Pore structure and gate residues, which are highly conserved, thus should be sensitive to environmental changes such as low concentrations of chaotropes. We now demonstrate that urea or guanidine (1–10 mM), far below concentrations for global unfolding, induced multiphasic rate increases in Fe
2+
-bipyridyl formation similar to conservative substitutions of pore residues. Urea (1 M) or the nonconservative Leu/Pro substitution that fully unfolded pores without urea both induced monophasic rate increases in Fe
2+
chelation rates, indicating unrestricted access between mineral and reductant/chelator. The observation of low-melting ferritin subdomains by CD spectroscopy (melting midpoint 53°C), accounting for 10% of ferritin α-helices, is unprecedented. The low-melting ferritin subdomains are pores, based on percentage helix and destabilization by either very dilute urea solutions (1 mM) or Leu/Pro substitution, which both increased Fe
2+
chelation. Biological molecules may have evolved to control gating of ferritin pores in response to cell iron need and, if mimicked by designer drugs, could impact chelation therapies in iron-overload diseases.
Structural elements of metal selectivity in metal sensor proteins
Authors: Pennella Mario A. , Shokes Jacob E. , Cosper Nathaniel J. , Scott Robert A. , Giedroc David P.
Published: April 2003

Staphylococcus aureus
CzrA and
Mycobacterium tuberculosis
NmtR are homologous zinc/cobalt-responsive and nickel/cobalt-responsive transcriptional repressors
in vivo
, respectively, and members of the ArsR/SmtB superfamily of prokaryotic metal sensor proteins. We show here that Zn(II) is the most potent negative allosteric regulator of
czr
operator/promoter binding
in vitro
with the trend Zn(II)>Co(II)≫Ni(II), whereas the opposite holds for the binding of NmtR to the
nmt
operator/promoter, Ni(II)>Co(II)>Zn(II). Characterization of the metal coordination complexes of CzrA and NmtR by UV/visible and x-ray absorption spectroscopies reveals that metals that form four-coordinate tetrahedral complexes with CzrA [Zn(II) and Co(II)] are potent regulators of DNA binding, whereas metals that form five- or six-coordinate complexes with NmtR [Ni(II) and Co(II)] are the strongest allosteric regulators in this system. Strikingly, the Zn(II) coordination complexes of CzrA and NmtR cannot be distinguished from one another by x-ray absorption spectroscopy, with the best fit a His-3-carboxylate complex in both cases. Inspection of the primary structures of CzrA and NmtR, coupled with previous functional data, suggests that three conserved His and one Asp from the C-terminal α5 helix donate ligands to create a four-coordinate complex in both CzrA and NmtR, with NmtR uniquely capable of expanding its coordination number in the Ni(II) and Co(II) complexes by recruiting additional His ligands from a C-terminal extension of the α5 helix.
A solution to the worn tooth conundrum in primate functional anatomy
Authors: Ungar Peter S. , M'Kirera Francis
Published: April 2003

Worn teeth are a bane to paleobiologists interested in the diets of human ancestors and other fossil primates. Although worn teeth dominate fossil assemblages, their shapes are usually not used to reconstruct the diets of extinct species. The problem is that traditional studies of primate dental functional anatomy have focused on unworn morphology. This has limited most functional analyses to only a few well-represented fossil species. This paper introduces a method to characterize and compare worn occlusal morphology in primates using laser scanning and geographic information systems technologies. A study of variably worn chimpanzee and gorilla molars indicates that differences between these species in tooth shape remain consistent at given stages of wear. Although cusp slope decreases with wear in both taxa, angularity values remain unchanged. These results indicate that African ape teeth wear in a manner that keeps them mechanically efficient for fracturing specific foods. Studies of changes in tooth shape with wear add a new dimension to dental functional anatomy, and offer a more complete picture of dental-dietary adaptations. Also, given how rare unworn teeth are in the fossil record, the ability to include worn specimens in analyses opens the door to reconstructing the diets of many more extinct primate groups, allowing us to better understand the adaptive radiation of our order.
How does cyanide inhibit superoxide reductase? Insight from synthetic Fe
III
N
4
S model complexes
Authors: Shearer Jason , Fitch Sarah B. , Kaminsky Werner , Benedict Jason , Scarrow Robert C. , Kovacs Julie A.
Published: April 2003

Superoxide reductases (SORs) are nonheme iron-containing enzymes that reduce HO
2
to H
2
O
2
. Exogenous substrates such as N

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and CN
−
have been shown to bind to the catalytic iron site of SOR, and cyanide acts as an inhibitor. To understand how these exogenous ligands alter the physical and reactivity properties of the SOR iron site, acetate-, azide-, and cyanide-ligated synthetic models of SOR have been prepared. The x-ray crystal structures of azide-ligated [Fe
III
(S
Me2
N
4
(tren))(N
3
)]
+
(3), dimeric cyanide-bridged ([Fe
III
(S
Me2
N
4
(tren))]
2
-μ-CN)
3+
(5), and acetate-ligated [Fe
III
(S
Me2
N
4
(tren))(OAc)]
+
(6) are described, in addition to x-ray absorption spectrum-derived and preliminary crystallographic structures of cyanide-ligated [Fe
III
(S
Me2
N
4
(tren))(CN)]
+
(4). Cyanide coordination to our model (4) causes the redox potential to shift anodically by 470 mV relative to acetate-ligated 6 and 395 mV relative to azide-ligated 3. If cyanide coordination were to cause a similar shift in redox potential with SOR, then the reduction potential of the catalytically active Fe
3+
center would fall well below that of its biological reductants. These results suggest therefore that cyanide inhibits SOR activity by making the Fe
2+
state inaccessible and thus preventing the enzyme from turning over. Cyanide inhibits activity in the metalloenzyme superoxide dismutase via a similar mechanism. The reduced five-coordinate precursor to 3, 4, and 6 [Fe
II
(S
Me2
N
4
(tren))]
+
(1) was previously shown by us to react with superoxide to afford H
2
O
2
via an [Fe
III
(S
Me2
N
4
(tren))(OOH)]
+
intermediate. Cyanide and azide do not bind to 1 and do not prevent 1 from reducing superoxide.
Microsecond electrophoresis
Authors: Plenert Matthew L. , Shear Jason B.
Published: April 2003

Although analysis strategies exist for probing a diverse array of molecular properties, most of these approaches are not amenable to the study of reaction intermediates and other transient species. Separations in particular can provide detailed information on attributes not readily measured by spectroscopy but typically are performed over time scales much longer than the life span of highly unstable compounds. Here we report the development of an electrophoretic strategy that dramatically extends the practical speed limit for fractionations and demonstrate its utility in examining transient hydroxyindole photoproducts. Fluorescent reaction intermediates are optically generated in femtoliter volumes within a flowing reagent stream and are differentially transported at velocities as large as 1.3 m⋅s
−1
, thereby minimizing band variance and allowing multicomponent reaction mixtures to be resolved over separation paths as short as 9 μm. Analyte migration times and band variances do not deviate significantly from basic theory for separations performed with fields that exceed 0.1 MV⋅cm
−1
, indicating that effects from Joule heating are minor. We demonstrate the feasibility of achieving baseline resolution of a binary mixture in <10 μs, nearly 100-fold faster than previously possible. Application of this approach to the study of a range of short-lived molecules should be feasible.
Synthetic modeling of zinc thiolates: Quantitative assessment of hydrogen bonding in modulating sulfur alkylation rates
Authors: Chiou Show-Jen , Riordan Charles G. , Rheingold Arnold L.
Published: April 2003

A series of mononuclear zinc thiolate complexes have been prepared and fully characterized. The reactions of the complexes with alkyl halides, leading to zinc halides and the corresponding thioethers, have been examined by kinetic methods. In toluene, the reactions obey a second-order rate law displaying activation parameters consistent with a S
N
2 attack of the zinc-bound thiolate on the carbon electrophile. Intramolecular hydrogen bonding of an amide N—H to the thiolate sulfur reduces the nucleophilicity and consequently, the rate of alkylation more than 30-fold at 25°C. The H-bonding shows an inverse H/D isotope effect of 0.33 (60°C) ascribed to differential H-bonding for the two isotopomers due to zero point energy differences. These model studies provide quantitative evaluation of H-bonding on reaction rates relevant to zinc thiol-activating proteins.
Kinetics of proton-coupled electron-transfer reactions to the manganese-oxo “cubane” complexes containing the Mn
4
O\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation*}{\mathrm{_{4}^{6+}}}\end{equation*}\end{document} and Mn
4
O\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation*}{\mathrm{_{4}^{7+}}}\end{equation*}\end{document} core types
Authors: Maneiro Marcelino , Ruettinger Wolfgang F. , Bourles Emilie , McLendon George L. , Dismukes G. Charles
Published: April 2003

The kinetics of proton-coupled electron-transfer (pcet) reactions are reported for Mn
4
O
4
(O
2
PPh
2
)
6
, 1, and [Mn
4
O
4
(O
2
PPh
2
)
6
]
+
, 1
+
, with phenothiazine (pzH). Both pcet reactions form 1H, by H transfer to 1 and by hydride transfer to 1
+
. Surprisingly, the rate constants differ by only 25% despite large differences in the formal charges and driving force. The driving force is proportional to the difference in the bond-dissociation energies (BDE >94 kcal/mol for homolytic, 1H → H + 1, vs. ≈127 kcal/mol for heterolytic, 1H → H
−
+ 1
+
, dissociation of the O—H bond in 1H). The enthalpy and entropy of activation for the homolytic reaction (ΔH
‡
= −1.2 kcal/mol and ΔS
‡
= −32 cal/mol⋅K; 25–6.7°C) reveal a low activation barrier and an appreciable entropic penalty in the transition state. The rate-limiting step exhibits no H/D kinetic isotope effect (
k
H
/
k
D
= 0.96) for the first H atom-transfer step and a small kinetic isotope effect (1.4) for the second step (1H + pzH → 1H
2
+ pz
•
). These lines of evidence indicate that formation of a reactive precursor complex before atom transfer is rate-limiting (conformational gating), and that little or no N—H bond cleavage occurs in the transition state. H-atom transfer from pzH to alkyl, alkoxyl, and peroxyl radicals reveals that BDEs are not a good predictor of the rates of this reaction. Hydride transfer to 1
+
provides a concrete example of two-electron pcet that is hypothesized for the O—H bond cleavage step during catalysis of photosynthetic water oxidation.
A macrocyclic zinc(II) fluorophore as a detector of apoptosis
Authors: Kimura Eiichi , Aoki Shin , Kikuta Emiko , Koike Tohru
Published: April 2003

Our originally designed dansylamidoethylcyclen 4 as a biomimetic Zn
2+
-selective fluorophore has been demonstrated to be a good detector of the apoptosis (induced by an anticancer agent, etoposide, and H
2
O
2
) in cancer cells such as HeLa and HL60 cells. The macrocyclic Zn
2+
ligand 4 (mostly as a deprotonated form) is cell-permeable to show weak fluorescence (emission at 550 nm), which forms a strong fluorescent 1:1 Zn
2+
complex 5 (emission at 530 nm) when Zn
2+
is incorporated into the cells by a zinc(II) ionophore pyrithione. Thus formed, Zn
2+
complex 5 is cell-impermeable and remains intact over a few hours. When apoptosis in HeLa or HL60 cells is artificially induced, 4 selectively and strongly stains apoptotic cells only at early stages, which was verified by using the conventional apoptosis detection probe annexin V-Cy3. Detection of the apoptotic cells by 4 was perhaps due to significantly increased free Zn
2+
flux at early stages of apoptosis. Apoptotic detection by 4 has been compared with a presently available Zn
2+
fluorophore, Zinquin 1. We present that 4 has advantages in detection of apoptosis over annexin V-Cy3 and Zinquin 1.
The protein-folding speed limit: Intrachain diffusion times set by electron-transfer rates in denatured Ru(NH
3
)
5
(His-33)-Zn-cytochrome
c
Authors: Chang I-Jy , Lee Jennifer C. , Winkler Jay R. , Gray Harry B.
Published: April 2003

The kinetics of electron transfer from the triplet-excited Zn-porphyrin to a Ru(NH
3
)
5
(His-33)
3+
complex have been measured in Zn-substituted ruthenium-modified cytochrome
c
under denaturing conditions. In the folded protein, the electron-tunneling rate constant is 7.5 × 10
5
s
−1
. As the protein is denatured with guanidine hydrochloride, a faster adiabatic electron-transfer reaction appears (4.0 × 10
6
s
−1
, [guanidine hydrochloride] = 5.4 M) that is limited by the rate of intrachain diffusion to bring the Zn-porphyrin and Ru complex into contact. The 250-ns contact time for formation of a 15-residue loop in denatured cytochrome
c
is in accord with a statistical model developed by Camacho and Thirumalai [Camacho, C. J. & Thirumalai, D. (1995)
Proc. Natl. Acad. Sci. USA
92, 1277–1281] that predicts that the most probable transient loops formed in denatured proteins are comprised of 10 amino acids. Extrapolation of the cytochrome
c
contact time to a 10-residue loop sets the folding speed limit at ≈10
7
s
−1
.
Dealing with iron: Common structural principles in proteins that transport iron and heme
Authors: Baker Heather M. , Anderson Bryan F. , Baker Edward N.
Published: April 2003

Iron is essential to life, but poses severe problems because of its toxicity and the insolubility of hydrated ferric ions at neutral pH. In animals, a family of proteins called transferrins are responsible for the sequestration, transport, and distribution of free iron. Comparison of the structure and function of transferrins with a completely unrelated protein hemopexin, which carries out the same function for heme, identifies molecular features that contribute to a successful protein system for iron acquisition, transport, and release. These include a two-domain protein structure with flexible hinges that allow these domains to enclose the bound ligand and provide suitable chemistry for stable binding and an appropriate trigger for release.
The retinitis pigmentosa GTPase regulator (RPGR)- interacting protein: Subserving RPGR function and participating in disk morphogenesis
Authors: Zhao Yun , Hong Dong-Hyun , Pawlyk Basil , Yue Guohua , Adamian Michael , Grynberg Marcin , Godzik Adam , Li Tiansen
Published: April 2003

Retinitis pigmentosa is a photoreceptor degenerative disease leading to blindness in adulthood. Leber congenital amaurosis (LCA) describes a more severe condition with visual deficit in early childhood. Defects in the retinitis pigmentosa GTPase regulator (RPGR) and an RPGR-interacting protein (RPGRIP) are known causes of retinitis pigmentosa and LCA, respectively. Both proteins localize in the photoreceptor connecting cilium (CC), a thin bridge linking the cell body and the light-sensing outer segment. We show that RPGR is absent in the CC of photoreceptors lacking RPGRIP, but not vice versa. Mice lacking RPGRIP elaborate grossly oversized outer segment disks resembling a cytochalasin D-induced defect and have a more severe disease than mice lacking RPGR. Mice lacking both proteins are phenotypically indistinguishable from mice lacking RPGRIP alone.
In vitro
, RPGRIP forms homodimer and elongated filaments via interactions involving its coiled-coil and C-terminal domains. We conclude that RPGRIP is a stable polymer in the CC where it tethers RPGR and that RPGR depends on RPGRIP for subcellular localization and normal function. Our data suggest that RPGRIP is also required for disk morphogenesis, putatively by regulating actin cytoskeleton dynamics. The latter hypothesis may be consistent with a distant homology between the C-terminal domain of RPGRIP and an actin-fragmin kinase, predicted by fold recognition algorithms. A defect in RPGRIP encompasses loss of both functions, hence the more severe clinical manifestation as LCA.
Structure and membrane affinity of a suite of amphiphilic siderophores produced by a marine bacterium
Authors: Martinez Jennifer S. , Carter-Franklin Jayme N. , Mann Elizabeth L. , Martin Jessica D. , Haygood Margo G. , Butler Alison
Published: April 2003

Iron concentrations in the ocean are low enough to limit the growth of marine microorganisms, which raises questions about the molecular mechanisms these organisms use to acquire iron. Marine bacteria have been shown to produce siderophores to facilitate iron(III) uptake. We describe the structures of a suite of amphiphilic siderophores, named the amphibactins, which are produced by a nearshore isolate, γ
Proteobacterium
,
Vibrio
sp. R-10. Each amphibactin has the same Tris-hydroxamate-containing peptidic headgroup composed of three ornithine residues and one serine residue but differs in the acyl appendage, which ranges from C-14 to C-18 and varies in the degree of saturation and hydroxylation. Although amphiphilic siderophores are relatively rare, cell-associated amphiphilic siderophores are even less common. We find that the amphibactins are cell-associated siderophores. As a result of the variation in the nature of the fatty acid appendage and the cellular location of the amphibactins, the membrane partitioning of these siderophores was investigated. The physiological mixture of amphibactins had a range of membrane affinities (3.8 × 10
3
to 8.3 × 10
2
M
−1
) that are larger overall than other amphiphilic siderophores, likely accounting for their cell association. This cell association is likely an important defense against siderophore diffusion in the oceanic environment. The phylogenetic affiliation of
Vibrio
sp. R-10 is discussed, as well as the observed predominance of amphiphilic siderophores produced by marine bacteria in contrast to those produced by terrestrial bacteria.
Antibody-enhanced pneumococcal adherence requires IgA1 protease
Authors: Weiser Jeffrey N. , Bae Deborah , Fasching Claudine , Scamurra Ronald W. , Ratner Adam J. , Janoff Edward N.
Published: April 2003

IgA, the major class of Ig in secretions, classically functions by interfering with microbial attachment to host tissues. Many mucosal pathogens, includingStreptococcus pneumoniae, express an IgA1 protease that may circumvent the protective effects of this Ig subclass. Because these proteases are specific for human IgA1, we generated human mAbs to the major surface antigen of the pneumococcus, its capsular polysaccharide, and tested their effect in a colonization model of bacterial adherence to respiratory epithelial cells in culture. Rather than inhibiting adherence, type-specific IgA1 markedly enhanced bacterial attachment to host cells, but only when cleaved by IgA1 protease. Neither antibodies of protease-insensitive subclasses (IgA2 and IgG) nor those directed against heterologous capsules had such activity. The adherence-promoting properties of cleaved antibodies correlated with the cationic characteristics of their variable segments, suggesting that bound Fab fragments may neutralize the inhibitory effect of negatively charged capsules on adhesive interaction with host cells. Coating of pneumococci with anticapsular polysaccharide antibody unmasked the bacterial phosphorylcholine ligand, allowing for increased adherence mediated by binding to the platelet activating factor receptor on epithelial cells. In addition, our findings provide evidence for a novel function of bacterial IgA1 proteases. These enzymes may enable pathogens to subvert the antigen specificity of the humoral immune response to facilitate adhesive interactions and persistence on the mucosal surface.
Global and specific transcriptional repression by the histone H3 amino terminus in yeast
Authors: Sabet Nevin , Tong Fumin , Madigan James P. , Volo Sam , Smith M. Mitchell , Morse Randall H.
Published: April 2003

The yeast
CHA1
promoter is activated in the presence of serine or threonine. Activation requires the Cha4p activator, and it results in perturbation of a nucleosome that incorporates the TATA element under noninducing conditions. We show that in yeast lacking the amino terminus of histone H3, the promoter is constitutively active and the chromatin is concomitantly perturbed. This derepression occurs in the absence of elevated intracellular levels of serine or threonine and is not observed in cells lacking Rpd3p, Tup1p, or the amino terminus of histone H4. Furthermore, derepression in the absence of the H3 amino terminus requires the primary activator of this promoter, Cha4p, which we show by chromatin immunoprecipitation to be constitutively bound to the
CHA1
promoter in WT yeast. Thus, the H3 amino terminus is required to prevent Cha4p from activating
CHA1
in the absence of inducer. We also present results of a microarray experiment showing that the H3 amino terminus has a substantial repressive effect on a genome-wide scale.
A regulatory protein that interferes with activator-stimulated transcription in bacteria
Authors: Nakano Shunji , Nakano Michiko M. , Zhang Ying , Leelakriangsak Montira , Zuber Peter
Published: April 2003

Transcriptional activator proteins in bacteria often operate by interaction with the C-terminal domain of the α-subunit of RNA polymerase (RNAP). Here we report the discovery of an “anti-α” factor Spx in
Bacillus subtilis
that blocks transcriptional activation by binding to the α-C-terminal domain, thereby interfering with the capacity of RNAP to respond to certain activator proteins. Spx disrupts complex formation between the activator proteins ResD and ComA and promoter-bound RNAP, and it does so by direct interaction with the α-subunit. ResD- and ComA-stimulated transcription requires the proteolytic elimination of Spx by the ATP-dependent protease ClpXP. Spx represents a class of transcriptional regulators that inhibit activator-stimulated transcription by interaction with α.