bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene expression profiles of human breast cancer progression
Authors: Ma Xiao-Jun , Salunga Ranelle , Tuggle J. Todd , Gaudet Justin , Enright Edward , McQuary Philip , Payette Terry , Pistone Maria , Stecker Kimberly , Zhang Brian M. , Zhou Yi-Xiong , Varnholt Heike , Smith Barbara , Gadd Michelle , Chatfield Erica , Kessler Jessica , Baer Thomas M. , Erlander Mark G. , Sgroi Dennis C.
Published: May 2003

Although distinct pathological stages of breast cancer have been described, the molecular differences among these stages are largely unknown. Here, through the combined use of laser capture microdissection and DNA microarrays, we have generated
in situ
gene expression profiles of the premalignant, preinvasive, and invasive stages of human breast cancer. Our data reveal extensive similarities at the transcriptome level among the distinct stages of progression and suggest that gene expression alterations conferring the potential for invasive growth are already present in the preinvasive stages. In contrast to tumor stage, different tumor grades are associated with distinct gene expression signatures. Furthermore, a subset of genes associated with high tumor grade is quantitatively correlated with the transition from preinvasive to invasive growth.
The Na/K pump, Cl ion, and osmotic stabilization of cells
Authors: Armstrong Clay M.
Published: May 2003

An equation for membrane voltage is derived that takes into account the electrogenicity of the Na/K pump and is valid dynamically, as well as in the steady state. This equation is incorporated into a model for the osmotic stabilization of cells. The results emphasize the role of the pump and membrane voltage in lowering internal Cl
−
concentration, thus making osmotic room for vital substances that must be sequestered in the cell.
Catechol
O
-methyltransferase
val

158

-met
genotype and individual variation in the brain response to amphetamine
Authors: Mattay Venkata S. , Goldberg Terry E. , Fera Francesco , Hariri Ahmad R. , Tessitore Alessandro , Egan Michael F. , Kolachana Bhaskar , Callicott Joseph H. , Weinberger Daniel R.
Published: May 2003

Monamines subserve many critical roles in the brain, and monoaminergic drugs such as amphetamine have a long history in the treatment of neuropsychiatric disorders and also as a substance of abuse. The clinical effects of amphetamine are quite variable, from positive effects on mood and cognition in some individuals, to negative responses in others, perhaps related to individual variations in monaminergic function and monoamine system genes. We explored the effect of a functional polymorphism (

val
158
-met

) in the catechol
O
-methyltransferase gene, which has been shown to modulate prefrontal dopamine in animals and prefrontal cortical function in humans, on the modulatory actions of amphetamine on the prefrontal cortex. Amphetamine enhanced the efficiency of prefrontal cortex function assayed with functional MRI during a working memory task in subjects with the high enzyme activity
val
/
val
genotype, who presumably have relatively less prefrontal synaptic dopamine, at all levels of task difficulty. In contrast, in subjects with the low activity
met
/
met
genotype who tend to have superior baseline prefrontal function, the drug had no effect on cortical efficiency at low-to-moderate working memory load and caused deterioration at high working memory load. These data illustrate an application of functional neuroimaging in pharmacogenomics and extend basic evidence of an inverted-“U” functional-response curve to increasing dopamine signaling in the prefrontal cortex. Further, individuals with the
met
/
met
catechol
O
-methyltransferase genotype appear to be at increased risk for an adverse response to amphetamine.
Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs
Authors: Torchilin Vladimir P. , Lukyanov Anatoly N. , Gao Zhonggao , Papahadjopoulos-Sternberg Brigitte
Published: May 2003

To prepare immunomicelles, new targeted carriers for poorly soluble pharmaceuticals, a procedure has been developed to chemically attach mAbs to reactive groups incorporated into the corona of polymeric micelles made of polyethylene glycol–phosphatidylethanolamine conjugates. Micelle-attached antibodies retained their ability to specifically interact with their antigens. Immunomicelles with attached antitumor mAb 2C5 effectively recognized and bound various cancer cells
in vitro
and showed an increased accumulation in experimental tumors in mice when compared with nontargeted micelles. Intravenous administration of tumor-specific 2C5 immunomicelles loaded with a sparingly soluble anticancer agent, taxol, into experimental mice bearing Lewis lung carcinoma resulted in an increased accumulation of taxol in the tumor compared with free taxol or taxol in nontargeted micelles and in enhanced tumor growth inhibition. This family of pharmaceutical carriers can be used for the solubilization and enhanced delivery of poorly soluble drugs to various pathological sites in the body.
Cloning of an
Alpha-TFEB
fusion in renal tumors harboring the t(6;11)(p21;q13) chromosome translocation
Authors: Davis Ian J. , Hsi Bae-Li , Arroyo Jason D. , Vargas Sara O. , Yeh Y. Albert , Motyckova Gabriela , Valencia Patricia , Perez-Atayde Antonio R. , Argani Pedram , Ladanyi Marc , Fletcher Jonathan A. , Fisher David E.
Published: May 2003

MITF, TFE3, TFEB, and TFEC comprise a transcription factor family (MiT) that regulates key developmental pathways in several cell lineages. Like MYC, MiT members are basic helix-loop-helix-leucine zipper transcription factors. MiT members share virtually perfect homology in their DNA binding domains and bind a common DNA motif. Translocations of
TFE3
occur in specific subsets of human renal cell carcinomas and in alveolar soft part sarcomas. Although multiple translocation partners are fused to
TFE3
, each translocation product retains TFE3's basic helix–loop–helix leucine zipper. We have identified the genes fused by the chromosomal translocation t(6;11)(p21.1;q13), characteristic of another subset of renal neoplasms. In two primary tumors we found that
Alpha
, an intronless gene, rearranges with the first intron of
TFEB
, just upstream of TFEB's initiation ATG, preserving the entire TFEB coding sequence. Fluorescence
in situ
hybridization confirmed the involvement of both
TFEB
and
Alpha
in this translocation. Although the
Alpha
promoter drives expression of this fusion gene, the Alpha gene does not contribute to the ORF. Whereas TFE3 is typically fused to partner proteins in subsets of renal tumors, we found that wild-type, unfused TFE3 stimulates clonogenic growth in a cell-based assay, suggesting that dysregulated expression, rather than altered function of TFEB or TFE3 fusions, may confer neoplastic properties, a mechanism reminiscent of MYC activation by promoter substitution in Burkitt's lymphoma.
Alpha-TFEB
is thus identified as a fusion gene in a subset of pediatric renal neoplasms.
Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate
Authors: Chen Lan , Walker Deborah , Sun Binyuan , Hu Yanan , Walker Suzanne , Kahne Daniel
Published: May 2003

Bacterial transglycosylases are enzymes that couple the disaccharide subunits of peptidoglycan to form long carbohydrate chains. These enzymes are the target of the pentasaccharide antibiotic moenomycin as well as the proposed target of certain glycopeptides that overcome vancomycin resistance. Because bacterial transglycosylases are difficult enzymes to study, it has not previously been possible to evaluate how moenomycin inhibits them or to determine whether glycopeptide analogues directly target them. We have identified transglycosylase assay conditions that enable kinetic analysis of inhibitors and have examined the inhibition of
Escherichia coli
penicillin-binding protein 1b (PBP1b) by moenomycin as well as by various glycopeptides. We report that chlorobiphenyl vancomycin analogues that are incapable of binding substrates nevertheless inhibit
E. coli
PBP1b, which shows that these compounds interact directly with the enzyme. These findings support the hypothesis that chlorobiphenyl vancomycin derivatives overcome vanA resistance by targeting bacterial transglycosylases. We have also found that moenomycin is not competitive with respect to the lipid II substrate of PBP1b, as has long been believed. With the development of suitable methods to evaluate bacterial transglycosylases, it is now possible to probe the mechanism of action of some potentially very important antibiotics.
Reverse engineering gene networks: Integrating genetic perturbations with dynamical modeling
Authors: Tegnér Jesper , Yeung M. K. Stephen , Hasty Jeff , Collins James J.
Published: May 2003

While the fundamental building blocks of biology are being tabulated by the various genome projects, microarray technology is setting the stage for the task of deducing the connectivity of large-scale gene networks. We show how the perturbation of carefully chosen genes in a microarray experiment can be used in conjunction with a reverse engineering algorithm to reveal the architecture of an underlying gene regulatory network. Our iterative scheme identifies the network topology by analyzing the steady-state changes in gene expression resulting from the systematic perturbation of a particular node in the network. We highlight the validity of our reverse engineering approach through the successful deduction of the topology of a linear
in numero
gene network and a recently reported model for the segmentation polarity network in
Drosophila melanogaster
. Our method may prove useful in identifying and validating specific drug targets and in deconvolving the effects of chemical compounds.
Digital genotyping and haplotyping with polymerase colonies
Authors: Mitra Robi D. , Butty Vincent L. , Shendure Jay , Williams Benjamin R. , Housman David E. , Church George M.
Published: May 2003

Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two single- nucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.
Postinsult treatment with lithium reduces brain damage and facilitates neurological recovery in a rat ischemia/reperfusion model
Authors: Ren Ming , Senatorov Vladimir V. , Chen Ren-Wu , Chuang De-Maw
Published: May 2003

Lithium has long been a primary drug used to treat bipolar mood disorder, even though the drug's therapeutic mechanisms remain obscure. Recent studies demonstrate that lithium has neuroprotective effects against glutamate-induced excitotoxicity in cultured neurons and
in vivo
. The present study was undertaken to examine whether postinsult treatment with lithium reduces brain damage induced by cerebral ischemia. We found that s.c. injection of lithium dose dependently (0.5–3 mEq/kg) reduced infarct volume in the rat model of middle cerebral artery occlusion/reperfusion. Infarct volume was reduced at a therapeutic dose of 1 mEq/kg even when administered up to 3 h after the onset of ischemia. Neurological deficits induced by ischemia were also reduced by daily administration of lithium over 1 week. Moreover, lithium treatment decreased the number of neurons showing DNA damage in the ischemic brain. These neuroprotective effects were associated with an up-regulation of cytoprotective heat shock protein 70 (HSP70) in the ischemic brain hemisphere as determined by immunohistochemistry and Western blotting analysis. Lithium-induced HSP70 up-regulation in the ischemic hemisphere was preceded by an increase in the DNA binding activity of heat shock factor 1, which regulates the transcription of HSP70. Physical variables and cerebral blood flow were unchanged by lithium treatment. Our results suggest that postinsult lithium treatment reduces both ischemia-induced brain damage and associated neurological deficits. Moreover, the heat shock response is likely to be involved in lithium's neuroprotective actions. Additionally, our studies indicate that lithium may have clinical utility for the treatment of patients with acute stroke.
SHED: Stem cells from human exfoliated deciduous teeth
Authors: Miura Masako , Gronthos Stan , Zhao Mingrui , Lu Bai , Fisher Larry W. , Robey Pamela Gehron , Shi Songtao
Published: May 2003

To isolate high-quality human postnatal stem cells from accessible resources is an important goal for stem-cell research. In this study we found that exfoliated human deciduous tooth contains multipotent stem cells [
s
tem cells from
h
uman
e
xfoliated
d
eciduous teeth (SHED)]. SHED were identified to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts. After
in vivo
transplantation, SHED were found to be able to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Here we show that a naturally exfoliated human organ contains a population of stem cells that are completely different from previously identified stem cells. SHED are not only derived from a very accessible tissue resource but are also capable of providing enough cells for potential clinical application. Thus, exfoliated teeth may be an unexpected unique resource for stem-cell therapies including autologous stem-cell transplantation and tissue engineering.
Adeno-associated viruses undergo substantial evolution in primates during natural infections
Authors: Gao Guangping , Alvira Mauricio R. , Somanathan Suryanarayan , Lu You , Vandenberghe Luk H. , Rux John J. , Calcedo Roberto , Sanmiguel Julio , Abbas Zahra , Wilson James M.
Published: May 2003

Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.
MmpL8 is required for sulfolipid-1 biosynthesis and
Mycobacterium tuberculosis
virulence
Authors: Converse Scott E. , Mougous Joseph D. , Leavell Michael D. , Leary Julie A. , Bertozzi Carolyn R. , Cox Jeffery S.
Published: May 2003

Mycobacterium tuberculosis
, the causative agent of human tuberculosis, is unique among bacterial pathogens in that it displays a wide array of complex lipids and lipoglycans on its cell surface. One of the more remarkable lipids is a sulfated glycolipid, termed sulfolipid-1 (SL-1), which is thought to mediate specific host-pathogen interactions during infection. However, a direct role for SL-1 in
M. tuberculosis
virulence has not been established. Here we show that MmpL8, a member of a large family of predicted lipid transporters in
M. tuberculosis
, is required for SL-1 production. The accumulation of an SL-1 precursor, termed SL
1278
, in
mmpL8
mutant cells indicates that MmpL8 is necessary for an intermediate step in the SL-1 biosynthesis pathway. We use a novel fractionation procedure to demonstrate that SL-1 is present on the cell surface, whereas SL
1278
is found exclusively in more internal layers. Importantly, we show that
mmpL8
mutants are attenuated for growth in a mouse model of tuberculosis. However, SL-1
per se
is not required for establishing infection as
pks2
mutants, which are defective in an earlier step in SL-1 biosynthesis, have no obvious growth defect. Thus, we hypothesize that either MmpL8 transports molecules in addition to SL-1 that mediate host-pathogen interactions or the accumulation of SL
1278
in
mmpL8
mutant cells interferes with other pathways required for growth during the early stages of infection.
Protein–protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces
Authors: Ma Buyong , Elkayam Tal , Wolfson Haim , Nussinov Ruth
Published: May 2003

Polar residue hot spots have been observed at protein–protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface. Consequently, hot spots can be used to define binding epitopes. We further show a correspondence between energy hot spots and structurally conserved residues. The number of structurally conserved residues, particularly of high ranking energy hot spots, increases with the binding site contact size. This finding may suggest that effectively dispersing hot spots within a large contact area, rather than compactly clustering them, may be a strategy to sustain essential key interactions while still allowing certain protein flexibility at the interface. Thus, most conserved polar residues at the binding interfaces confer rigidity to minimize the entropic cost on binding, whereas surrounding residues form a flexible cushion. Furthermore, our finding that similar residue hot spots occur across different protein families suggests that affinity and specificity are not necessarily coupled: higher affinity does not directly imply greater specificity. Conservation of Trp on the protein surface indicates a highly likely binding site. To a lesser extent, conservation of Phe and Met also imply a binding site. For all three residues, there is a significant conservation in binding sites, whereas there is no conservation on the exposed surface. A hybrid strategy, mapping sequence alignment onto a single structure illustrates the possibility of binding site identification around these three residues.
Dynamic assembly of surface structures in living cells
Authors: Gorelik Julia , Shevchuk Andrew I. , Frolenkov Gregory I. , Diakonov Ivan A. , Lab Max J. , Kros Corné J. , Richardson Guy P. , Vodyanoy Igor , Edwards Christopher R. W. , Klenerman David , Korchev Yuri E.
Published: May 2003

Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary “building blocks” for the assembly of specialized structures on the cell surface.
Bacteria are not too small for spatial sensing of chemical gradients: An experimental evidence
Authors: Thar Roland , Kühl Michael
Published: May 2003

By analyzing the chemotactic behavior of a recently described marine bacterial species, we provide experimental evidence that bacteria are not too small for sensing chemical gradients spatially. The bipolar flagellated vibrioid bacteria (typical size 2 × 6 μm) exhibit a unique motility pattern as they translate along as well as rotate around their short axis, i.e., the pathways of the cell poles describe a double helix. The natural habitat of the bacteria is characterized by steep oxygen gradients where they accumulate in a band at their preferred oxygen concentration of ≈2 μM. Single cells leaving the band toward the oxic region typically return to the band within 16 s following a U-shaped track. A detailed analysis of the tracks reveals that the cells must be able to sense the oxygen gradient perpendicular to their swimming direction. Thus, they can detect oxygen gradients along a distance of ≈5 μm corresponding to the extension of their long axis. The observed behavior can be explained by the presence of two independent sensor regions at either cell pole that modulate the rotation speed of the polar flagellar bundles, i.e., the flagellar bundle at the cell pole exposed to higher oxygen concentration is rotating faster than the other bundle. A mathematical model based on these assumptions reproduces the observed swimming behavior of the bacteria.
Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: A cause of vitamin D resistance
Authors: Chen Hong , Hewison Martin , Hu Bing , Adams John S.
Published: May 2003

In previous studies, we have shown that steroid hormone resistance in New World primates occurs in the absence of abnormal expression of cognate nuclear receptors. Rather, these animals have elevated levels of heterogeneous nuclear ribonucleoproteins (hnRNPs) that act as hormone response element-binding proteins and attenuate target gene transactivation. Here we present evidence for a similar mechanism in humans via a patient with resistance to the active form of vitamin D [1,25-dihydroxyvitamin D
3
(1,25(OH)
2
D
3
)] who presented with normal vitamin D receptor (VDR) expression. Initial cotransfection studies showed that the cells of the patient suppressed basal and hormone-induced transactivation by wild-type VDR. Electrophoretic mobility-shift assays and Western/Southwestern blot analyses indicated that this suppressive effect was due to overexpression of a nuclear protein that specifically interacts with a DNA response element known to bind retinoid X receptor–VDR heterodimers. Ab blocking in electrophoretic mobility-shift assays indicated that this dominant-negative acting protein was in the hnRNPA family of nucleic acid-binding proteins. Further studies have shown that several members of this family, most notably hnRNPA1, were able to suppress basal and 1,25(OH)
2
D
3
-induced luciferase activity. We therefore propose that this case of vitamin D resistance in a human subject is similar to that previously described for New World primates in which abnormal expression of a hormone response element-binding protein can cause target cell resistance to 1,25(OH)
2
D
3
. That this protein is a member of the hnRNP family capable of interacting with double-stranded DNA highlights a potentially important new component of the complex machinery required for steroid hormone signal transduction.
Amyloid-β peptide levels in brain are inversely correlated with insulysin activity levels
in vivo
Authors: Miller Bonnie C. , Eckman Elizabeth A. , Sambamurti Kumar , Dobbs Nicole , Chow K. Martin , Eckman Christopher B. , Hersh Louis B. , Thiele Dwain L.
Published: May 2003

Factors that elevate amyloid-β (Aβ) peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Aβ degradation based on its hydrolysis of Aβ peptides
in vitro
. In this study,
in vivo
levels of brain Aβ40 and Aβ42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the γ-secretase-generated C-terminal fragment of the Aβ precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased ≈50%, brain Aβ peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between
in vivo
insulysin activity levels and brain Aβ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease.
Role of backbone solvation and electrostatics in generating preferred peptide backbone conformations: Distributions of phi
Authors: Avbelj Franc , Baldwin Robert L.
Published: May 2003

The “coil library,” consisting of the φ, ψ values of residues outside secondary structure in high-resolution protein structure
s
, has chiefly the β, α
R
, α
L
, and polyproline II backbone conformations. In denatured proteins, the 20 aa have different average values of the
3
J
HN
α
coupling constant, related to the backbone angle φ by the Karplus relation. Average
3
J
HN
α
values obtained from the distributions of φ,
g
(φ), of the coil library agree with NMR results, and so the coil library can be and is being used to model denatured proteins. Here, Monte Carlo simulations of backbone conformations in denatured proteins are used to test two physics-based models: the random coil model of Brant and Flory [(1965)
J. Am. Chem. Soc.
87, 2788–2791 and 2791–2800] and an electrostatic screening model (ESM) that includes electrostatic solvation. The random coil model represents hindered rotation about φ and ψ backbone angles, nonbonded interactions, and dipole–dipole interactions. In the ESM, the nonbonded interactions term is replaced by the use of hard sphere repulsion and allowed regions in the Ramachadran maps. These models were tested by using the amino acid sequences of three small proteins. There are two main conclusions: (
i
) The
g
(φ) distributions of the coil library contain detailed, specific information, so that prediction of the
g
(φ) distributions of the different amino acids is a demanding test of the energy function. (
ii
) The ESM is partly successful in predicting the
g
(φ) distributions. Electrostatic solvation is primarily responsible, and steric clash between pairs of atoms connected by torsion angles is not responsible.
Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and
in vivo
inhibitor treatment
Authors: Wainwright Mark S. , Rossi Janet , Schavocky James , Crawford Susan , Steinhorn David , Velentza Anastasia V. , Zasadzki Magdalena , Shirinsky Vladimir , Jia Yuzhi , Haiech Jacques , Van Eldik Linda J. , Watterson D. Martin
Published: May 2003

Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal
in vivo
role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.
Mesenchymal progenitor self-renewal deficiency leads to age-dependent osteoporosis in Sca-1/Ly-6A null mice
Authors: Bonyadi Mortaza , Waldman Stephen D. , Liu Danmei , Aubin Jane E. , Grynpas Marc D. , Stanford William L.
Published: May 2003

The cellular and molecular mechanisms that underlie age-dependent osteoporosis, the most common disease in the Western Hemisphere, are poorly understood in part due to the lack of appropriate animal models in which to study disease progression. Here, we present a model that shows many similarities to the human disease. Sca-1, well known for its expression on hematopoietic stem cells, is present on a subset of bone marrow stromal cells, which potentially include mesenchymal stem cells. Longitudinal studies showed that
Sca-1
−/−
mice undergo normal bone development but with age exhibit dramatically decreased bone mass resulting in brittle bones.
In vivo
and
in vitro
analyses demonstrated that Sca-1 is required directly for the self-renewal of mesenchymal progenitors and indirectly for the regulation of osteoclast differentiation. Thus, defective mesenchymal stem or progenitor cell self-renewal may represent a previously uncharacterized mechanism of age-dependent osteoporosis in humans.
The initiation–elongation transition: Lateral mobility of RNA in RNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23
Authors: Pal Mahadeb , Luse Donal S.
Published: May 2003

RNA polymerase II transcription complexes stalled shortly after initiation over a repetitive segment of the template can undergo efficient transcript slippage, during which the 3′ end of the RNA slides upstream and then re-pairs with the template, allowing transcription to continue. In the present study, we have used transcript slippage as an assay to identify possible structural transitions that occur as the polymerase passes from the initiation to the elongation phase of transcription. We reasoned that transcript slippage would not occur in fully processive complexes. We constructed a series of templates that allowed us to stall RNA polymerase II after the synthesis of a repetitive sequence (5′-CUCUCU-3′) at varying distances downstream of +1. We found that polymerase must synthesize at least a 23-nt RNA to attain resistance to transcript slippage. The ability to undergo slippage was lost in two discrete steps, suggestive of two distinct transitions. The first transition is the formation of the 8- to 9-bp mature RNA–DNA hybrid, when slippage abruptly dropped by 10-fold. However, easily detectable slippage continued until 14 more bonds were made. Thus, although the transcript becomes tightly constrained within the transcription complex once the hybrid reaches its final length, much more RNA synthesis is required before the RNA is no longer able to slip upstream along the template. This last point may reflect an important stabilizing role for the interaction of the polymerase with the transcript well upstream of the RNA–DNA hybrid.
Tuning activation of the AMPA-sensitive GluR2 ion channel by genetic adjustment of agonist-induced conformational changes
Authors: Armstrong Neali , Mayer Mark , Gouaux Eric
Published: May 2003

The (
S
)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazole) propionic acid (AMPA) receptor discriminates between agonists in terms of binding and channel gating; AMPA is a high-affinity full agonist, whereas kainate is a low-affinity partial agonist. Although there is extensive literature on the functional characterization of partial agonist activity in ion channels, structure-based mechanisms are scarce. Here we investigate the role of Leu-650, a binding cleft residue conserved among AMPA receptors, in maintaining agonist specificity and regulating agonist binding and channel gating by using physiological, x-ray crystallographic, and biochemical techniques. Changing Leu-650 to Thr yields a receptor that responds more potently and efficaciously to kainate and less potently and efficaciously to AMPA relative to the WT receptor. Crystal structures of the Leu-650 to Thr mutant reveal an increase in domain closure in the kainate-bound state and a partially closed and a fully closed conformation in the AMPA-bound form. Our results indicate that agonists can induce a range of conformations in the GluR2 ligand-binding core and that domain closure is directly correlated to channel activation. The partially closed, AMPA-bound conformation of the L650T mutant likely captures the structure of an agonist-bound, inactive state of the receptor. Together with previously solved structures, we have determined a mechanism of agonist binding and subsequent conformational rearrangements.
A TyrCD1/TrpG8 hydrogen bond network and a TyrB10—TyrCD1 covalent link shape the heme distal site of
Mycobacterium tuberculosis
hemoglobin O
Authors: Milani Mario , Savard Pierre-Yves , Ouellet Hugues , Ascenzi Paolo , Guertin Michel , Bolognesi Martino
Published: May 2003

Truncated hemoglobins (Hbs) are small hemoproteins, identified in microorganisms and in some plants, forming a separate cluster within the Hb superfamily. Two distantly related truncated Hbs, trHbN and trHbO, are expressed at different developmental stages in
Mycobacterium tuberculosis
. Sequence analysis shows that the two proteins share 18% amino acid identities and belong to different groups within the truncated Hb cluster. Although a specific defense role against nitrosative stress has been ascribed to trHbN (expressed during the
Mycobacterium
stationary phase), no clear functions have been recognized for trHbO, which is expressed throughout the
Mycobacterium
growth phase. The 2.1-Å crystal structure of
M. tuberculosis
cyano-met trHbO shows that the protein assembles in a compact dodecamer. Six of the dodecamer subunits are characterized by a double conformation for their CD regions and, most notably, by a covalent bond linking the phenolic O atom of TyrB10 to the aromatic ring of TyrCD1, in the heme distal cavity. All 12 subunits display a cyanide ion bound to the heme Fe atom, stabilized by a tight hydrogen-bonded network based on the (globin very rare) TyrCD1 and TrpG8 residues. The small apolar AlaE7 residue leaves room for ligand access to the heme distal site through the conventional “E7 path,” as proposed for myoglobin. Different from trHbN, where a 20-Å protein matrix tunnel is held to sustain ligand diffusion to an otherwise inaccessible heme distal site, the topologically related region in trHbO hosts two protein matrix cavities.
The loss of the hemoglobin H
2
S-binding function in annelids from sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection
Authors: Bailly Xavier , Leroy Riwanon , Carney Susan , Collin Olivier , Zal Franck , Toulmond André , Jollivet Didier
Published: May 2003

The hemoglobin of the deep-sea hydrothermal vent vestimentiferan
Riftia pachyptila
(annelid) is able to bind toxic hydrogen sulfide (H
2
S) to free cysteine residues and to transport it to fuel endosymbiotic sulfide-oxidising bacteria. The cysteine residues are conserved key amino acids in annelid globins living in sulfide-rich environments, but are absent in annelid globins from sulfide-free environments. Synonymous and nonsynonymous substitution analysis from two different sets of orthologous annelid globin genes from sulfide rich and sulfide free environments have been performed to understand how the sulfide-binding function of hemoglobin appeared and has been maintained during the course of evolution. This study reveals that the sites occupied by free-cysteine residues in annelids living in sulfide-rich environments and occupied by other amino acids in annelids from sulfide-free environments, have undergone positive selection in annelids from sulfide-free environments. We assumed that the high reactivity of cysteine residues became a disadvantage when H
2
S disappeared because free cysteines without their natural ligand had the capacity to interact with other blood components, disturb homeostasis, reduce fitness and thus could have been counterselected. To our knowledge, we pointed out for the first time a case of function loss driven by molecular adaptation rather than genetic drift. If constraint relaxation (H
2
S disappearance) led to the loss of the sulfide-binding function in modern annelids from sulfide-free environments, our work suggests that adaptation to sulfide-rich environments is a plesiomorphic feature, and thus that the annelid ancestor could have emerged in a sulfide-rich environment.
Distinct response to dioxin in an arylhydrocarbon receptor (AHR)-humanized mouse
Authors: Moriguchi Takashi , Motohashi Hozumi , Hosoya Tomonori , Nakajima Osamu , Takahashi Satoru , Ohsako Seiichiroh , Aoki Yasunobu , Nishimura Noriko , Tohyama Chiharu , Fujii-Kuriyama Yoshiaki , Yamamoto Masayuki
Published: May 2003

There are large inter- and intraspecies differences in susceptibility to dioxin-induced toxicities. A critical question in risk assessment of dioxin and related compounds is whether humans are sensitive or resistant to their toxicities. The diverse responses of mammals to dioxin are strongly influenced by functional polymorphisms of the arylhydrocarbon receptor (AHR). To characterize responses mediated by the human AHR (hAHR), we generated a mouse possessing hAHR instead of mouse AHR. Responses of these mice to 2,3,7,8-tetrachlorodibenzo-
p
-dioxin (TCDD) and 3-methylcholanthrene were compared with the responses of naturally sensitive (C57BL/6J) and resistant (DBA/2) mice. Mice homozygous for h
AHR
exhibited weaker induction of AHR target genes such as
cyp1a1
and
cyp1a2
than did C57BL/6J (
Ahr
b-1/b-1
) mice. DBA/2 (
Ahr
d/d
) mice were less responsive to induction of
cyp
genes than C57BL/6J mice. hAHR and DBA/2 AHR exhibit similar ligand-binding affinities and homozygous h
AHR
and
Ahr
d/d
mice displayed comparable induction of AHR target genes by 3-methylcholanthrene. However, when TCDD was administered, a greatly diminished response was observed in homozygous h
AHR
mice compared with
Ahr
d/d
mice, indicating that hAHR expressed in mice is functionally less responsive to TCDD than DBA/2 AHR. After maternal exposure to TCDD, homozygous h
AHR
fetuses developed embryonic hydronephrosis, but not cleft palate, whereas fetuses possessing
Ahr
b-1
or
Ahr
d
developed both anomalies. These results suggest that hAHR may define the specificity of the responses to various AHR ligands. Thus, the hAHR knock-in mouse is a humanized model mouse that may better predict the biological effects of bioaccumulative environmental toxicants like TCDD in humans.
Oxidative folding intermediates with nonnative disulfide bridges between adjacent cysteine residues
Authors: Čemažar Maša , Zahariev Sotir , Lopez Jakob J. , Carugo Oliviero , Jones Jonathan A. , Hore P. J. , Pongor Sándor
Published: May 2003

The oxidative folding of the Amaranthus α-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied
in vitro
in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (
MFI
), was further analyzed by
1
H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in
MFI
may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.
An Anionic Ryanoid, 10-O-succinoylryanodol, Provides Insights into the Mechanisms Governing the Interaction of Ryanoids and the Subsequent Altered Function of Ryanodine-receptor Channels
Authors: Tanna Bhavna , Welch William , Ruest Luc , Sutko John L. , Williams Alan J.
Published: May 2003

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.
Extracellular Ca2+ Modulates the Effects of Protons on Gating and Conduction Properties of the T-type Ca2+ Channel α1G (CaV3.1)
Authors: Talavera Karel , Janssens Annelies , Klugbauer Norbert , Droogmans Guy , Nilius Bernd
Published: May 2003

Since Ca2+ is a major competitor of protons for the modulation of high voltage–activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel α1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.
Potentiated L-type Ca2+ Channels Rectify
Authors: Leuranguer Valérie , Dirksen Robert T. , Beam Kurt G.
Published: May 2003

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type α1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-α1C) or repeat III (E3K-α1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-α1C and E3K-α1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (−)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-α1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-α1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.
Phylogenetically Older Introns Strongly Correlate With Module Boundaries in Ancient Proteins
Authors: Fedorov Alexei , Roy Scott , Cao Xiaohong , Gilbert Walter
Published: May 2003

The hypothesis that some (but not all) introns were used to construct ancient genes by exon shuffling of modules at the earliest stages of evolution is supported by the finding of an excess of phase-zero intron positions in the boundary regions of such modules in 276 ancient proteins (defined as common to eukaryotes and prokaryotes). Here we show further that as phase-zero intron positions are shared by distant taxa, and thus are truly phylogenetically ancient, their excess in the boundaries becomes greater, rising to an 80% excess if shared by four out of the five taxa: vertebrates, invertebrates, fungi, plants, and protists.