bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the human GLUT4 gene promoter: Interaction between a transcriptional activator and myocyte enhancer factor 2A
Authors: Knight John B. , Eyster Craig A. , Griesel Beth A. , Olson Ann Louise
Published: December 2003

The GLUT4 gene is subject to complex tissue-specific and metabolic regulation, with a profound impact on insulin-mediated glucose disposal. We have shown, by using transgenic mice, that the human GLUT4 promoter is regulated through the cooperative function of two distinct regulatory elements, domain 1 and the myocyte enhancer factor 2 (MEF2) domain. The MEF2 domain binds transcription factors MEF2A and MEF2D
in vivo
. Domain I binds a transcription factor, GLUT4 enhancer factor (GEF). In this report, we show a restricted pattern of GEF expression in human tissues, which overlaps with MEF2A only in tissues expressing high levels of GLUT4, suggesting the hypothesis that GEF and MEF2A function together to activate GLUT4 transcription. Data obtained from transiently transfected cells support this hypothesis. Neither GEF nor MEF2A alone significantly activated GLUT4 promoter activity, but increased promoter activity 4- to 5-fold when expressed together. Deletion of the GEF-binding domain (domain I) and the MEF2-binding domain prevented activation, strengthening the conclusion that promoter regulation occurs through these elements. GEF and MEF2A, isolated from nuclei of transfected cells, bound domain I and the MEF2 domain, respectively, which is consistent with activation through these regulatory elements. Finally, GEF and MEF2A coimmunoprecipitated
in vivo
, strongly supporting a mechanism of GLUT4 transcription activation that depends on this protein–protein interaction.
Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer
Authors: Ying Liming , Green Jeremy J. , Li Haitao , Klenerman David , Balasubramanian Shankar
Published: December 2003

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 ± 0.4 kcal·mol
–1
and –52.3 ± 1.4 cal·mol
–1
·K
–1
, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 ± 0.2 kcal·mol
–1
and –23.0 ± 0.8 cal·mol
–1
·K
–1
, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15
k
B
T
based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.
Cu nanocrystal growth on peptide nanotubes by biomineralization: Size control of Cu nanocrystals by tuning peptide conformation
Authors: Banerjee Ipsita A. , Yu Lingtao , Matsui Hiroshi
Published: December 2003

With recent interest in seeking new biologically inspired device-fabrication methods in nanotechnology, a new biological approach was examined to fabricate Cu nanotubes by using sequenced histidine-rich peptide nanotubes as templates. The sequenced histidine-rich peptide molecules were assembled as nanotubes, and the biological recognition of the specific sequence toward Cu lead to efficient Cu coating on the nanotubes. Cu nanocrystals were uniformly coated on the histidine-incorporated nanotubes with high packing density. In addition, the diameter of Cu nanocrystal was controlled between 10 and 30 nm on the nanotube by controlling the conformation of histidine-rich peptide by means of pH changes. Those nanotubes showed significant change in electronic structure by varying the nanocrystal diameter; therefore, this system may be developed to a conductivity-tunable building block for microelectronics and biological sensors. This simple biomineralization method can be applied to fabricate various metallic and semiconductor nanotubes with peptides whose sequences are known to mineralize specific ions.
Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide
Authors: Eu Jerry P. , Hare Joshua M. , Hess Douglas T. , Skaf Michel , Sun Junhui , Cardenas-Navina Isabella , Sun Qi-An , Dewhirst Mark , Meissner Gerhard , Stamler Jonathan S.
Published: December 2003

It is generally accepted that inhibition of nitric oxide synthase (NOS) facilitates, and thus nitric oxide (NO) inhibits, contractility of skeletal muscle. However, standard assessments of contractility are carried out at a nonphysiological oxygen tension [partial pressure of oxygen (pO
2
)] that can interfere with NO signaling (95% O
2
). We therefore examined, in normal and neuronal NOS (nNOS)-deficient mice, the influence of pO
2
on whole-muscle contractility and on myocyte calcium flux and sarcomere shortening. Here, we demonstrate a significant enhancement of these measures of muscle performance at low physiological pO
2
and an inhibitory influence at higher physiological pO
2
, which depend on endogenous nNOS. At 95% O
2
(which produces oxidative stress; muscle core pO
2
≈400 mmHg), force production is enhanced but control of contractility by NO/nitrosylation is greatly attenuated. In addition, responsivity to pO
2
is altered significantly in nNOS mutant muscle. These results reveal a fundamental role for the concerted action of NO and O
2
in physiological regulation of skeletal muscle contractility, and suggest novel molecular aspects of myopathic disease. They suggest further that the role of NO in some cellular systems may require reexamination.
Lesion bypass DNA polymerases replicate across non-DNA segments
Authors: Maor-Shoshani Ayelet , Ben-Ari Vered , Livneh Zvi
Published: December 2003

A critical feature of the robustness of the DNA replication machinery is the ability to complete its task in the presence of interfering DNA damage. A key mechanism responsible for this task is translesion replication (also termed translesion synthesis), carried out by specialized lesion bypass DNA polymerases of the Y superfamily. Here we show that in
Escherichia coli
, plasmids can be replicated across a segment of foreign non-DNA material, consisting of hydrocarbon chains of 3 or 12 methylene residues. This replication is carried out by DNA polymerase V and proceeds by at least two mechanisms: (
i
) Editing out the foreign insert, by polymerase “hopping” across it, which can be mediated by looping out of the insert, leading to its deletion, while preserving the DNA sequence. (
ii
) DNA synthesis through the insert, which occurs by incorporating one or two nucleotides opposite the hydrocarbon chain, yielding a net increase in the length of the DNA sequence. The remarkable ability of DNA polymerase V to insert nucleotides opposite a hydrocarbon chain shows that DNA synthesis can occur in a region of the template strand, which lacks all fundamental features of DNA, including its purine, pyrimidine, sugar, and phosphate moieties, and its hydrophilic and ionic nature. This bypass ability reflects a striking robustness of the translesion replication apparatus and is likely to contribute to its effectiveness in maintaining genome stability.
NMR-detected hydrogen exchange and molecular dynamics simulations provide structural insight into fibril formation of prion protein fragment 106–126
Authors: Kuwata Kazuo , Matumoto Tomoharu , Cheng Hong , Nagayama Kuniaki , James Thomas L. , Roder Heinrich
Published: December 2003

PrP106–126, a peptide corresponding to residues 107–127 of the human prion protein, induces neuronal cell death by apoptosis and causes proliferation and hypertrophy of glia, reproducing the main neuropathological features of prion-related transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. Although PrP106–126 has been shown to form amyloid-like fibrilsin vitro, their structural properties have not been elucidated. Here, we investigate the conformational characteristics of a fibril-forming fragment of the mouse prion protein, MoPrP106–126, by using electron microscopy, CD spectroscopy, NMR-detected hydrogen–deuterium exchange measurements, and molecular dynamics simulations. The fibrils contain ≈50% β-sheet structure, and strong amide exchange protection is limited to the central portion of the peptide spanning the palindromic sequence VAGAAAAGAV. Molecular dynamics simulations indicate that MoPrP106–126 in water assumes a stable structure consisting of two four-stranded parallel β-sheets that are tightly packed against each other by methyl–methyl interactions. Fibril formation involving polyalanine stacking is consistent with the experimental observations.
DNA bending and unbending by MutS govern mismatch recognition and specificity
Authors: Wang Hong , Yang Yong , Schofield Mark J. , Du Chunwei , Fridman Yonatan , Lee Susan D. , Larson Erik D. , Drummond James T. , Alani Eric , Hsieh Peggy , Erie Dorothy A.
Published: December 2003

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base–base mismatches and insertion/deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS–mismatch complex but not in the MutS–homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS–DNA complexes by using atomic force microscopy. Interestingly, MutS–DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (
i
) binds to DNA nonspecifically and bends it in search of a mismatch; (
ii
) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (
iii
) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity.
A microfluidic model for single-cell capillary obstruction by
Plasmodium falciparum
-infected erythrocytes
Authors: Shelby J. Patrick , White John , Ganesan Karthikeyan , Rathod Pradipsinh K. , Chiu Daniel T.
Published: December 2003

Severe malaria by
Plasmodium falciparum
is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage.
In vitro
techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-μm widths were readily traversed by the 8 μm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-μm channels; some that passed through the 4-μm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-μm channels, trophozoites mimicked “pitting,” a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-μm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-μm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.
Adhesive–cohesive model for protein compressibility: An alternative perspective on stability
Authors: Dadarlat Voichita M. , Post Carol Beth
Published: December 2003

As a dynamic property of folded proteins, protein compressibility provides important information about the forces that govern structural stability. We relate intrinsic compressibility to stability by using molecular dynamics to identify a molecular basis for the variation in compressibility among globular proteins. We find that excess surface charge accounts for this variation not only for the proteins simulated by molecular dynamics but also for a larger set of globular proteins. This dependence on charge distribution forms the basis for an adhesive–cohesive model of protein compressibility in which attractive forces from solvent compete with tertiary interactions that favor folding. Further, a newly recognized correlation between compressibility and the heat capacity of unfolding infers a link between compressibility and the enthalpy of unfolding. This linkage, together with the adhesive–cohesive model for compressibility, leads to the conclusion that folded proteins can gain enthalpic stability from a uniform distribution of charged atoms, as opposed to partitioning charge to the protein surface. Whether buried charged groups can be energetically stabilizing is a fundamental, yet controversial, question regarding protein structure. The analysis reported here implies that one mechanism to gain enthalpic stability involves positioning charge inside the protein in an optimal structural arrangement.
Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease
Authors: Ban Yoshiyuki , Greenberg David A. , Concepcion Erlinda , Skrabanek Lucy , Villanueva Ronald , Tomer Yaron
Published: December 2003

The 8q24 locus, which contains the thyroglobulin (Tg) gene, was previously shown to be strongly linked with autoimmune thyroid disease (AITD). We sequenced all 48 exons of the Tg gene and identified 14 single-nucleotide polymorphisms (SNPs). Case control association studies demonstrated that an exon 10-12 SNP cluster and an exon 33 SNP were significantly associated with AITD (P< 0.01). Haplotype analysis demonstrated that the combination of these two SNP groups was more significantly associated with AITD (P< 0.001). Gene-gene interaction studies provided evidence for an interaction between HLA-DR3 and the exon 33 SNP, giving an odds ratio of 6.1 for Graves' disease. We then sequenced exons 10,12, and 33 of the mouse Tg gene in 19 strains of mice. Fifty percent of the strains susceptible to thyroiditis had a unique SNP haplotype at exons 10 and 12, whereas none of the mouse strains that were resistant to thyroiditis had this SNP haplotype (P= 0.01). We concluded that Tg is a susceptibility gene for AITD, both in humans in and in mice. A combination of at least two Tg SNPs conferred susceptibility to human AITD. Moreover, the exon 33 SNP showed evidence for interaction with HLA-DR3 in conferring susceptibility to Graves' disease.
The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart
Authors: Stieber Juliane , Herrmann Stefan , Feil Susanne , Löster Jana , Feil Robert , Biel Martin , Hofmann Franz , Ludwig Andreas
Published: December 2003

Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed I
f
or I
h
, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, I
f
in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4
-/-
mice, cardiac cells with “primitive” pacemaker action potentials could be found. However, cardiac cells with “mature” pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.
Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells
Authors: Brock Sean C. , Goldenring James R. , Crowe James E.
Published: December 2003

Respiratory syncytial virus (RSV) is the major viral cause of serious lower respiratory tract illness in infants and young children worldwide. RSV infection is limited to the superficial layers of the respiratory epithelium in immunocompetent individuals. Consistent with this
in vivo
observation, we and others have found that RSV buds preferentially from the apical surface of infected polarized epithelial cells. In contrast, directional budding is not observed in nonpolarized human epithelial cells. These findings suggest that RSV uses specific cellular trafficking pathways to accomplish viral replication. The host cell proteins that regulate directional budding of RSV are undefined. Apical sorting of cellular proteins in polarized epithelial cells involves the apical recycling endosome (ARE). To investigate whether ARE-mediated protein sorting plays a role during RSV replication, we expressed a fragment of the myosin Vb tail that functions as a dominant negative inhibitor of ARE-mediated protein sorting in polarized Madin-Darby canine kidney cells. When these cells were infected with RSV, a >9,000-fold reduction in viral yield was observed. A similar effect on virus replication was observed when a carboxyl-terminal fragment of another ARE-associated protein, the Rab11 family interacting protein 1, was expressed in Madin-Darby canine kidney cells. These data suggest that RSV requires proper ARE-mediated protein sorting for efficient egress from the apical surface of polarized epithelial cells.
Mineralized matrix deposition by marrow stromal osteoblasts in 3D perfusion culture increases with increasing fluid shear forces
Authors: Sikavitsas Vassilios I. , Bancroft Gregory N. , Holtorf Heidi L. , Jansen John A. , Mikos Antonios G.
Published: December 2003

In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant. This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant. Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype. Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds. The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment.
Hematopoietic progenitors express neural genes
Authors: Goolsby James , Marty Marie C. , Heletz Dafna , Chiappelli Joshua , Tashko Gerti , Yarnell Deborah , Fishman Paul S. , Dhib-Jalbut Suhayl , Bever Christopher T. , Pessac Bernard , Trisler David
Published: December 2003

Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after
in vitro
treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of
ex vivo
bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2′,3′ cyclic nucleotide 3′-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34
+
, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34
+
cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45
+
, CD34
+
, Sca-1
+
, AA4.1
+
, cKit
+
, GATA-2
+
, and LMO-2
+
). The neuronal and oligodendroglial genes expressed in
ex vivo
bone marrow also were expressed in all cultured CD34
+
cells, and GFAP was not observed. After CD34
+
cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34
+
cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells.
Redox regulation of surface protein thiols: Identification of integrin α-4 as a molecular target by using redox proteomics
Authors: Laragione Teresa , Bonetto Valentina , Casoni Filippo , Massignan Tania , Bianchi Giancarlo , Gianazza Elisabetta , Ghezzi Pietro
Published: December 2003

Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1–2 h) of cells with 0.1–5 mMN-acetyl-l-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiolN-acetyl-d-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin α-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and β-actin. NAC pretreatment augmented integrin α-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.
Food-web constraints on biodiversity–ecosystem functioning relationships
Authors: Thébault Elisa , Loreau Michel
Published: December 2003

The consequences of biodiversity loss for ecosystem functioning and ecosystem services have aroused considerable interest during the past decade. Recent work has focused mainly on the impact of species diversity within single trophic levels, both experimentally and theoretically. Experiments have usually showed increased plant biomass and productivity with increasing plant diversity. Changes in biodiversity, however, may affect ecosystem processes through trophic interactions among species as well. An important current challenge is to understand how these trophic interactions affect the relationship between biodiversity and ecosystem functioning. Here we present a mechanistic model of an ecosystem with multiple trophic levels in which plants compete for a limiting soil nutrient. In contrast to previous studies that focused on single trophic levels, we show that plant biomass does not always increase with plant diversity and that changes in biodiversity can lead to complex if predictable changes in ecosystem processes. Our analysis demonstrates that food-web structure can profoundly influence ecosystem properties.
Myeloperoxidase up-regulates the catalytic activity of inducible nitric oxide synthase by preventing nitric oxide feedback inhibition
Authors: Galijasevic Semira , Saed Ghassan M. , Diamond Michael P. , Abu-Soud Husam M.
Published: December 2003

Kinetic and structure analysis of inducible nitric oxide synthase (iNOS) revealed that, in addition to the increase of iNOS expression in inflamed areas, the major pathway causing overproduction of NO is destabilization of the iNOS–nitrosyl complex(es) that form during steady-state catalysis. Formation of such a complex allows iNOS to operate at only a fraction (20–30%) of its maximum activity. Thus, bioavailability of NO scavengers at sites of inflammation may play an essential role in up-regulation of the catalytic activity of iNOS, by preventing the catalytic activity inhibition that is attributed to nitrosyl complex formation. Myeloperoxidase (MPO), a major NO scavenger, is a pivotal enzyme involved in leukocyte-mediated host defenses. It is thought to play a pathogenic role under circumstances such as acute inflammatory tissue injury and chronic inflammatory conditions. However, a detailed understanding of the interrelationship between iNOS and MPO at sites of inflammation is lacking. We used direct spectroscopic, HPLC, and selective NO-electrode measurements to determine the interdependent relationship that exists between iNOS and MPO and the role of the MPO/H2O2system in up-regulating the catalytic activity of iNOS that occurs at sites of inflammation. Scavenging free NO from the iNOS milieu by the MPO/H2O2system subsequently restores the full capacity of iNOS to convertl-aginine to product (NO), as judged by the increase in the rates of citrulline and nitrite/nitrate production. Studies of iNOS catalytic mechanisms and function are essential to a more fundamental understanding of these factors, which govern iNOS-dependent processes in human health and disease.
Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H
2
O
2
in NO-independent relaxations of rabbit arteries
Authors: Chaytor Andrew T. , Edwards David H. , Bakker Linda M. , Griffith Tudor M.
Published: December 2003

We have compared the contributions of gap junctional communication and chemical signaling via H
2
O
2
to NO-independent relaxations evoked by the Ca
2+
ionophore A23187 and acetylcholine (ACh) in rabbit ilio-femoral arteries. Immunostaining confirmed the presence of connexins (Cxs) 37 and 40 in the endothelium and Cxs 40 and 43 in smooth muscle. Maximal endothelium-dependent subintimal smooth muscle hyperpolarizations evoked by A23187 and ACh were equivalent (≈20 mV) and almost abolished by an inhibitory peptide combination targeted against Cxs 37, 40, and 43. However, maximal NO-independent relaxations evoked by A23187 were unaffected by such peptides, whereas those evoked by ACh were depressed by ≈70%. By contrast, the enzyme catalase, which destroys H
2
O
2
, attenuated A23187-induced relaxations over a broad range of concentrations, but only minimally depressed the maximum response to ACh. Catalase did not affect A23187- or ACh-evoked hyperpolarizations. After loading with an H
2
O
2
-sensitive probe, A23187 caused a marked increase in endothelial fluorescence that correlated temporally with relaxation, whereas only a weak delayed increase was observed with ACh. In arteries without endothelium, the H
2
O
2
-generating system xanthine/xanthine oxidase induced a catalase-sensitive relaxation that mimicked the gap junction-independent response to A23187 as it was maximally equivalent to ≈80% of induced tone, but associated with a smooth muscle hyperpolarization <5 mV. We conclude that myoendothelial gap junctions underpin smooth muscle hyperpolarizations evoked by A23187 and ACh, but that A23187-induced relaxation is dominated by extracellular release of H
2
O
2
. Endothelium-derived H
2
O
2
may thus be regarded as a relaxing factor, but not a hyperpolarizing factor, in rabbit arteries.
Evaluating plague and smallpox as historical selective pressures for theCCR5-Δ32HIV-resistance allele
Authors: Galvani Alison P. , Slatkin Montgomery
Published: December 2003

The high frequency, recent origin, and geographic distribution of theCCR5-Δ32deletion allele together indicate that it has been intensely selected in Europe. Although the allele confers resistance against HIV-1, HIV has not existed in the human population long enough to account for this selective pressure. The prevailing hypothesis is that the selective rise ofCCR5-Δ32to its current frequency can be attributed to bubonic plague. By using a population genetic framework that takes into account the temporal pattern and age-dependent nature of specific diseases, we find that smallpox is more consistent with this historical role.
Map-based isolation of the leaf rust disease resistance gene
Lr10
from the hexaploid wheat (
Triticum aestivum
L.) genome
Authors: Feuillet Catherine , Travella Silvia , Stein Nils , Albar Laurence , Nublat Aurélie , Keller Beat
Published: December 2003

More than 50 leaf rust resistance (
Lr
) genes against the fungal pathogen
Puccinia triticina
have been identified in the wheat gene pool, and a large number of them have been extensively used in breeding. Of the 50
Lr
genes, all are known only from their phenotype and/or map position except for
Lr21
, which was cloned recently. For many years, the problems of molecular work in the large (1.6 × 10
10
bp), highly repetitive (80%), and hexaploid bread wheat (
Triticum aestivum
L.) genome have hampered map-based cloning. Here, we report the isolation of the
Lr
gene
Lr10
from hexaploid wheat by using a combination of subgenome map-based cloning and haplotype studies in the genus
Triticum. Lr10
is a single-copy gene on chromosome 1AS. It encodes a CC-NBS-LRR type of protein with an N-terminal domain, which is under diversifying selection. When overexpressed in transgenic wheat plants,
Lr10
confers enhanced resistance to leaf rust.
Lr10
has similarities to
RPM1
in
Arabidopsis thaliana
and to resistance gene analogs in rice and barley, but is not closely related to other wheat
Lr
genes based on Southern analysis. We conclude that map-based cloning of genes of agronomic importance in hexaploid wheat is now feasible, opening perspectives for molecular bread wheat improvement trough transgenic strategies and diagnostic allele detection.
A peptide switch regulates DNA polymerase processivity
Authors: López de Saro Francisco J. , Georgescu Roxana E. , O'Donnell Mike
Published: December 2003

Chromosomal DNA polymerases are tethered to DNA by a circular sliding clamp for high processivity. However, lagging strand synthesis requires the polymerase to rapidly dissociate on finishing each Okazaki fragment. The
Escherichia coli
replicase contains a subunit (τ) that promotes separation of polymerase from its clamp on finishing DNA segments. This report reveals the mechanism of this process. We find that τ binds the C-terminal residues of the DNA polymerase. Surprisingly, this same C-terminal “tail” of the polymerase interacts with the β clamp, and τ competes with β for this sequence. Moreover, τ acts as a DNA sensor. On binding primed DNA, τ releases the polymerase tail, allowing polymerase to bind β for processive synthesis. But on sensing the DNA is complete (duplex), τ sequesters the polymerase tail from β, disengaging polymerase from DNA. Therefore, DNA sensing by τ switches the polymerase peptide tail on and off the clamp and coordinates the dynamic turnover of polymerase during lagging strand synthesis.
The RNA polymerase III transcriptome revealed by genome-wide localization and activity–occupancy relationships
Authors: Roberts Douglas N. , Stewart Allen J. , Huff Jason T. , Cairns Bradley R.
Published: December 2003

RNA polymerase III (Pol III) transcribes small untranslated RNAs, such as tRNAs. To define the Pol III transcriptome in
Saccharomyces cerevisiae
, we performed genome-wide chromatin immunoprecipitation using subunits of Pol III, TFIIIB and TFIIIC. Virtually all of the predicted targets of Pol III, as well as several novel candidates, were occupied by Pol III machinery. Interestingly, TATA box-binding protein occupancy was greater at Pol III targets than virtually all Pol II targets, and the highly occupied Pol II targets are generally strongly transcribed. The temporal relationships between factor occupancy and gene activity were then investigated at selected targets. Nutrient deprivation rapidly reduced both Pol III transcription and Pol III occupancy of both a tRNA gene and
RPR1
. In contrast, TFIIIB remained bound, suggesting that TFIIIB release is not a critical aspect of the onset of repression. Remarkably, TFIIIC occupancy increased dramatically during repression. Nutrient addition generally reestablished transcription and initial occupancy levels. Our results are consistent with active Pol III displacing TFIIIC, and with inactivation/release of Pol III enabling TFIIIC to bind, marking targets for later activation. These studies reveal new aspects of the kinetics, dynamics, and targets of the Pol III system.
Crystal structure of the complete core of archaeal signal recognition particle and implications for interdomain communication
Authors: Rosendal Ken R. , Wild Klemens , Montoya Guillermo , Sinning Irmgard
Published: December 2003

Targeting of secretory and membrane proteins by the signal recognition particle (SRP) is evolutionarily conserved, and the multidomain protein SRP54 acts as the key player in SRP-mediated protein transport. Binding of a signal peptide to SRP54 at the ribosome is coordinated with GTP binding and subsequent complex formation with the SRP receptor. Because these functions are localized to distinct domains of SRP54, communication between them is essential. We report the crystal structures of SRP54 from the Archaeon
Sulfolobus solfataricus
with and without its cognate SRP RNA binding site (helix 8) at 4-Å resolution. The two structures show the flexibility of the SRP core and the position of SRP54 relative to the RNA. A long linker helix connects the GTPase (G domain) with the signal peptide binding (M) domain, and a hydrophobic contact between the N and M domains relates the signal peptide binding site to the G domain. Hinge regions are identified in the linker between the G and M domains (292-LGMGD) and in the N-terminal part of the M domain, which allow for structural rearrangements within SRP54 upon signal peptide binding at the ribosome.
A coarse-grained model for a nanometer-scale molecular pump
Authors: Hod Oded , Rabani Eran
Published: December 2003

A theory for a nanometer-scale pump based on the ratchet concept is presented. A lattice gas model with a set of moves that satisfy hydrodynamic equations is used to describe an asymmetric nanometer channel connecting two reservoirs of fluid. The channel, which is coupled to an external oscillatory (or stochastic) driving force, pumps fluid from one reservoir to the other. The frequency of the external driving force, the fluid density, and the channel dimensions are used to control the fluid flow. We observe a nonmonotonic behavior of the flow with respect to some model parameters and discuss the efficiency of the device.
Converting conformational changes to electrostatic energy in molecular motors: The energetics of ATP synthase
Authors: S̆trajbl Marek , Shurki Avital , Warshel Arieh
Published: December 2003

F
1
-ATPase is the catalytic component of the ATP synthase molecular machine responsible for most of the uphill synthesis of ATP in living systems. The enormous advances in biochemical and structural studies of this machine provide an opportunity for detailed understanding of the nature of its rotary mechanism. However, further quantitative progress in this direction requires development of reliable ways of translating the observed structural changes to the corresponding energies. This requirement is particularly challenging because we are dealing with a large system that couples major structural changes with a chemical process. The present work provides such a structure–function correlation by using the linear response approximation to describe the rotary mechanism. This approach allows one to evaluate the energy of transitions between different conformational states by considering only the changes in the corresponding electrostatic energies of the ligands. The relevant energetics are also obtained by calculating the linear response approximation-based free energies of transferring the ligands from water to the different sites of F
1
-ATPase in their different conformational states. We also use the empirical valence bond approach to evaluate the actual free-energy profile for the ATP synthesis in the different conformational states of the system. Integrating the information from the different approaches provides a semiquantitative structure–function correlation for F
1
-ATPase. It is found that the conformational changes are converted to changes in the electrostatic interaction between the protein and its ligands, which drives the ATP synthesis.
Rapamycin is active against B-precursor leukemiain vitroandin vivo, an effect that is modulated by IL-7-mediated signaling
Authors: Brown Valerie I. , Fang Junjie , Alcorn Keith , Barr Rosalind , Kim Jenny M. , Wasserman Robert , Grupp Stephan A.
Published: December 2003

A balance between survival and apoptotic signals regulates B cell development. These signals are tightly regulated by a host of molecules, including IL-7. Abnormal signaling events may lead to neoplastic transformation of progenitor B cells. Signal transduction inhibitors potentially may modulate these abnormal signals. Inhibitors of the mammalian target of rapamycin (mTOR) such as rapamycin have been used as immunosuppressive agents. We hypothesized that rapamycin might demonstrate activity against B-precursor acute lymphoblastic leukemia. We have found that rapamycin inhibited growth of B-precursor acute lymphoblastic leukemia linesin vitro, with evidence of apoptotic cell death. This growth inhibition was reversible by IL-7. One candidate as a signaling intermediate cross-regulated by rapamycin and IL-7 was p70 S6 kinase. Rapamycin also demonstratedin vivoactivity in Eμ-ret transgenic mice, which develop pre-B leukemia/lymphoma: Eμ-ret transgenic mice with advanced disease treated daily with rapamycin as a single agent showed a >2-fold increase in length of survival as compared with symptomatic littermates who received vehicle alone. These results suggest that mammalian target of rapamycin inhibitors may be effective agents against leukemia and that one of the growth signals inhibited by this class of drugs in precursor B leukemic cells may be IL-7-mediated.
Site-specific recombination by the DDE family member mobile element IS30transposase
Authors: Kiss János , Szabó Mónika , Olasz Ferenc
Published: December 2003

DNA rearrangements carried out by site-specific recombinases and transposases (Tpases) show striking similarities despite the wide spectrum of the catalytic mechanisms involved in the reactions. Here, we show that the bacterial insertion sequence (IS)30element can act similarly to site-specific systems. We have developed an inversion system using IS30Tpase and a viable λ phage, where the integration/excision system is replaced with IS30. Both models have been proved to operate analogously to their natural counterpart, confirming that a DDE family Tpase is able to fulfill the functions of site-specific recombinases. This work demonstrates that distinction between transposition and site-specific recombination becomes blurred, because both functions can be fulfilled by the same enzyme, and both types of rearrangements can be achieved by the same catalytic mechanisms.
Mucosal tolerance to E-selectin provides cell-mediated protection against ischemic brain injury
Authors: Chen Yong , Ruetzler Christl , Pandipati Sruthi , Spatz Maria , McCarron Richard M. , Becker Kyra , Hallenbeck John M.
Published: December 2003

We have demonstrated that induction of mucosal tolerance to E-selectin, a cytokine-inducible adhesion molecule restricted to activating blood vessels, prevents ischemic and hemorrhagic stroke in spontaneously hypertensive, genetically stroke-prone (SHR-SP) rats. We now examine whether mucosal tolerance to E-selectin has protective effects in ischemic brain damage after permanent middle cerebral artery occlusion (MCAO) in SHR-SP rats and whether these effects are related to generation of regulatory T cells. Rats were exposed to intranasal administration of E-selectin every other day for 10 days (single tolerization group) or on two tolerization schedules separated by 11 days (booster tolerization group). Control groups received PBS on corresponding schedules. MCAO was performed 48 h after the last dose of E-selectin or PBS. There were 45.8% and 37.9% (P< 0.05) decreases of infarction volume in the E-selectin booster group compared with the PBS group at 6 and 48 h, respectively. Single tolerization with E-selectin had only a slight trend toward a decrease in infarction volume (6.3%). CD8-positive cells were decreased in brains of E-selectin booster animals (46.6%,P< 0.01) compared with controls; splenocyte-culture supernatant levels of IL-10 were increased (59.3%,P< 0.05) in E-selectin booster animals. A decrease of infarction volume (34%,P< 0.05) was also observed in SHR-SP rats subjected to MCAO after adoptive transfer of splenocytes from E-selectin-tolerized compared with PBS-tolerized donors. The results indicate that, in addition to preventing stroke, mucosal tolerance to E-selectin is cytoprotective. Thus, immunomodulation targeted to activated blood vessel segments can both reduce stroke occurrence and attenuate brain damage if a stroke supervenes.
Reduction of lipocalin-type prostaglandin D synthase in the preoptic area of female mice mimics estradiol effects on arousal and sex behavior
Authors: Mong Jessica A. , Devidze Nino , Goodwillie Andrew , Pfaff Donald W.
Published: December 2003

In female rodents, sleep and activity levels fluctuate over the estrous cycle. When estradiol (E
2
) levels are highest, sleep is reduced whereas locomotion is increased. The preoptic area (POA) is a key site for estrogenic regulation of these functions. However, molecular mechanisms by which E
2
acts to reduce sleep and increase activity are unclear. Recently, we demonstrated a twofold reduction in lipocalin-type prostaglandin D synthase (L-PGDS) transcript levels, after E
2
treatment, in the ventrolateral POA (VLPO), a putative sleep-active nucleus. Catalytic activity of L-PGDS produces PGD
2
, an endogenous somnogen. Thus, we hypothesized that decreases in PGD
2
in the VLPO may contribute to the generalized arousal mediated by estrogens. To test this, we infused (
i
) antisense oligonucleotides (oligos), containing locked nucleic acid moieties (an improved technology), targeted to L-PGDS mRNA, (
ii
) scrambled sequence control oligos, or (
iii
) saline vehicle into the VLPO of ovariectomized female mice treated with E
2
or oil. Arousal states and activity levels were assessed in response to a series of sensory stimuli (vestibular, olfactory, and somatosensory). The vestibular stimulus, which was administered first, resulted in the strongest responses and elicited significantly different responses among the groups: all groups in the E
2
cohort demonstrated increases in overall home cage activity and duration of that activity compared with the oil-treated control groups. As predicted from E
2
suppression of L-PGDS transcript levels, the responses of the locked nucleic acid antisense oligo-treated animals from the oil cohort did not differ from the E
2
-treated groups, such that they also demonstrated increases in activity and duration of activity compared with their controls. Thus, reducing L-PGDS in the VLPO of oil-treated females mimicked the effect of E
2
on activity and arousal and represents a unique molecular pathway through which E
2
may modulate these functions.
Notch signaling controls multiple steps of pancreatic differentiation
Authors: Murtaugh L. Charles , Stanger Ben Z. , Kwan Kristen M. , Melton Douglas A.
Published: December 2003

Multiple cell types of the pancreas appear asynchronously during embryogenesis, which requires that pancreatic progenitor cell potential changes over time. Loss-of-function studies have shown that Notch signaling modulates the differentiation of these progenitors, but it remains unclear how and when the Notch pathway acts. We established a modular transgenic system to heritably activate mouse Notch1 in multiple types of progenitors and differentiated cells. We find that misexpression of activated Notch in
Pdx1
-expressing progenitor cells prevents differentiation of both exocrine and endocrine lineages. Progenitors remain trapped in an undifferentiated state even if Notch activation occurs long after the pancreas has been specified. Furthermore, endocrine differentiation is associated with escape from this activity, because
Ngn3
-expressing endocrine precursors are susceptible to Notch inhibition, whereas fully differentiated endocrine cells are resistant.